Renal Ischemia/Reperfusion

Injury in Diabetes
Experimental Studies in the Rat
BY
JAN MELIN
Comprehensive Summaries oI Uppsala Dissertations
Irom the Faculty oI Medicine 1131
Renal Ischemia/ReperIusion Injury in
Diabetes
Experimental Studies in the Rat
BY
JAN MELIN
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2002
Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Internal
Medicine presented at Uppsala University 2002.
ABSTRACT
Melin, J. 2002. Renal Ischemia/Reperfusion Injury in Diabetes
Experimental Studies in the Rat. Acta Universitatis Upsaliensis. Comprehensive Summaries
of Uppsala Dissertations from the Faculty of Medicine 1131. 55 pp. Uppsala ISBN 91-554-
5264-7
Diabetes mellitus (DM) is one of the leading causes of end stage renal failure. An increased
susceptibility to renal ischemia/reperfusion (I/R)-injury was found in DM rats. Unilateral
renal ischemia for as short as 20 minutes led to an irreversible progressive injury in DM
kidneys, whereas the injury in non-DM kidneys was almost reversible. The renal I/R injury
was characterized by anuria, infiltration of inflammatory cells, tubular atrophy, dilation of the
remaining tubuli and tubulointerstitial fibrosis. Necrotic areas were found in the inner parts of
the outer medulla and in the papilla. The renal medulla was the most vulnerable part of the
kidney. This was seen both by the extent of fibrosis four and eight weeks after I/R and by the
presence of TUNEL-positive (apoptotic) cells 6h after ischemia. Increased accumulation of
HA and enhanced CD44 expression was seen after I/R in DM kidneys.
Treatment with long acting insulin 7-14 days before I/R, decreased the number of
apoptotic cells in the renal medulla and protected renal function and morphology after the
insult, while insulin treatment after the injury did not have any protective effect. Short acting
insulin given 2-6 hours before I/R partially protected renal function but did not improve the
morphological picture.
Treatment with the angiotensin II receptor type 1 blocker candesartan, the PAF-
antagonist UR-12670, the immunosuppressive agents tacrolimus and cyclosporin A, or
prednisolone did not improve the outcome of the renal I/R injury in DM. Injection of cobalt
protoporphyrin (CoPP) intraperitoneally in order to induce an over-expression of heme
oxygenase-1 (HO-1) resulted in a trend towards a better function in DM kidneys after I/R.
However, the induction of HO-1 by intraperitoneal CoPP injection was not achieved in all
rats, when examined by western blot.
In conclusion, unilateral renal I/R leads to a severe progressive injury in DM
kidneys. Insulin treatment before ischemia, but not after, reduces the renal injury in DM rats.
Studies using a more reliable administration of CoPP are required to decide if induction of
HO-1 protects against renal I/R injury in DM
Key words: diabetes mellitus, ischemia/reperfusion, rat, end-stage renal disease,
inflammation, fibrosis, hyaluronan, CD44, treatment.
Jan Melin, Department of Medical Sciences, Uppsala University, University Hospital, SE-751
85, Uppsala, Sweden
© Jan Melin, 2002
ISSN 0282-7476
ISBN 91-554-5264-7
Printed in Sweden by Akdemitryck AB, Edsbruk 2002
To Camilla, Erik and Amanda
PAPERS
This thesis is based on the following papers, which will be referred to in the text
by their roman numerals:
I Melin J, Hellberg O, Aküyrek ML, Källskog Ö, Larsson E and Fellström B:
Ischemia Causes Rapidly Progressive Nephropathy in the Diabetic Rat
Kidney Int (1997) 52:985-991
II Melin J, Hellberg O, Larsson E, Zezina L, and Fellström BC:
Protective Effect of Insulin on Ischemic Renal Injury in Diabetes Mellitus
Kidney Int, in press (2002) 61(4)
III Melin J, Hellberg O, Funa K, Hällgren R, Larsson E and Fellström BC:
Ischemia-Induced Renal Expression of Hyaluronan and CD44 in Diabetic
Rats
Manuscript
IV Melin J, Larsson E, and Fellström BC:
Pharmacological Interventions in Renal Ischemia/Reperfusion Injury in
Diabetes
Manuscript
Reprints were made with permission from Kidney International.
TABLE OF CONTENTS
ABBREVIATIONS 6
BACKGROUND 7
Renal disease and diabetes 7
Pathophysiology of diabetic nephropathy 7
Animal models of DM-nephropathy 10
Treatment of DM nephropathy 11
Pathophysiology of ischemic renal failure 11
Hyaluronan in renal disease 15
AIMS OF THE INVESTIGATION 16
MATERIAL AND METHODS 17
Induction of diabetes (Studies I-IV) 17
Renal artery clamping (Studies I-IV) 17
Inulin clearance (Studies I, II and IV) 17
Histological evaluation (Studies I-IV) 18
Evaluation of tubular atrophy and glomerular injury (Study I) 18
Insulin treatment (Study II) 18
TUNEL (Study II) 19
Hyaluronan staining (Study III) 20
Immunohistochemistry (Study III) 20
Hyaluronan and water content measurements (Study III) 21
Pharmacological treatments (Study IV) 21
Western blot against HO-1 (Study IV) 22
Statistical analysis (Study I-IV) 22
RESULTS 23
Study I 23
Study II 24
Study III 25
Study IV 27
GENERAL DISCUSSION 30
GENERAL SUMMARY 41
ACKNOWLEDGEMENTS 42
REFERENCES 44
6
ABBREVIATIONS
ACE Angiotensin converting enzyme
AGE Advanced glycosylation end products
Ang II Angiotensin II
AT-1/2 Angiotensin II receptor type-1/type-2
b. w. Body weight
BGC Blood glucose concentration
BSA Bovine serum albumin
C
In
Inulin clearance
CoPP Cobalt IX protoporphyrin chloride
CsA Cyclosporin A
DM Diabetes mellitus
EM Extracellullar matrix
ESRD End stage renal disease
GFR Glomerular filtration rate
HA Hyaluronan (Hyaluronic acid)
HO-1/2 Heme oxygenase-1/2
HSP Heat shock protein
I/R Ischemia/reperfusion
IARF Ischemic acute renal failure
ICAM-1 Intercellular adhesion molecule-1
IGF-1 Insulin like growth factor - 1
iNOS Inducible-nitric oxide synthase
MAP Mean arterial pressure
NOS Nitric-oxide synthase
PAF Platelet activating factor
RAS Renin angiotensin system
RNS Reactive nitrogen species
ROS Reactive oxygen species
RVR Renal vascular resistance
STZ Streptozotocin
TGF-β Transforming growth factor beta
TUNEL Terminal deoxynucleotidyl transferase (TdT) mediated nick-end-
labeling
VCAM-1 Vascular cell adhesion molecule-1
7
BACKGROUND
Renal disease in diabetes
Diabetes mellitus (DM) is one of the leading causes of end stage renal disease
(ESRD), and in 1999 31.8% of all registered ESRD cases in the US were
attributed diabetic nephropathy [1]. The mechanisms behind the injury in
diabetic nephropathy are not fully understood despite intense research. The
frequency of diabetic nephropathy in patients with type-1 DM has decreased
significantly in recent years probably due to improvements in the treatment [2].
With a good metabolic control and aggressive treatment against hypertension,
diabetic nephropathy could be prevented or at least delayed in many DM type-1
patients [3]. In type-2 DM the picture is different [4], and the number of patients
with ESRD because of DM type-2 increases steadily. The main reason is the
steep increase in the number of patients with DM type-2, in combination with
ignorance of the condition by the patients. The incidence of nephropathy was
higher in type-2 diabetics than in DM type-1 patients in a recent study from
Japan [4]. Without proper treatment DM leads to progressive renal injury with
increasing proteinuria and ESRD in many patients.
Glomerular changes are mostly studied in diabetic renal disease. A typical
finding is thickening of the basal membranes in the glomeruli [5]. Two typical
patterns of glomerular changes have been described in diabetes. The nodular
Kimelstiel-Wilson lesion and the diffuse mesangial lesion. The Kimmelstiel-
Wilson lesion is characterized by nodular aggregates deposited in the
glomerulus [6, 7]. In the diffuse diabetic glomerular lesion a widespread
accumulation of extracellular matrix is observed throughout the glomerulus [6].
However, tubulointerstitial lesions are also prominent in DM. Interestingly, the
correlation between the renal function and the cortical tubulointerstitial changes
are at least as good, if not better, than the correlation between function and
glomerular changes [6, 8]. Interstitial inflammation, tubulointerstitial fibrosis
and tubular atrophy are, along with the glomerular changes, important features
of the progression of diabetic nephropathy towards ESRD [6] and similar
findings are seen in other renal diseases [9]. The pathways leading to this
scarring process are not fully elucidated. TGF-β has been suggested to be
involved in glomerular matrix accumulation [10]. The importance of
inflammation in DM nephropathy has been demonstrated, as in several other
chronic renal diseases [6, 9, 11].
Pathophysiology of diabetic nephropathy
Hemodynamic mechanisms
Some authors have claimed that increased renal microcirculation is the main
cause of DM-nephropathy [12]. DM rats have an elevated GFR compared to
8
non-DM rats [13]. Increased mesangial thickening has been demonstrated in
uninephrectomized DM rats compared to intact DM rats three and six months
after the operation, indicating that an increased workload is harmful to the DM
kidney [14]. Furthermore treatment with the ACE inhibitor enalapril protects
against glomerular injury and albuminuria in DM rats possibly by reducing the
glomerular capillary pressure [15]. Increased renal plasma flow (RPF) has
resulted in renal injury in other models. Protein loading, which elevated
glomerular plasma flow and GFR in DM rats after 2-10 weeks, resulted in a
higher number of sclerotic glomeruli and increased proteinuria after 11-13
months [16]. Glomerular and tubulointerstitial injury has been demonstrated in
the 5/6 nephrectomized rat, and in this model increased angiotensin II (Ang II)
expression, which can cause injury via a TGF-β dependent pathway has been
suggested to be involved in the pathogenesis [17]. Upregulated expression of the
PDGF-B chain has been advocated to be important in the renal injury in the 5/6
nephrectomized rat [18]. A dysfunction of the dilatory response of the afferent
arterioles in diabetic kidneys has been suggested to cause increased glomerular
capillary flow in DM kidneys [19, 20]. Insulin treatment has been shown to
lower the elevated GFR in DM rats [21].
Hyperglycemia per se leads to elevated blood pressure in humans. Using a
glycemic clamp in type-1 DM subjects it was found that the mean arterial
pressure (MAP) and renal vascular resistance (RVR) was higher during
hyperglycemia (BCG 9 to 11 mmol/L) compared to euglycemia (BGC 4 to 6
mmol/L) [22]. In this study the ACE inhibitor enalapril lowered the MAP and
the RVR in the hyperglycemic situation but not in euglycemia. In the same study
it was also found that Ang II infusion caused an increase of MAP and RVR both
in the euglycemic and hyperglycemic situation and that the increase was greater
under normoglycemic conditions. The author concluded that these findings
support the involvement of the RAS system in propagating injury due to
hyperglycemia. ACE-inhibition is the established antihypertensive treatment in
diabetes. ACE-inhibitors may protect against diabetic nephropathy in other ways
than merely by lowering the blood pressure such as decrease the profibrogenic
effect of Ang II [23]. However, the mechanisms of Ang II involvement in
diabetic renal disease are far from clear. In most studies the systemic levels of
angiotensin and renin are lower in diabetes, but increased intrarenal RAS
activation has been found by some authors [24].
Angiotensin II
There are two kinds of Ang II receptors, type 1 and type 2 (AT-1

and

AT-2).
They have been found to have profoundly different effects in the kidney. The
AT-1

is mainly blamed to be involved in renal pathology, while activation of
9
AT-2 has antihypertensive and anti-proliferative effects in some models, and
may thus be renoprotective [25]. It has recently been reported that the renal
expression of AT-2 is downregulated in STZ-DM and the authors suggest that
an imbalance between AT-1

and

AT-2 could contribute to the renal injury [26].
The downregulation of AT-2 is partially restored by insulin treatment.
Ang II has been proposed to stimulate fibrosis via TGF-β [27]which has been
implicated in the development of diabetic nephropathy [10]. Chronic Ang II
infusion for two weeks caused a significant renal injury in rats despite only a
moderate increase in the MAP [28]. In 1998 a new model for diabetic
nephropathy was introduced: the STZ-DM (mRen-2)27 rat (TGR). In the TGR
an additional mouse renin gene has been transfected into the genome of
Sprague-Dawley rats. Glomerular changes very similar to human diabetic
nephropathy along with tubulointerstitial lesions and decreased GFR have
developed 12 weeks after induction of DM in the STZ-DM TGR-rat [29]. The
findings in the STZ-DM TGR-rat support the involvement of RAS in DM
nephropathy.
Advanced glycosylation end products
High BGC causes proteins to be non-enzymatically glycosylated. These
reactions occur in several steps leading eventually to the formation of advanced
glycosylation end products (AGE). AGE proteins have been suggested to play a
role in the development of DM-nephropathy. AGE can affect the kidney in
several ways such as causing oxidative stress [30], initiate expression of
potentially harmful adhesion molecules [31], by inducing growth factors and
stimulating extracellular matrix synthesis[32]. AGE has also been found to
increase vascular tone by quenching NO [33]. The levels of AGEs in the
circulation can be measured by an ELISA [34]. Several well-defined AGEs have
been synthesized but these are not identical to what is found in vivo [34]. Serum
levels of AGE are elevated in DM and Non-DM patients with ESRD but not in
DM patients with normal renal function [35].
Oxidative stress
There is evidence for increased lipid peroxidation in experimental DM [36].
However, the importance of oxidative stress in the early stages of human DM
has been challenged. Baynes and co-workers [37] suggest that the signs
indicating oxidative stress in DM are in fact due to insufficient detoxification of
AGEs which they called carbonyl stress. The authors did not, however, exclude
oxidative stress locally in tissues, or the role of oxidative stress in DM when
advanced complications had developed. However, the link between oxidative
stress and renal injury in DM rats remains rather strong. For instance, enalapril
10
treatment decreases tubulointerstitial injury and levels of oxidative stress
markers in the DM kidney [38].
Metabolic disturbances
In diabetes several metabolic disturbances take place in the cells. One example
is the polyol pathway, which is enhanced in DM. In the polyol pathway glucose
is reduced to sorbitol by aldose reductase and further dehydrogenated to fructose
by sorbitol dehydrogenase. One effect of the increased polyol pathway could be
creatin an increased NADH/NAD+ ratio and cause a pseudohypoxic state [39].
The influence of two enzymes involved in the polyol pathway, aldose reductase
and sorbitol dehydrogenase, on the progression of DM nephropathy has recently
been studied. Increased mRNA expression of aldose reductase and sorbitol
dehydrogenase, was only seen in cells from patients with DM nephropathy and
not in cells from DM patients without renal disease or healthy controls, when
peripheral blood mononuclear cells were grown in high glucose media [40]. The
authors suggest that disturbed expression of enzymes in the polyol pathway may
be involved in the development of DM nephropathy. Furthermore this study also
showed a possible link between certain genotypes for these enzymes and
susceptibility to DM nephropathy.
Shortage of C-peptide.
For a long time C-peptide was viewed as an unimportant by-product. This view
has recently been challenged, and some studies have shown that C-peptide has
important effects by itself. For instance C-peptide decreases the hyperfiltration
in DM-rats [41].
Animal models of DM-nephropathy
Several animal models have been developed for investigating the mechanisms
behind diabetic complications. Diabetes can be induced chemically by injection
of streptozotocin [42] or alloxan [43]. Decreased renal function at the age of 60
weeks, has been demonstrated in obese Zucker rats [44]. In Zucker rats DM
develops spontaneously, and the model is generally considered to represent DM
type-2. However, Zucker rats are only moderately hyperglycemic and were
initially recognized as a model for hyperlipidemia [44, 45]. STZ-DM rats
develop typical diabetic lesions early, such as hyperfiltration, progressive
basement membrane thickening, albuminuria and renal hypertrophy. However,
classical signs of DM glomerulosclerosis never develop [46]. Tubulointerstitial
signs of injury in STZ-DM are more pronounced. They start with vacuolization
of tubular cells and subsequently tubular atrophy, infiltration of inflammatory
cells and fibrosis after several months [38, 47]. Tubular vacuolization has been
described in man, but is probably rare nowadays because of insulin therapy [48].
11
Some tubular and glomerular damage has been observed in aging non-DM rats.
These changes are strain dependent and become even more prominent in
uninephrectomized rats [49]. In order to get a more severe and rapid injury STZ-
DM has been combined with other modifications, such as salt loading[50] or
introduction of an additional murine renin gene in the genome of rats that are
subsequently made diabetic[29]. In order to achieve a more severe renal injury
in DM we combined DM with unilateral ischemia.
Treatment of diabetic nephropathy
In recent years substantial improvement has been made in the treatment of DM
nephropathy especially in type-1 DM patients. The basis for the treatment is to
normalize metabolic and hemodynamic disturbances thought to be involved in
the pathophysiology. So far ACE-inhibitors have been the established
antihypertensive therapy in DM. However, AT-1 blockers have also been shown
to have renoprotective effects in DM type 2 patients [51-53]. Prevention of
nephropathy is more difficult in DM type-2 than in type-1, since a good
metabolic control is more difficult to achieve in type-2 patients due to insulin
resistance. DM Type-2 patients are as a rule older and have more concomitant
diseases. Experimental evidence suggests the possibilty that renal sensitivity to
insults such as I/R increase with age [54]. The number of sclerotic glomeruli
increases with age [55] and in most patients GFR also decreases with age [56].
Pathophysiology of ischemic acute renal failure
Despite tremendous efforts the pathophysiology of ischemic acute renal failure
(IARF) remains elusive. IARF has mostly been studied in different rodent
models but they are not identical to IARF in man. In rodents models several
factors ameliorating the injury have been identified. It seems clear that the
etiology of renal I/R injury is multi-factorial. An important factor when
evaluating the results of treatment studies concerning renal I/R is the
observation time. Some treatments have a protective effect on renal function in
the acute phase, while they do not change the outcome in the long run. One
example is that treatment with SOD and sucrose decreased erythrocyte trapping
and tubular cast formation in the acute phase of renal I/R injury, without
changing the long-term outcome [57]. Damage to peritubular capillaries could
be present in kidneys after I/R despite normal inulin clearance and only modest
morphological signs of renal injury [58]. This could mean that kidneys that
appear to have recovered almost completely might harbor injuries that will lead
to progressive injury in the end. No curative treatment for ischemic IARF has
been established in man to date, and dialysis still remains the only way to treat
the condition [59].
12
ATP-depletion
The shortage of oxygen during ischemia leads to a rapid fall in the intracellular
levels of ATP [60]. Shortage of ATP can cause both necrosis and apoptosis in
tubular cells [61]. ATP is first dephosphorylated to ADP and AMP. AMP is then
degraded to adenosine, and further to xanthine via inosine and hypoxanthine.
The step from inosine to hypoxanthine is irreversible while inosine could be
transformed back to adenosine if the oxygen supply to the cell is restored. The
reaction from hypoxanthine produces reactive oxygen species (ROS) if it takes
place in an aerobic environment such as during reperfusion. After reoxygenation
the ATP levels are restored in a biphasic manner [62]. First the available AMP is
phosphorylated to ATP and then new ATP is synthesized from inosine de novo.
ATP does not reach normal levels immediately and there is a correlation
between the duration of the ischemia and the decrease in ATP during the
recovery. Furthermore, the ATP concentration after 120 minutes of reperfusion
was correlated to renal function after 24 hours [62]. The basal levels of ATP,
ADP and AMP in the kidney are not altered in STZ-DM [63].
Medullary hypoxia
Low oxygen tension has been documented in the renal medulla, and the
medullary oxygen partial pressure is increased when loop diuretics are
administrated, suggesting active reabsorbtion in the medullary thick ascending
limb as the main reason for the hypoxia [64]. Brezis and colleagues have
proposed that the renal sensitivity to ischemia is due to the hypoxic conditions in
the renal medulla [64]. In diabetes, the hypoxia in the medulla might be even
more pronounced due to the increased work of the Na
+
,K
+
-ATPase [65]. This
could thus be a possible explanation for the increased sensitivity to ischemia in
DM rats [66].
Oxidative and nitrosative stress
ROS has been implicated as a mediator of renal I/R in by numerous authors. The
increased levels of ROS found in the kidneys after I/R have been explained in
several ways. Hypoxanthine accumulates in the kidney during ischemia, and in
the presence of oxygen, such as during reperfusion, the degradation of
hypoxanthine to xanthine generates ROS. Since the level of hypoxanthine
correlates with the duration of ischemia [60] it seems possible that reperfusion
injury, at least in part, is caused by ROS produced by this reaction. However, the
role of this mechanism is controversial. Paller and co-workers found that several
anti-oxidative agents including allopurinol had protective effects against renal
I/R injury [67]. Paller et. al. Gave, however, the control animals dextrose which
could possibly be deleterious for the kidneys in I/R by increasing BGC. In
another study a dose-dependent protective effect against renal I/R injury by a
13
different xanthine-oxidase inhibitor, oxypurinol, was found, and surprisingly
oxypurinol was renoprotective even when given 40 minutes after I/R [68].
However, in another study no protection against I/R no beneficial effect of
treatment with allopurinol or oxypurinol could be shown, ann this study the
effectiveness of the xanthine-oxidase inhibitors was confirmed by nucleotide
measurements [69]. Treatment with SOD, and SOD and sucrose, improved the
function in the acute phase after I/R but no functional improvement after one or
four weeks [57]. Taken together these studies indicate that ROS from the
degradation of hypoxanthine to xanthine is of some but limited importance in
renal I/R. Evidence for oxidative stress and generation of reactive nitrogen
species already during ischemia has recently been presented and these authors
found no evidence for further increased levels of ROS and RNS during
reperfusion[70]. Other authors have also found evidence for RNS in renal I/R.
Peroxynitrite (ONOO
-
) a highly reactive species formed by NO and superoxide
has been implied in renal I/R injury [71]. Treatment with SOD, an iNOS
inhibitor or the combination of SOD and the iNOS inhibitor, ameliorated
ischemic renal failure and decreased nitrostyrosine formation, lipid peroxidation
and DNA damage. A scavenger of ONOO
-
, ebselen, had exactly the same
effects indicating a role for ONOO
-
in renal I/R injury [71]. Another and less
controversial source of ROS in I/R injury is the influx of inflammatory cells
especially neutrophils, which can release ROS. Several studies have indicated
the importance of neutrophils in renal failure after I/R [72, 73]. Increased levels
of ROS have been associated with increased apoptosis in proximal tubuli but not
in distal tubuli [74].
Erythrocyte trapping
Trapping of erythrocytes in the renal medulla after I/R has been demonstrated
and the trapping increase with the duration of the ischemia [75]. Erythrocyte
trapping could result in secondary ischemia, which could affect the degree of
renal I/R injury. Hemodilution has been found to decrease the trapping and
improve long-term outcome while hemoconcentration resulted in a more
pronounced trapping and worse long-term outcome. [75].
Apoptosis
Necrosis was until recently considered the only important mean of cell death in
IARF, however, new evidence suggests a role also for apoptosis in this process
[76]. TUNEL-positive cells have been demonstrated repeatedly 4-48 hours after
I/R in kidneys [74, 77-79]. The basis for TUNEL-positivity is nuclear DNA
fragmentation[80], which is considered a hallmark of apoptosis. A correlation
between the incidence of apoptosis and the severity of the renal I/R injury has
been shown [74]. Positive TUNEL-staining does not exclude necrosis, and DNA
14
fragmentation by an endonuclease without signs of apoptosis after I/R has been
observed in vitro, and in this study endonuclease inhibitors increased cell
survival. [81]. Other evidence of apoptosis than TUNEL-positivity in the acute
phase after renal I/R has been reported. DNA fragmentation was evident already
2 hours after ischemia in mice. Decreased DNA fragmentation after I/R in
animals treated with α
1
-antitrypsin and α
1
-acid glycoprotein was associated with
improved renal function. [82]. 48 hours after I/R apoptosis was confirmed in the
kidney by electron microscopy [83]. In several studies anti-apoptotic agents
such as IGF-1 and hepatocyte growth factor (HGF) have decreased apoptosis
and the injury after renal I/R [77, 83]. Tacrolimus and cyclosporin CsA also
decrease both the injury and the degree of apoptosis if administrated before I/R
[84]. The number of apoptotic cells could be reduced and the renal function
improved by guanosine supplementation in mice subjected to renal I/R, whereas
no improvement in the morphological appearance of the injury in kidneys from
the treated animals was detected [85].
Inflammation
The inflammatory response is a prominent feature after I/R injury. Infiltrating
inflammatory cells have also been described in human acute tubular necrosis
[86]. Several pro-inflammatory cytokines such as interferon-γ, interlukin-2 (IL-
2), IL-10, granulocyte-macrophage colony stimulating factor and TGF-β are
induced by renal I/R [87]. Further evidence for the role of leukocytes in renal
I/R is provided by the finding that antibodies against ICAM-1 have a protective
effect against renal I/R [88]. T-cells have also been implied in the acute phase of
the renal I/R injury in DM since T-cell deficient mice were partially protected
from I/R injury[89]. However, using knock out mice deficient in the
recombination activating gene, which are defect in T and B-lymphocytes and do
not produce immunoglobulins or T-cell receptor protein, Park and co-workers
claimed that renal I/R-injury is independent of immunoglobulins and T-
lymphocytes. However, in this study the animals were only observed for 30
hours [90]. The inflammatory response to renal I/R in non-DM animals appears
to be transient and occurs in several waves. First an increase in the number of
neutrophils is observed, and some authors claim that this wave peaks at three
hours after ischemia and is almost gone after 24 hours [91]. The number of
monocytes and macrophages started to increase two days after I/R and peaked
after eight days in another study. [78]. However, when observed long enough a
new wave of inflammatory cells is found after several weeks and this was
enhanced when renal I/R was combined with uninephrectomy [92, 93].
15
Hyaluronan in renal diseases
Hyaluronan is a large polysaccharide consisting of repeated glucuronic acid N-
acetylglucosamine disaccharides, which has a unique ability to bind water [94].
HA is ubiquitously expressed in areas of inflammation [95]. In the healthy
kidney the papilla contains a very high concentration of HA, while the renal
cortex is almost free of HA [96, 97]. HA has been suggested to be involved in
the control of water handling in the kidney. Increased HA accumulation in the
renal cortex has been described in several human and experimental renal
diseases. Both acutely and chronically rejected kidneys show an increased HA
expression in the renal cortex [97-99]. In autoimmune glomerulonephritis in rats
HA is present in the crescents [100, 101]. In a mouse model of interstitial
nephritis increased HA expression was also observed [102]. An increased renal
HA content has been demonstrated in DM patients [103], and after renal I/R
[104]. HA can bind to the CD44 receptor [105], and renal CD44 expression is
enhanced by I/R [106]. Interaction between CD44 positive leukocytes and
interstitial HA has been suggested as a mechanism for the infiltration of
inflammatory cells in renal injury [107]. Fragmented HA may exert a pro-
inflammatory effect by inducing ICAM-1 [108], chemokines [109] and
monocyte chemoattractant protein-1 [110].
16
AIMS OF THE INVESTIGATION:
• To study the effects of renal ischemia/reperfusion injury diabetic rats and to
evaluate if this combination could be a tool in the study of DM-nephropathy.
• To examine the effects of insulin treatment before or after I/R on the
outcome of renal I/R injury in diabetic rats.
• To study the role of hyaluronan and hyaluronan/CD44 interaction on renal
I/R injury in DM.
• To investigate if immunomodulating therapy or induction of protective genes
could reduce the renal injury after I/R in DM kidneys.
17
MATERIAL AND METHODS
Induction of diabetes (Studies I-IV)
The animals were rendered diabetic by a single injection of STZ (Zanosar,
Upjohn, Kalamazoo, MI; 0.55 mg/ kg body weight). The solution contained 11
mg/ml of STZ in saline with a sodium citrate buffer, pH 4.0. Food and water
consumption, weight gain and the blood glucose levels were recorded to monitor
the degree of diabetes. In study I and III, blood taken from the femoral vein was
analyzed by the glucose dehydrogenase method (Granutest 250; Merck,
Whitehouse Station, NJ, USA). In study II and IV blood obtained from the tip of
the tails was analyzed by a modified glucose dehydrogenase method (Hemocue,
Ängelholm, Sweden). The rats were considered diabetic if the BGC (blood
glucose concentration) was above 20 mmol/L or if they drank at least 100
mL/day. At least two weeks elapsed between the induction of diabetes and
ischemic injury.
Renal artery clamping (Studies I-IV)
The animals were anesthetized by an intraperitoneal injection of Eqviticin (a
mixture of chloral hydrate 183 mg/kg

body weight, magnesium sulfate 0.09
mg/kg body weight and thiobarbital sodium 42 mg kg/body weight,
Apoteksbolaget, Umeå, Sweden) In the experiments where the effects of CoPP
were studied the rats were anesthetized by an intraperitoneal bolus of equal parts
fentanyl and fluanisone (Hypnorm, Janssen) and midazolam (Dormicum 5
mg/ml, Roche). During the operation the animals were placed on a
servocontrolled heating pad keeping the body temperature at 37.5 °C. A midline
incision was made, and the left renal artery was located and dissected free from
its surrounding structures. After a recovery period of 10 minutes, renal ischemia
was achieved by clamping of the left renal artery for 30 minutes. Subsequently
the abdomen was sutured, and the animals returned to their cages.
Inulin clearance (Studies I,II and IV)
GFR was estimated from the clearance of inulin (C
In
). The animals were
anesthetized by Inactin (Byk Gulden, Konstanz, Germany) or Eqviticin and put
on an electric heating pad as described above and tracheostomized. Catheters
were inserted into both ureters through a supravesical incision. The right femoral
artery was catheterized for blood pressure monitoring and withdrawal of blood
samples, and the femoral vein for infusion of saline (5 ml/kg

body weight/h,
non-diabetic rats and rats treated with insulin after I/R, and 10 ml/kg

body
weight/h diabetic rats); After the operation the infusion was switched to a saline
containing 5 mCi/ml
3
H-inulin(American Radiolabeled Chemicals Inc, St Louis,
MO). After a bolus injection of 1 ml an equilibration period of 45 minutes
followed. Urine was then collected separately from both kidneys for 30 minutes.
18
In the middle of this period a reference blood sample was drawn. The
3
H
activity in plasma and urine was measured by the liquid scintillation technique.
Histological evaluation (Studies I-IV)
After the completion of functional studies both kidneys were removed and the
upper third of each kidney was put into a buffered 4% formalin fixation solution
with 1% N-Cetylpyridinium chloride and processed for histological
examination. The sections were subjected to Periodic Acid Shiff-staining for
detection of basal membranes and cell nuclei, and Picro-Sirius staining for the
investigation of fibrosis. The histological changes were blindly evaluated on
coded samples. On the arbitrary scale used, 0 indicated no sign of inflammation,
tubular dilation or fibrosis, 1 indicated mild changes, 2 moderate damage, and 3
severe lesions.
Evaluation of degree of tubular atrophy and glomerular injury (Study I)
In order to evaluate the degree of tubular atrophy and/or glomerular injury, the
number of glomeruli in four randomly selected visual fields (magnification
x160) in the cortex of each kidney was counted. To estimate the relative
glomerular size, two photographs were taken of the cortex on sections from
kidney. The sizes were then estimated by weighing the glomeruli cut out from
the photographs. The average size of the glomeruli in the non-ischemic, non-
DM group observed for four weeks was put at 1 and the values in the other
groups were normalized to this value.
Insulin treatment (Study II)
Insulatard MC insulin (NOVO - Nordisk, Denmark) was used and the treatment
started with a dose of 8 U for three days. Thereafter the dose was adjusted in
order to keep the BGC between 5 and 12 mmol/L. If the BGC fell below 5
mmol/L the dose was decreased by 1 U and if the BGC rose above 12 mmol/L
the dose was increased 1 U. This protocol was followed both for treatment with
long acting insulin both before and after ischemia. Treatment prior to ischemia
was started 7-14 days before the insult and treatment after ischemia was started
in the afternoon on the day after renal artery clamping.
19
In a separate series of experiments short acting insulin was used. The animals
were treated with 10-25 units of Actrapid MC (NOVO Nordisk, Denmark) 2-6h
before renal artery clamping. The efficacy of short acting insulin varied
considerably between the animals. Only if the animals had a BGC below 10,5
mmol/L prior to anesthesia were they admitted into the study. Some animals that
received short acting insulin but had a BGC above 10,5 mmol/L after treatment
were subsequently included into the study either as untreated controls or as
treated animals with a higher dose of insulin.
TUNEL (Study II)
The effect of insulin treatment on the development of apoptosis was evaluated
six hours after ischemia/reperfusion on snap frozen specimens. The animals
were divided into three groups: Non-diabetic, untreated diabetic, and diabetic
animals treated with long acting insulin for at least one week before I/R. (N=6 in
each group). The procedure was otherwise similar to what is described above.
Apoptotic cells were detected by terminal deoxynucleotidyl transferase (TdT)
mediated nick-end-labeling (TUNEL) assay (Roche Diagnostics Scandinavia
AB, Bromma, Sweden) according to the recommendations of the manufacturer.
Five µm thick frozen sections of the kidneys were fixed in 4%
paraformaldehyde in PBS, 20 min at RT, and permeabilized by 0.1% triton in
0.1% sodium citrate for 5 min on ice. Labeling mixture ( TdT and fluoroscein-
dUTP, 1:9) was applied, 50 µl/section, and the sections were incubated 1 hour at
37°C. Sections were finally rinsed in PBS, mounted with Vectashield (Vector
- 2 w
4 w
- 1 w 0
3 w
2 w 1 w
Daily insulin injections
Untreated DM
I / R Inulin clearance
STZ
Insulin before I/R
Insulin after I/R
Figure 1. Design of treatment with long acting insulin.
20
Laboratories Inc., Burlingame, CA), and analyzed under fluorescence
microscope coupled to an image analysis system Leica Qwin (Leica Imaging
Systems, Cambridge, UK). The apoptotic cells were defined by intensive green
fluorescence of the nuclei, and the number of positive cells was counted at 200x
magnification in five fields, separately in the medulla and the cortex. Results are
presented as a number of positive cells per mm
2
in medulla or cortex
respectively.
Hyaluronan staining (Study III)
Five µm formalin fixed sections were used. After rehydration and blocking of
the endogenous peroxidase by 0.3% H
2
O
2
for 30 minutes the samples were
incubated with 2% bovine serum albumin fraction V (BSA, Sigma St Louis MO,
USA) in phosphate buffered saline (PBS) for 30 minutes at room temperature.
The superfluous liquid was dried off and a biotinylated HABP (Hyaluronan
Binding Protein) in PBS [111] with 0.1% BSA was added and the incubation
continued for 14 hours at 4°C. Following rinsing, an avidin-biotin complex
coupled with peroxidase was added for 30 minutes (Dako, Glostrup, Denmark).
The specimens were developed with di-amino benzidine 0.3 mg/ml in PBS and
to which 0.005% H
2
O
2
was added. Finally the specimens were counterstained
with Meyer’s hematoxylin. The specimens were evaluated independently by two
investigators on coded samples. In the arbitrary scale used 0 meant no or
negligible HA staining, 1 some positive areas, 2 roughly 50% of the area was
stained and 3 HA positive areas dominating. Perivascular HA in the cortex was
disregarded in the evaluation.
Immunohistochemistry (Study III)
Five µm cryosections were put into 4°C acetone for ten minutes and then
allowed to dry. After washing in PBS (phosphate buffered saline) three times,
they were immersed into 0.3% H
2
O
2
for 15 minutes to eliminate endogenous
peroxidase. They were then incubated with 10% normal goat serum in PBS with
1% BSA for at least five minutes. Antibodies diluted in PBS with 1% BSA were
then added and incubated for 30 minutes, and the specimens were subsequently
rinsed in PBS and a goat anti-mouse-Ig antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA; 1:100) in PBS with 1% BSA was applied for 30
minutes. Following rinsing, mouse-PAP (Dako, Glostrup, Denmark; 1:250) in
PBS was added. The specimens were developed with di-amino benzidine 0.3
mg/ml in PBS and to which 0.005% H
2
O
2
was added. Finally, the specimens
were counterstained with Meyer’s hematoxylin (Histolab, Göteborg, Sweden).
For smooth muscle α-actin clone 1A4 (dilution 1:1400; Dako, Glostrup,
Denmark) was used and for CD44 the clone Ox50 (dilution 1:50) was used.
CD44 staining was graded on a scale from 0-3 with steps of 0.5 by two
21
investigators together. Myofibroblasts were evaluated as 1 when present or 0
when absent. The following antibodies were used when characterizing the
inflammatory cells: ED1 (dilution 1:400) recognizing monocytes and
macrophages, R73 (dilution 1:200) binding to the alpha/beta T-cell receptor
which is a marker for T-lymphocytes, W3/25 recognizing CD4 (dilution 1:50)
and Ox33 (dilution 1:250) for staining of B-cells. All antibodies, except the one
against α-actin were from Serotec, Oxford, England.
Hyaluronan and water content measurements (Study III)
After removal of the kidney, the middle third was resected and immediately
placed on a filter paper for 1 and 1/2 minutes on each cutting edge and
subsequently weighed (wet weight, WW). Specimens were then stored at -20°C
until lyophilization over night. The samples were weighed again (dry weight,
DW) and ground. Water content in percent was calculated using the formula:
100 x (WW-DW)/WW. In order to extract HA, 2 ml of 0.5M NaCl was added to
20 mg of the ground tissue. After 16 hours the sample was centrifuged 15
minutes at 2000 g. The HA content of the supernatant was then measured using
a radiometric assay (Pharmacia HA test, Pharmacia, Uppsala, Sweden). 100 µl
of the supernatant was mixed with 200 µl of
125
I-labeled HABP and incubated
for 60 minutes at 4-7°C. Subsequently, 100 µl of HA-sepharose was added and
the incubation continued for 45 minutes at the same temperature. Then, 2 ml of
the decanting solution was added and the sample centrifuged for 10 minutes at
2000 g and the liquid was decanted before the radioactivity in the pellet was
counted in a gamma counter. A standard curve was prepared using known
amounts of HA.
Pharmacological treatments (Study IV)
CoPP was given i.p., 5 mg/kg 24 hours prior to the ischemic injury or 15 mg/ kg
48 hours before the ischemic injury. The PAF-antagonist UR-12670 was
administrated by gavage 20mg/kg b. w. The first dose was given 30 minutes
before the start of the renal clamping operation and the drug was then given
once daily until the end of the experiments. Candesartan Cilexitil was
administrated in the drinking water (10.2 ± 2.2 mg/kg b.w.) In the first rats
candesartan was given directly after the clamping but due to high mortality
(about 50%) the initiation of candesartan treatment was delayed until two days
post-ischemia. 3/7 four-week rats and 2/7 eight-week rats received candesartan
beginning on the day of I/R. In 4/7 four-week-rats and 5/7 eight-week rats
candesartan treatment was delayed until two days after I/R. Prednisolone (10
mg/kg b.w.) was administrated intravenously in the tail vein three hours before
ischemia and (5 mg/kg b.w.) daily subcutaneously for five days after ischemia.
Tacrolimus (7 mg/kg b.w.) was given orally three hours before ischemia and
22
then daily (3,2 mg/kg b.w.) for five days. Cyclosporin A (CsA, 10 mg/kg b.w.)
was given orally three hours before ischemia and then daily (5 mg/kg b.w.) for
five days. Cobalt protopophyrin was injected i. p. 24 hours (5 mg/kg b.w.) or 48
hours (15 mg/kg b.w.) before ischemia. An i.p. injection of 0.9% NaCl was
given to DM and non-DM serving as control for CoPP.
Western blot against HO-1 (Study IV)
24 hours after injection of NaCl or CoPP three rats in each group were
anesthetized by Inactin using dose described above and the kidneys were
removed for further analyses. The kidneys were cut and the pieces intended for
Western blot were snap-frozen into isopentane chilled with dry ice in acetone.
Roughly 1/4 of each kidney was homogenized in a Dounce homogenizer
together with three times the weight of a lysis buffer (50 mM Tris pH 8.0, 150
mM NaCl, 5 mM EDTA, 0,5% NP-40, 50mM NaF and Complete protease
inhibitor (Roche, Bromma Sweden)). After 20 minutes on ice the samples were
spun at 20 000 X g for ten minutes and stored at –70° C until use. Protein
content was measured using the Bradford micro-assay (Bio-Rad, Hercules, CA).
20 µg of protein was run on a 12% polyacrylamide gel containing SDS. After
electrophoresis the proteins was transferred to a Hybond-P membrane
(Amersham Biosciences, Uppsala, Sweden) over night using a wet method.
Unspecific protein binding was blocked by 5% dry milk (Semper, Stockholm),
in PBS for one hour. HO-1 was detected by incubation with a polyclonal rabbit
anti HO-1 antibody (StressGen Biotechnologies Corp., Vancouver, British
Colombia, Canada) diluted 1:2000 in PBS with 5% dry milk for two hours.
After rinsing with 5% dry milk in PBS a biotinylated donkey anti-rabbit
antibody (Amersham Biosciences, Uppsala, Sweden) was applied in a dilution
of 1:2000 for 30 minutes. Subsequently the membrane was rinsed with 0,5%
Tween-20 (Bio-Rad, Hercules, CA). The blots were developed using ECL-plus
reagents (Amersham Biosciences, Uppsala, Sweden).
Statistical analyses
In the experiments with long acting insulin, kidneys subjected to the same
treatment (i. e. ischemia or no ischemia) from different groups were compared.
Kruskal-Wallis test was used to compare urinary volume, inulin clearance and
results from morphological and immunohistochemical evaluations. When
significant differences were found Mann-Whitney U-test was used to evaluate
differences between the different groups. Kidney weights, BGC and blood
pressure were compared by a one way ANOVA with Scheffe´s post hoc test. In
the study with short acting insulin student’s t-test was used instead of ANOVA
since only two groups was compared.
23
RESULTS
Study I
Renal function
The renal function in the experiments in study I are shown in table 1. In the DM
animals, ischemia caused substantial decrease in C
In
to less than 20% of that in
non-DM after four weeks. The dramatic loss of renal function led to complete
anuria within eight weeks post-ischemia. A compensatory increase in GFR in
the contralateral non-ischemic kidney was found in DM animals.
Table 1. Renal function in study I
Non-diabetic Diabetic
Observation time 4 weeks
(N=8)
8 weeks
(N=8)
4 weeks
(N=7)
8 weeks
(N=8)
I/R 6.1 ±1.1 3.8 ± 1.1 4.2 ± 3.5 0
c
Urinary Flow
L/min Non-I/R 7.6 ± 2.1 5.7 ± 1.6 33.7 ± 9.7
a
47.4 ± 4.7
c
C
In
mL/min I/R 0.74 ± 0.15 0.56 ± 0.16 0.14 ± 0.13
b
0
b
Non-I/R 0.86 ± 0.12 0.76 ± 0.18 1.44 ± 0.38 1.85 ± 0.16
b
Values are ±1 SEM .
P<0.05 DM vs. non-DM in corresponding kidneys
b) P<0.01 DM vs. non-DM in corresponding kidneys
c) p<0.001 vs. non-DM in corresponding kidneys
Morphology
Marked scaring was observed on the surface of post-ischemic DM kidneys four
weeks after I/R, and this was even more pronounced after eight weeks. In the
DM kidneys subjected to I/R numerous inflammatory cells were found in all
renal compartments except the glomeruli. The inflammation was extensive both
in the cortex and medulla and seemed to increase over time. In the papilla
inflammatory cells were less frequent. In non-diabetic post-ischemic kidneys
were found only occasionally. In the post-ischemic DM kidneys extensive
fibrosis was observed both in the cortex and outer medulla. Large areas of theses
kidneys were fibrotic and filled with inflammatory cells, and in these areas
tubular structures could not be clearly identified. Eight weeks after the injury
there was a marked increase in the number of glomeruli per visual field in the
DM kidneys subjected to ischemia, indicating substantial tubular atrophy.
Tubular casts were occasionally seen. In all DM kidneys subjected to I/R
substantial tubular dilation was evident.
In the post-ischemic DM kidneys the glomeruli appeared heterogeneous. At four
weeks post-ischemia some glomeruli were atrophic and sclerotic but most of
them appeared almost normal apart from thickening of Bowman’s capsule. Eight
24
weeks after the ischemic insult the number of small and sclerotic glomeruli had
increased. The relative glomerular size in the non-ischemic DM kidneys had
increased by roughly one third after four weeks and by two thirds after eight
weeks. In contrast, the glomeruli of the DM kidneys subjected to ischemia
showed no hypertrophy at four weeks, and after eight weeks they were
decreased in size.
Study II
Untreated animals
In DM rats without insulin treatment similar findings as in study I were seen.
The DM kidneys subjected to I/R were virtually anuric four weeks after the
insult whereas the contralateral kidney showed a high urinary output and inulin
clearance. The histopathological lesions correlated with renal function. Severe
lesions were seen in the kidneys with poor function. The dominating features of
the lesions were tubular atrophy and dilation, infiltration of inflammatory cells
and fibrosis. Papillary necrosis was present in most of the ischemic kidneys in
the untreated group. The glomeruli were comparatively spared. In rats not
receiving insulin treatment, hypertrophy was seen in the contralateral non-
ischemic kidneys. Compared to animals pre-treated with insulin the weights of
the non-ischemic kidneys were higher in the untreated group. The weight ratio
between ischemic and non-ischemic kidneys was also lower in untreated rats
compared to the animals pre-treated with long acting insulin.
Long acting insulin before or after ischemia
At the time of I/R BGC in untreated DM rats was 37.2 ± 0.7 and rats treated
with insulin after I/R

36.9 ± 0.7

and in treated with long acting insulin before
I/R BGC was 2.8 ± 0.2 at the time of renal I/R. No difference in urinary
production or inulin clearance was found between ischemic and non-ischemic
kidneys in the rats which had been treated with long acting insulin before the
ischemic insult. The inulin clearance and urinary production was significantly
increased in the post-ischemic kidneys treated with long acting insulin before
the insult compared to untreated rats and rats treated with insulin after ischemia.
There was almost no evidence of structural renal damage. Neither could a
decrease in the size of ischemic kidneys four weeks after ischemia be observed.
Treatment with insulin beginning one day after ischemia did not produce any
improvement of the lesions. The same functional pattern was seen in these rats
as in the untreated rats i.e. no urine production in the injured kidney and
increased function on the non-ischemic side. The morphological findings were
in accordance with the functional differences.
25
Short acting insulin before ischemia
Treatment with short acting insulin before renal I/R decreased the loss of renal
function. Inulin clearance in the post-ischemic DM kidneys were approximately
50% of the contralateral kidneys. However in the morphological evaluation no
improvement could be seen after treatment with short acting insulin. The same
morphological features were present such as tubular atrophy and dilation,
infiltration of inflammatory cells, fibrosis and papillary necrosis. However, there
was a difference in the weight of the ischemic kidneys The BGC after ischemia
was 10.3 ± 0.7 mmol/l for the animals treated with short acting insulin and 41.0
± 1.1 mmol/l for the untreated rats.
TUNEL
The apoptotic cells were defined by intensive green fluorescence of the nuclei.
In the untreated diabetic animals high numbers of positive nuclei could be
detected in the tubular cells of the outer medulla, whereas only single positive
cells were found throughout the cortex. The positive nuclei of the tubular cells
were mostly rounded and some of them were clearly reduced in size. Positive
nuclei were also found in the lumen of the tubuli and of the collecting duct.
Some nuclei were swollen, which suggests that they might rather be necrotic.
Cells with positive nuclei were also seen in the interstitium. Significantly less
positive nuclei could be detected in the outer medulla of the kidneys from the
diabetic rats pre-treated with long acting insulin (median 11 nuclei/mm
2
, 0- 280
nuclei/mm
2
) compared to the untreated DM animals (median 332 nuclei/mm
2
,
54-504 nuclei/mm
2
) P<0.05. The median number of nuclei in the medulla in the
non-DM group was 22 nuclei/mm
2
, 3-834 nuclei/mm
2
. At least four non-
ischemic kidneys were evaluated from each group and all of them had less than
four positive nuclei per mm
2
. In the TUNEL experiments BGC before ischemia
in non-DM rats was 7.4 ± 0.4 mmol/l, in insulin treated DM rats 2.7 ± 0.2
mmol/l, and in untreated DM rats 45.9 ± 3.0 mmol/l (P<0.001 compared to the
non-DM and insulin treated groups). Body weight at the time of clamping was in
untreated DM rats 232 ± 9 g. Non-DM rats weighed 286 ± 13 g (P<0.01 vs.
untreated DM rats). In the rats treated with long acting insulin the body weight
was 300 ± 7 g (P=0.001 vs. untreated DM rats).
Study III
Renal HA content
The ANOVA-analysis demonstrated that the HA-content was significantly
higher both in non-ischemic (p<0.001) and post-ischemic (p<0.001) DM-
kidneys than in non-DM kidneys. Time elapsed after the ischemic insult also
significantly affected the HA-levels in the kidneys in post-ischemic (p<0.01) as
well as non-ischemic kidneys (p<0.05). Furthermore, using an ANOVA split for
26
observation time it was seen that in kidneys subjected to ischemia the renal HA-
content was increased significantly after one week, two weeks and eight weeks
in DM animals compared to non diabetic rats. In kidneys not subjected to
ischemia the DM kidneys only had a higher HA-content at two weeks (p<0.05).
Renal water content
Measurements of the renal water content demonstrated a significant increase in
the ischemic diabetic kidneys compared to non-DM kidneys (p<0.001). In the
non-ischemic kidneys no significant difference in water content was found
between non-DM and DM animals.
Localization of HA
In non-ishemic kidneys and at two or 24 hours after ischemia, no cortical HA
could be demonstrated in any of the experimental groups. In some kidneys
subjected to the combination of ischemia and diabetes, a positive HA-staining in
the renal cortex was found at one and two weeks of observation time, and at
eight weeks after I/R all DM kidneys were HA-positive in the cortex. In the
larger group of diabetic rats studied four and eight weeks after ischemia,
significant increases in HA staining could be seen in the cortex, medulla and
papilla at both four and eight weeks. The accumulation of HA was not
homogeneous in the kidneys. The areas where inflammation and tubular dilation
were most obvious also showed the most pronounced HA staining.
Morphology
Severe renal damage was observed in most DM kidneys subjected to 30 minutes
of ischemia followed by reperfusion. Early signs of ischemic injury could be
observed in the renal medulla in two out of three DM kidneys after two hours of
reperfusion. The injury included tubular cell swelling, some pycnotic nuclei and
hyperemia. All DM kidneys subjected to ischemia showed signs of injury in the
renal medulla 24 hours after I/R and tubular casts were present in variable
numbers. The structure of the tubuli was disrupted. Hyperemia was present.
Also the papilla showed signs of ischemic injury. Tubular dilation was observed.
Infiltrating inflammatory cells were few. The renal cortex was relatively
preserved during the early stages. Tubular atrophy and dilation as well as
pronounced inflammation with mainly mononuclear cells also characterized the
post-ischemic DM kidneys, in particular when observed for eight weeks. The
accumulation of HA was not homogeneous in the kidneys. The areas where
inflammation and tubular dilation were most obvious also showed the most
pronounced HA staining.
27
CD44 staining
The tubular expression of CD44 was significantly higher in the cortex of
ischemic DM kidneys compared to ischemic non-DM kidneys at one, two and
eight weeks. In the medulla of ischemic kidneys tubular CD44 expression was
increased at one and eight weeks in DM-kidneys. CD44 positive infiltrating cells
were significantly increased in the cortex of ischemic DM kidneys after eight
weeks and in the medulla after one, two and eight weeks. In the medulla of non-
ischemic kidneys tubular CD44 expression was increased in DM animals
compared to non-DM animals at 24 hours, two weeks and eight weeks. CD44
positive infiltrating cells were not increased in non-ischemic kidneys of DM
animals.
Characterization of inflammatory cells eight weeks after I/R
In an attempt to characterize the infiltrating inflammatory cells seen in this
model immunohistochemical stainings with antibodies against cell-type specific
antigens were performed on kidneys from ischemic DM-kidneys and on non-
DM non-ischemic kidneys eight weeks after the insult. There were few
infiltrating cells of any kind in the non-DM non-ischemic kidneys. In the
ischemic DM-kidneys numerous infiltrating cells were seen. A large number of
the infiltrating cells were positive for CD4 indicating that they were
macrophages or T-helper cells. T-lymphocytes were frequent but not as
numerous as the CD4-positive cells. Both lymphocytes and CD4-positive cells
tended to appear in clusters. ED1-positive cells, monocytes and macrophages,
were also quite common. However, the monocytes and macrophages were not as
frequent as the T-lymphocytes and did not show as much accumulation in
clusters. Few B-cells were observed in these kidneys.
Study IV
Untreated DM-animals
All post-ischemic kidneys in DM animals had greatly decreased function and
showed severe injuries morphologically. A common pattern was observed which
included tubular dilation in the outer part of the renal medulla and in the cortex.
In the inner part of the renal medulla a zone with severe injury was seen where
hardly any tubular structures could be distinguished. Inflammation was present
in the outer part of the medulla and to a lesser extent in the cortex. Fibrosis was
a main feature and it was most pronounced in the renal medulla. In the
contralateral kidneys of DM animals vacuolization was present in many tubular
cells.
28
Candesartan and UR-12670
Candesartan gave a significantly lower mean arterial blood pressure after eight
weeks of treatment. After four weeks there was a trend towards a lower blood
pressure. No improvement of renal function in the ischemic kidney could be
observed between the different treatment groups. The morphological picture was
not improved in the treated groups. Compared to untreated animals candesartan
cilexitil caused an increased tubular dilation in the renal cortex both four and
eight weeks after ischemia and increased fibrosis in the renal medulla after four
weeks. UR-12670 treatment resulted in a more pronounced tubular dilation eight
weeks after ischemia. Eight weeks after renal clamping the weight of the
postichemic kidneys was higher than in the DM rats treated with UR-12670 than
in untreated DM kidneys. In this study two different treatments with candesartan
was tried. Treatment with candesartan starting immediately after ischemia
caused 50 % mortality. The surviving rats drank more water the first day after
the ischemic insult than the rats that died (104 ml ± 21 ml vs. 30 ml ± 11 ml
P<0.05). The mortality was 10% in the group that received candesartan two days
after I/R.
Prednisolone, Cyclosporin A and Tacrolimus
The ischemic kidneys from all the DM groups, except prednisolone treatment,
had all significantly lower inulin clearance than non-DM kidneys regardless of
treatment. Treatment with prednisolone, CsA or tacrolimus did not have any
protective effect on renal function in DM animals subjected to renal I/R. There
was no improvement of the renal histology by immunosuppressive treatment.
No difference in weight in the ischemic kidneys was observed between the
different groups.
CoPP
Induction of HO-1 by CoPP resulted in a trend to an improved renal function
two weeks after I/R in DM rats. Three out of seven rats which received CoPP
5mg/kg b.w., and one out of four rats that received CoPP 15 mg/kg b.w. showed
signs of preserved renal function on the ischemic side two weeks after ischemia.
Three out of seven rats, which received CoPP 15 mg/kg, had an intraperitoneal
exudation and these rats never woke up after anesthesia during the renal
clamping. In these experiments non-DM animals had better function in the post-
ischemic kidney than both CoPP-treated and untreated DM animals. The post-
ischemic kidneys in the DM + NaCl groups weighed less than the corresponding
kidneys in the non-DM groups.
29
Western blot against HO-1
In animals that received CoPP 5mg /kg body weight HO-1 was detected in one
out of three animals and in rats receiving CoPP 15mg /kg body weight HO-1
was detected in two out of three animals. The bands were stronger in the rats
that received the higher dose of CoPP. No HO-1 was detected in DM or non-
DM animals that received NaCl i.p.
30
GENERAL DISCUSSION
In this thesis a model of ESRD due to the combination of unilateral I/R and DM
is presented. Increased sensitivity to I/R in DM rats has also been shown
previously [66, 112-114]. This model can be useful to study the influnce
hyperglycemia and shortage of insulin on acute renal failure, and in the study of
the progression of DM nephropathy. The importance of hyperglycemia and
insulin treatment in acute renal failure has recently been highlighted [115]. Both
the mortality and incidence of acute renal failure can be reduced by
approximately 40% when blood glucose is kept between 4.6 mmol/L and 6.1
mmol/L by continuos infusion of insulin in intensive care unit patients [115].
Furthermore there is a correlation between the BGC in the post-operative phase
and the frequency of acute rejections after renal transplantation in DM patients
[116]. The progression towards ESRD show similarities in many renal diseases,
including DM nephropathy [6]. Some common features are tubular atrophy,
interstitial fibrosis and infiltration of inflammatory cells [6], all of which are
present in our model. Untreated diabetic animals develop changes such as
vacuolization of tubular cells and tubular atrophy [47]. Tubular vacuolization
has also been reported in patients dying from diabetic coma [48]. The validity of
the present model in the study of diabetic nephropathy could of course be
questioned. DM nephropathy is a chronic disease, which takes years to develop.
No initiating event such as ischemia is present in diabetic nephropathy.
Glomerular changes are rather modest in the present model whereas they are
prominent in DM nephropathy [5]. However, considering the importance of the
tubulointerstitial lesions in the progression of DM nephropathy, this model
could be of value in the study of advanced diabetic nephropathy.
The findings in the present investigations indicate that the medulla is the most
sensitive part of the kidney during I/R injury in DM. Swollen tubular cells, and
pycnotic nuclei were observed in the medulla after two hours of reperfusion.
The large number of TUNEL-positive nuclei in the renal medulla at six hours
after ischemia, seen in Study II, suggests that apoptosis is present in this model.
Secondary ischemia due to erythrocyte trapping could not be excluded in this
model, since hyperemia was observed 2 and 24 hours after renal ischemia in DM
kidneys in Study III. Tubular casts were observed in some of the DM kidneys at
2 h, 24 h and one week after I/R. Shedding of tubular cells has been described in
renal I/R and could result in impaired tubular flow [117].
Death of tubular cells and infiltration of inflammatory cells cause, tubular
atrophy, fibrosis and renal scarring and once established this process might be
self-perpetuating. The injury in the medulla subsequently spreads to the cortex.
It is likely that obstructed tubular flow was involved in the progression of the
31
injury in the cortex, since extremely dilated tubuli dominated the morphological
picture.
In Study I we found a correlation between renal morphology and renal function.
Non-DM I/R kidneys had good function four and eight weeks after ischemia,
and showed little morphological evidence of injury, while DM kidneys
subjected to I/R had poor function and severe injury could be demonstrated
morphologically. The same pattern was seen when we studied the effects of long
acting insulin. Severe damage was seen in kidneys with pronounced functional
impairment. However, in kidneys subjected to I/R in DM rats treated with short
acting insulin, inulin clearance was restored to about 50% of that of non-I/R
kidneys, but no morphological improvement was observed.
In all studies the same investigators performed all morphological scoring, except
evaluation of the presence of myofibroblasts, blindly on coded samples.
Difference in opinion between the investigators was rarely more than one step
on the arbitrary semiquantitative scale used. The parameters, fibrosis, infiltration
of inflammatory cells and tubular dilation were chosen since they are typical
findings in the advanced stages of I/R injury in DM rats. One weakness of our
evaluation was that we made a total score for each compartment in the entire
kidney even though the lesions were not entirely homogenous. Here a refined
method of evaluation such as scoring a number of randomly selected fields or
using an image analysis system might have revealed a difference. Possibly a
scale with more steps might also have sharpened the evaluation.
In study II non-DM I/R kidneys and DM I/R kidneys from animals pretreated
with long acting insulin recovered almost completely from the ischemic insult.
However, some recent research shows that kidneys which seem to have
recovered from I/R almost completely, with few morphological signs of injury
and almost normal function, might have microvascular changes that may cause
progressive functional and structural injury later [58].
In study I-III we used 30 minutes of ischemia, but in a small pilot study we
found that 20 minutes of ischemia was enough to create a severe I/R injury in
DM rats. We believed that a shorter ischemia would augment the chances of
seeing protective effects of different treatments in that were tried, and therefore
we used 20 minutes of ischemia in the last study. The importance of the
ischemic duration is well established, and it has previously been shown that a
longer period of ischemia causes a more severe injury [75, 118]. The
degradation of ATP to hypoxanthine and xanthine via inosin is enhanced with
the duration of ischemia [60]. The restoration of ATP levels during reperfusion
32
is slower after prolonged ischemia [62]. The rat strain might also be of
importance in determining the degree of renal I/R injury, and a difference
between rat strains in the expression of major histocompatibility complex
(MHC) class II between rat strains one week after ischemia has been reported
[119]. Temperature is a critical factor in ischemic injury. Hyperthermia,
especially during the ischemic phase, leads to a more severe renal I/R injury.
Raising the temperature from 37 °C to 39.5 °C during ischemia leads to a 100%
increase in blood urea nitrogen (BUN) in a model using 30 minutes of ischemia
and uninephrectomy [120]. In order to keep the temperature constant we used a
servo controlled heating pad that kept the temperature in the rat at 37.5°.
STZ-DM in rats is a well-established model of diabetes type-1. STZ-DM rats
have greatly elevated blood glucose levels, some of our rats had a BGC over 60
mmol/L. However, despite the extreme hyperglycemia metabolic acidosis was
not observed in the DM rats. DM patients rarely have as high BGC as STZ-DM
rats even though very high BGC sometimes occur in poorly controlled type 2
diabetics and newly diagnosed DM type-1 patients. The high BGC in the STZ-
rat is of course a reason for caution when interpreting the findings in this model.
However, there might be differences between species regarding the sensitivity to
a particular BGC. The biological significance of a BGC of 30 mmol/L may not
be the same in rats as it is in humans.
STZ is nephrotoxic and causes tubular injury in humans, but STZ does not
induce DM in man in doses used for chemotherapy [121-123]. However,
glycosuria without hyperglycemia was observed in 14 out of 18 patients after
repeated STZ-administration [123]. Tubular and glomerular injuries have been
described along with tumor formation in STZ-treated rats[47, 124, 125], and
insulin treatment can reverse the typical lesions seen in STZ-DM rats [21, 125-
127]. Furthermore, Evan and colleagues have concluded that STZ is less
nephrotoxic than alloxan, which is also used to render rats diabetic [125, 128].
The increased renal susceptibility to I/R in STZ-DM rats is most likely not due
to STZ-toxicity, since treatment with long acting insulin before renal ischemia
abolished the increased renal sensitivity to I/R. Despite its shortcomings, STZ-
DM is a valuable model for the study of the disease.
Even though inulin clearance is the golden standard in determining GFR, the
applicability of the method could be discussed. Low blood pressure could
influence inulin clearance. In study I, MAP was equally low during inulin
clearance in all examined groups. There was no difference in MAP between the
groups. Average urinary flow was not depressed and inulin clearance was not
lower than in the following studies. The reason for the low MAP is most likely
33
Eqviticin, the anesthetic agent used, since similar values have been found in
other studies from our laboratory using the same drug [129]. In a few rats,
however, low blood pressure may have caused a decreased total inulin clearance
but we did not exclude any rats on this basis. Some rats had a low total inulin
clearance without being hypotensive. The reason for the low filtration in these
cases is not clear but volume depletion cannot be excluded. Eqviticin was also
used during the renal artery clamping in most of the present experiments. The
low MAP during Eqviticin anesthesia is not a likely cause for the increased
sensitivity to I/R in DM rats since there was no difference in MAP between DM
and non-DM animals.
An important question in this thesis is how the increased BGC level or shortage
of insulin could cause the increased sensitivity to renal I/R observed in DM
animals. Several possible explanations exist. The increased sensitivity to I/R
could be due to hyperglycemia per se. Shortage of insulin could also be involved
in the increased sensitivity to I/R. Secondary effects of hyperglycemia such as
formation of AGE, increased oxidative stress, hemodynamic alterations and
differences in the response and formation of NO could also be involved.
Table 2 Possible pathophysiological mechanisms in ischemia/reperfusion (I/R) injury in
diabetes
Initiating
events
• I/R-injury • Hyperglycemia • Hypoinsulinemia
Mediators • Oxidative stress • Increased renal
workload
• Apoptosis
• Release of cytokines
and inflammatory
mediators
• Advanced
glycosylation end
products.
• Enhanced CD44
expression
• Increased
inflammatory response
• Accumulation of HA
• Infiltration of
inflammatory cells
End result • Reduced GFR • Tubular atrophy • Tubulo-interstitial
fibrosis
• Tubular dilation
Hyaluronan (HA), Glomerular filtration rate (GFR)
In studies I-IV high BGC during I/R was associated with a severe renal injury,
and this finding supports the idea that elevated BGC during I/R could be
deleterious for the kidney. An increased acute sensitivity to ischemia has been
34
demonstrated when BGC was raised by dextrose infusion or intraperitoneal
glucose injection in combination with renal I/R in both rats and dogs [130, 131].
However, the long-term outcome of the combination hyperglycemia in non-DM
animals and I/R has not been investigated. The situation with elevated BGC in
non-DM animals differs from the situation in STZ-DM. The elevated BGC is
only present for a short time before renal I/R, which means that there might not
be enough time for secondary effects of hyperglycemia to arise. There is also no
shortage of insulin in hyperglycemic non-DM animals.
Numerous studies have investigated the influence of hyperglycemia and diabetes
in cerebral ischemia. Diabetes is associated with a worse outcome after stroke in
humans, and elevated blood glucose predisposes for a more severe cerebral
injury even in non-DM patients [132]. There are conflicting evidences regarding
the influence of hyperglycemia and diabetes on the degree of injury in
experimental cerebral ischemia. DM or hyperglycemia in non-DM animals
caused increased cerebral injury in most studies, especially when models with
reperfusion were used [133-142]. However, hyperglycemia without DM had a
protective effect against cerebral injury in some studies using permanent
vascular occlusion [143-145]. In a study where small microinfarctions were
induced in the rabbit brain hyperglycemia also exerted a protective effect [146].
Taken together these studies suggest a role for reperfusion in the harmful effect
of hyperglycemia in cerebral ischemic injury.
In study II rats treated with long-acting insulin before I/R had a very low BGC
before renal artery clamping, which was associated with an almost complete
protection against renal I/R injury. Therefore the question arises if sub-
physiological BGC could be protective against renal I/R. Anaerobic metabolism
is increased during ischemia when no oxygen is available. It has been
demonstrated that the BGC influences the renal lactate content during
ischemia[131]. High BGC induced by an intraperitoneal glucose injection before
I/R increased lactate concentration in the kidney and worsened the injury.
However, lowering the BCG below normal levels decreased lactate production,
but did not improve renal function after I/R [131]. Sub-physiological BGC is
thus less likely to protect against renal I/R.
Increased workload of proximal tubular cells (Fig. 2) due to the osmotic diuresis
may be involved in the increased renal sensitivity to I/R in DM [66]. The
activity of renal Na
+
,K
+
-ATPase is increased in the kidneys of STZ-DM rats
[65] and this increase can be reduced by insulin [147]. The hypothesis that the
level of energy demand is important in renal I/R is corroborated by the
protective effect by furosemide against renal I/R injury in rats [148]. The
35
decrease in oxygen consumption in renal tissue slices by furosemide, is greater
in DM rats than in non-DM rats, when measured in vitro [114]. Kuramochi and
Homma using the same method, found that oxygen consumption from the renal
medulla was decreased in DM rats but not in non-DM rats after 24 hours of
reperfusion following 30 minutes of ischemia [113]. Despite increasing the
sensitivity to renal I/R, DM protects the kidney against nephrotoxins such as
mercury chloride and gentamicin [66, 149-151]. The protective effect of DM
against renal gentamicin injury is reversed by insulin treatment indicating that
the protection is caused by DM [151]. It has been proposed that the protective
effect of DM against nephrotoxicity in rats could be explained by a decreased
accumulation of the toxins in the renal cells due to increased diuresis and GFR
[151].
Figure 2. High glucose concentration in the proximal tubuli enhances the influx of glucose
and Na
+
into the tubular cells via the Na
+
/glucose co-transporter. Elevated intracellular Na
+
concentration. activates the Na
+
/ K
+
-ATPase and causes increased energy demand in the
tubular cells.
The nitric oxide system may be involved in the increased sensitivity to I/R in
DM. There is evidence for increased NO-production in the STZ-DM kidney
[112, 152], and renal I/R in non-DM rats also has been reported to be associated
with increased NO production [112]. However, reduced production of NO as
well as a decreased vascular response to NO has been observed in DM rats after
renal I/R by Goor et. al.[112]. Goor and co-workers suggest that endothelial
injury may explain the lower NO production in DM kidneys after I/R. Decreased
response to NO in combination with reduced NO production could have caused
renal vasoconstriction and thus impaired renal blood flow during reperfusion in
DM kidneys
Na
+
K
+
ATP
K
+
Normoglycemia
Hyperglycmeia
Glucose + Na
+
Na
+
ATP
Glucose
+ Na
+
Tubular
cell
Tubular
cell
36
Treatment with long acting insulin 7-14 days before ischemia protected DM
kidneys from renal I/R and almost abolished the induction of apoptosis seen in
the renal medulla 6 hours after ischemia in DM animals. Insulin may affect the
DM kidney in several ways, but it is difficult to distinguish between direct
actions of insulin itself and what caused by its effects on BGC. DM leads to an
increased GFR during the early stages both in humans and rats [21, 153]. An
interesting finding is the fast effect of the BGC on the weight of the
kidneys[154]. A ten-minute infusion of glucose in non-DM rats gave a
significant increase in the weight of the kidney and insulin treatment reduces
kidney weight in DM animals within hours. The reason for these fast changes in
renal size is not established, but a role of GFR alterations could not be ruled
out[154].
The issue of insulin action on renal hemodynamics is complex. The renal
response to insulin is different in DM and non-DM during euglycemic clamping.
Hyperinsulinemia increases the renal blood flow under euglycemic conditions,
an effect, which has been suggested to be NO-dependent [155]. In DM rats
insulin decreased RPF, whereas in non-DM rats RPF increased [156]. Insulin
treatment also has been shown to normalize the renal hemodynamics in STZ-
DM rats. [21].
Insulin treatment decreased the number of apoptotic cells in the renal medulla
after I/R in study II. Insulin in high doses is able to activate the IGF-1 receptor
in vitro, but the effect is not as strong as that IGF-1 [157]. IGF-1 is a well-
known anti-apoptotic agent, and IGF-1 has been reported to protect kidneys
from apoptosis, inflammation and renal failure after I/R in mice [77, 158].
However, in another study IGF-1 increased the inflammation and renal I/R
injury in rats [159]. Insulin has an anti-apoptotic effect also on epithelial cells
from the mammary gland [160]. High doses of insulin protect cultured mouse
proximal tubular cells deprived of growth factors from apoptosis, however,
when apoptosis is caused by chemically induced ATP-depletion, insulin does
not have an anti-apoptotic effect [61]. Insulin decreases apoptosis in myocytes,
when added to the culture media during reoxygenation after ischemia [161].
Insulin can also counteract the expression of potentially harmful genes. For
example insulin can prevent an increased expression of the angiotensin gene in
cultured immortalized tubular cells grown in high glucose media [162]. There
are thus, several ways in which insulin per se may protect tubular cells from I/R
injury, other than lowering BGC.
Oxidative stress might play a role in renal I/R injury in DM rats given the
knowledge that oxidative stress is implicated both in the complications of DM
37
and renal I/R. Elevated oxidative stress has been demonstrated in cerebral [163]
and intestinal [164] I/R in DM rats and the levels of oxidative stress in the brain
could be lowered by pretreatment with dehydroepiandrosterone [163]. The
combined oxidative stress from two sources may thus increase the total level of
ROS. Different anti-oxidative strategies could be used to test this hypothesis and
one is to induce heme oxygenase-1 (HO-1), which have anti-oxidative and anti-
apoptotic effects[165, 166].
HO-1 is the first and rate-limiting enzyme in the degradation of heme. Induction
of HO-1 pharmacologically or by gene therapy has been shown to decrease the
injury after I/R or transplantation in several models [167-169]. HO-1 is
upegulated after renal I/R and inhibition of HO-1 activity increases renal I/R-
injury [170]. A number of different mechanisms have been proposed to explain
the protective effect of HO-1. HO-1 can act as an anti-oxidant by producing
scavengers such as biliverdin. [166] In the process of degrading heme to
biliverdin CO is released and CO has a vasodilatory effect similar to NO but
weaker. However, CO is a much more stable compound so the vasodilatory
effect may be more prolonged than that of NO [171]. CO also has anti-apoptotic
effects [165]. By degrading heme, which may be toxic in the tissue, HO-1 could
also protect from I/R-injury. HO-1 also has anti-inflammatory properties [172].
A case report demonstrated that tubular injury is an important feature in human
HO-1 deficiency [173].
HO-1 can be induced by cobalt protoporphyrin IX chloride (CoPP) [167]. In
study IV we gave CoPP intraperitoneally in order to induce HO-1. We failed to
achieve induction of HO-1 in all animals that received the injection, according
to the expression examined by Western. A possible explanation is that the CoPP
intended to be injected intraperitoneally was deposited in the intestine. There
was a trend towards an improved renal function in the postischemic kidneys
after CoPP injection. These results suggest that another route of administration
must be tried in order to evaluate the effect of HO-1 induction on renal I/R
injury in DM.
Infiltration of inflammatory cells is one of the main features of renal I/R injury
in DM rats. The infiltrate mainly consisted of cells identified as
macrophages/monocytes and T-lymphocytes by immunohistochemistry. The
infiltration of inflammatory cells persisted for at least eight weeks. This is in
contrast to studies of renal I/R injury in non-DM animals where a transient
inflammation has been observed [78]. The inflammatory response is increased
acutely after I/R of the intestines in DM animals [174]. After a brief ischemia of
the intestine ROS are also increased and the increase is more pronounced in
38
DM[164]. It is likely that inflammatory cells contribute to increased oxidative
stress in DM kidneys after I/R.
A possible mechanism responsible for the recruitment of inflammatory cells in
the present model is HA/CD44 interaction. In study III an increased HA
accumulation was observed in the I/R kidneys from DM-animals, and HA was
also expressed in the renal cortex which is almost free of HA in the healthy
kidney [97]. Increased HA content and cortical HA-expression in the kidney
after I/R has also been demonstrated in non-DM rats [104]. CD44 is an
important receptor for HA [105] and was upregulated both on tubular as well as
infiltrating cells after renal I/R in DM. Inflammatory cells often express CD44,
and it has been suggested that CD44 positive cells could use HA in the ECM for
disseminating in the tissue [100, 107]. Prolonged renal and cardiac allograft
survival was achieved by injection of low molecular weight HA (5-8
dissacharide units), probably through blocking CD44/HA interaction [175]. In
study III increased expression of HA and CD44 was demonstrated after I/R in
kidneys from DM rats. The HA-molecule can be depolymerized by ROS thereby
creating HA fragments [176]. Fragmented HA has been shown to act pro-
inflammatory by inducing production of chemoattractant protein-1 [110],
VCAM-1 and ICAM-1 in cultured tubular cells [108], and induce macrophages
to express chemokines in vitro [109].
Another way by which HA could promote the progression of the renal injury, is
to create an interstitial edema by its tremendous ability to bind water. In
transplantation models where treatment with hyaluronidase decreased HA
content, the interstitial pressure in the graft was reduced [177, 178]. Even if the
HA-concentrations were not as high in our study edema could hamper the renal
microcirculation locally and create secondary ischemia in certain parts of the
kidney.
Modulating the inflammatory response could be an option to counteract the I/R
injury in the present model. Several studies have shown that immunomodulating
treatments have protective effects in renal I/R. We therefore, examined the
effect of treatment with the cortico-steroid prednisolon and the
immunosuppressive substances tacrolimus or CsA. None of the treatments we
tried offered any clear protection of renal function and morphology. Several
causes for the lack of effect are possible. Even though we decreased the duration
of ischemia to 20 minutes the injury may still be so severe that it is difficult to
alter.
39
There is good reason to suspect that we did not administrate CsA and tacrolimus
at the optimal time point. According to Sakr et. al, pretreatment with a single i.v.
injection of tacrolimus 0.3 mg/kg b.w. 24 hours prior to 60 minutes of ischemia
was able to decreased the renal injury, and the effect was associated with
decreased levels of TNF-α. [179]. CsA and tacrolimus both decreased apoptosis
and renal I/R injury when administrated 6 hours before renal I/R [84]. The
authors of the latter study suggested the possibility that the protective effect of
CsA and tacrolimus against renal I/R-injury could be mediated through
upregulation of heat shock protein (HSP) 70. HSP 70 expression started at 2h
after injection and peaked at 6 hours after i.v. administration of CsA or
tacrolimus. We gave both CsA and tacrolimus by gavage 3 hours before
ischemia. Failure to induce HSP 70 before I/R could be one of the reasons for
the lack of protective effect from CsA and tacrolimus.
Nephrotoxic effects of the immunosupressants must also be considered. Daily
subcutaneous injections of CsA 10 mg/kg b.w were demonstrated to delay the
recovery after I/R in one rat model, despite decreased inflammation [180].
Tacrolimus also has documented nephrotoxic effects however not given orally to
rats in the doses used in this study [181, 182]. The oral tacrolimus dose used in
the present study was chosen from what is common after renal transplantation in
the rat[181]. Prednisolone given to the donor rats 24 hours and one hour before
transplantation has recently been shown to protect renal allograft function six
months after transplantation [183]. To our knowledge there are no nephrotoxic
effects of prednisolone. No decrease in the amount of inflammatory cells was
seen after prednisolone, tacrolimus or CsA treatment four weeks after I/R. This
may in part be explained by that we only gave the rats the drugs during the first
week of the observation period.
Tubulointerstitial fibrosis was a consistent finding in DM kidneys after I/R.
Macrophages are important mediators of renal fibrosis, for example, by
releasing cytokines and growth factors [184]. TGF-β is generally assumed to be
one of the main factors behind extracellular matrix accumulation and
tubulointerstitial fibrosis in DM nephropathy [185]. Platelet activating factor
(PAF) stimulates extracellular matrix accumulation in both mesangial and
tubular cells in vitro, by a TGF-β dependent pathway [186]. One possible
strategy to counteract the inflammatory response and fibrotic process could be to
use a PAF-antagonist. Various PAF-receptor antagonists improve function and
morphology after renal I/R injury [54, 187, 188]. PAF is an acetylated alkyl
phosphoglyceride with profound biological actions, it stimulates inflammation
and some authors suggest that PAF receptor antagonists may protect the kidney
against renal I/R injury by blocking proinflammatory effects. It has been
40
suggested that PAF play a role in the neutrophil mediated injury in kidneys after
cold ischemia [187]. Increased survival of the graft, and reduced number of
infiltrating leukocytes, has been demonstrated after PAF-antagonist treatment, in
a syngeneic kidney transplant model [189]. In study IV treatment with UR-
12670 did not improve renal function or morphology after renal I/R injury in
DM. UR-12670 has been shown to improve the long-term outcome after renal
I/R in rats when the same does was used as in study IV [190]. However, the
decrease in kidney size observed eight weeks after ischemia in DM-kidneys was
significantly less in the PAF-treated animals than in the untreated DM kidneys.
This suggests that UR-12670 in some way slowed down the sacring process.
In an attempt to reduce the progression of fibrosis, treatment with the AT-1
blocker candesartan cilexitil was tried in the present model. AT-1 blockers have
recently been shown to have a protective effect against nephropathy in DM type
2 [51-53]. Ang II can exert pro-fibrotic actions via the TGF-β pathway [185].
Candesartan reduces TGF-β expression in the vessel wall in a rat aorta
transplantation model, where it decreases graft arteriosclerosis[129]. Ang II
stimulates hypertrophy of proximal tubular cells in vitro [191] and continuos
Ang-II infusion causes tubular dilation and atrophy in the rat [28]. Treatment
with the AT-1 blocker losartan decreased serum createnine at 72 h following 60
minutes of bilateral ischemia in non-DM rats [192]. Candesartan did not protect
DM kidneys against I/R injury in study IV, and caused a more pronounced
tubular dilation in the renal cortex four and eight weeks after ischemia. About 50
% of the rats died when candesartan was added to the drinking water
immediately after ischemia. The rats that died drank less water than the
survivors. Dehydration may thus play a role in the deaths of the candesartan
treated rats. The high mortality was ameliorated by starting candesartan
treatment two days after I/R. since the rats that died drank less water in which
the drug was dissolved,and thus recieved less of the drug, pharmacological
effects of candesartan in itself is not a likely cause of mortality. Candesartan
treatment resulted in a lower MAP at the time of inulin clearance. The level of
candesartan in the circulation was not measured in this study, but the lower
MAP in the treated animals suggests that they received enough of the drug to
lower the blood pressure. Furthermore, graft arterioscleroses in a rat aorta
transplantation model was reduced by candesartan cilexitil in the same dose as
we used [129].
In this thesis a model for I/R injury in diabetes has been presented and
discussed. The model can be useful in the study mechanisms behind acute
ischemic renal failure in DM and the progression of diabetic nephropathy.
41
GENERAL SUMMARY
• Kidneys in DM rats have an increased susceptibility to renal I/R-injury.
Unilateral renal ischemia for as little as 20 minutes leads to an irreversible
progressive injury in DM kidneys, whereas the similar injury in non-DM is
reversible.
• The renal I/R injury is characterized by anuria, infiltration of inflammatory
cells, tubular atrophy, dilation of the remaining tubuli, and tubulointerstitial
fibrosis. The inner parts of the medulla and the papilla contained necrotic
areas.
• The renal medulla is the most vulnerable compartment of the kidney. This is
seen both by the extent of fibrosis four and eight weeks after I/R and by the
presence of apoptotic cells after six hours of reperfusion.
• Treatment with long acting insulin 7-14 days before I/R decreased the
number of apoptotic cells in the renal medulla and protected renal function
and morphology after the insult, while insulin treatment after the injury did
not have any beneficial effect. Treatment with short acting insulin 2-6 hours
before I/R partly preserved renal function but did not preserve the
morphological texture.
• Increased accumulation of HA, expression of CD44 and water content were
seen after I/R in DM kidneys and may be involved in the chemotaxis of
inflammatory cells and enhanced production of ECM.
• Treatment with the Ang II receptor type 1 blocker candesartan, the PAF
antagonist UR-12670, the immunosuppressive agents tacrolimus and
cyclosporin A or prednisolon did not improve the outcome of the renal I/R
injury in DM.
• Injection of cobalt protoporphyrin (CoPP) intraperitonealy in order to induce
HO-1 resulted in a trend towards a renoprotection. However, the induction of
HO-1 by intraperitoneal CoPP injection was uneven when examined by
Western blot. Further studies with a more reliable induction of HO-1 are
required to evaluate the protective capacity of HO-1 against renal I/R injury
in DM.
42
ACKNOWLEDGEMENTS
I wish to express my gratitude to everyone that has been involved in this work
and made it possible. I especially want to thank:
Bengt Fellström, my main-supervisor for his endless energy, encouragement
and support.
Olof Hellberg, my co-supervisor for initiating the project and for always having
something interesting to discuss.
Erik Larsson, for stimulating hours at the microscope and for always taking the
time for a little chat.
Örjan Källskog, my co-supervisor for encouragement and interesting
discussions.
Keiko Funa, my co-author for good advice.
Roger Hällgren, for all the help with the hyaluronan studies and sharing his
deep knowledge in the hyaluronan field.
Ulla Svensson, for doing so much of the experimental work and making our lab
such a friendly place.
Lilian Zezina, for all the help and encouragement during the years and
especially for the efforts to improve my writing skills.
Margus Annuk, for the excellent idea of having his disputation exactly one
month before me so I always knew what to do.
Fredrik Lennmyr, for friendship, lunches and valuable discussion and sharing
of the pre-disputation stress.
Levent Akyürek and Agneta Wallmon, for pleasant cooperation
Gunnar Westin and Peter Lillhager, for teaching me Western blot.
Margit Tjärnberg, for performing the HA analyses.
Angelika Fasching, Eva Jacobsson, Mikael Broman, Mats Sjöquist, Mats
Wolgast, Peter Hansell and all the others at the kidney research group at BMC
where my research once started.
43
Kjell Öberg, head of the department of medical sciences, and Agneta
Wahlström for providing excellent working conditions
Cecilia Johnsson, Anders Stenberg and Tomas Lorant in the transplantation
lab for friendship, valuable discussions and help in solving various small
practical problems.
Anne-Marie Westlund and Marianne Carlsson, our previous next door
neighbors in the lab for being excellent neighbors.
Majstin Wik, Elizabeth Dehlin, Elisabet Dew and Annette McLoughlin for
excellent administrative help
Sven-Olof and Elisabeth Granstam, for being good lab-partners and friends.
Margareta Eriksson, for always having some wise words to say.
Michael Björk, for solving various computer problems despite the fact that I’m
a Mac user.
My fellow Ph D. students during the years at the research department 2, Gennet
Gebre-Medhin, Filip Sköldberg, Pamela Correa, Eva Szabo, Olov Ekvall,
Helena Brändström, Andreas Kindmark, Håkan Hedstrand, Anna Schölin,
Mikael Kullin, Ulrika Segersten, Kenneth Johnsson, Juan Lopez Egido, and
Eva Bornefalk for making the lab a pleasant place.
My new roommates in the internal medicine department, Andreas Fugman and
Lotta Welsh, for vivid descriptions of their state of mind just before their
disputations.
My parents in law, Gun and Lasse, for all the help and love.
My mother and my late father for everything.
Erik and Amanda for just being my two little darlings.
Last but not least important, my wife Camilla, for suffering all the hardships of
being married to a laboratory rat.
Financial support for these studies were supplied by: Svenska Läkarsällskapet,
Svenska Diabetesförbundet, Njursjukas i CUWX-Länens Forskningsfond,
Svenska Sällskapet för Medicinsk Forskning, Medicinska Forskningsrådet,
Uppsala University and Förenade Livs Forskarmånader.
44
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