Chapter 3

Biochemistry and Pathophysiology

iabetic nephropathy appears to occur as a result of a complex

inter play between metabolic and haemodynamic factors. Glucose-

dependence increases in oxidative stress, formation of renal polyols and

the accumulation of advanced glycation endproducts (AGEs) occur in the

diabetic Kidney. Haemodynamic factors are also implicated in the

pathogenesis of diabetic nephropathy and include increased systemic and

intra glomerular pressure and activation of various vasoactive hormone

pathways including the reninangiotensin system (RAS) and

endothelin.These haemodynamic pathways, independently and in concert

with metabolic perturbations, activate various intracellular signaling

pathways and induce the production of cytokines that ultimately produce

the

classical structural and functional hallmarks of diabetic nephropathy,

including albuminuria and glomerular ultra structural injury.

With the onset of hyperglycaemia, structural and haemodynamic changes

are rapidly detectable with in the Kidney. The earliest changes include an
increase in the GFR of upto 150%. These changes occurs in parallel with
an increase in renal size resulting primarily from hypertrophy, but also
from an increase in cell proliferation. The next observable change is the
development of increased rates of urinary albumin excretion of 30-300
mg/day (microalbuminuria). Special assay techniques are required as this
level is below the detection level of conventional urinary dipsticks.
Persistent micro albuminuria is associated with changes both in
glomerular structure (mesangial matrix expansion and basement membrane
thickening) and permeability, and is often referred to as 'incipient
nephropathy’. These changes are present in 20-30% of people with type 2
diabetes at the time of initial diagnosis. The development of persistent
microalbuminuria is associated with a marked increased risk of
developing macro proteinuria and in the progression to ESRF. A
significant proportion of type 2 diabetic patients with microalbuminuria
will progress to nephrotic range proteinuria within 10 years, although
many will suffer from a Cardio vascular event in the intervening period.
Histologically glomeruli show GRM thickening and mesangial expansion
eventually resulting in diffuse and/or local nodular glomerulosclerosis,
aflerent and efferent arteriolar sclerosis and tubulointerstitial fibrosis
. Pathogenesis

Familial clustering of diabetic nephropathy suggests

a possible genetic basis for this complication, though no gene

has been clearly shown to determine susceptibility to diabetic

nephropathy. Some studies have proposed a link between

diabetic nephropathy and the double delection ACE genotype.

However not all studies have shown a significant association,

particularly with type 2 diabetes.

1 he mechanisms linking chronic hyperglycaemia and

hypertension to the development of diabetic nephropathy have

begun to unraveled. It was recognized at the Joslin clinic soon after

the introduction of insulin that poor glycaemic control and disease

duration were risk factors for the development of diabetic

nephropathy and this has now been firmly established by the

finding from the Diabetic control and Complication Trial (DCCT)

and in particular in type 2 diabetic patients from the UK

Prospective Diabetes Study (UKPDS). Numerous studies have

shown that control of systemic hypertension has a major effect on

reducing proteinuria and slowing progression to renal failure in

both type 1 and 2 diabetes.

Role of hyperglycaemia

Since diabetes is a state of chronic hyperglycaemia, it is

likely that glucose dependent processes participate in the genesis of

diabetic complications. A number of equally tenable hypothesis exist

for the mechanism of hyperglycaemia - medicated renal damage.

Traditionally

considered to be separate pathways, it has recently been suggested that

these metabolic alterations do not exist independently, but are

interlinked and stimulated collectively. In particular, the generation of

reactive oxygen species by glucose may act as a common intracellular

messenger system involved in the activation of several of these

pathways.

Metabolic alterations
 A. Advanced glycation

Advanced glycation is a non enzymatic post-translational

modification of proteins via the spontaneous reaction of reducing sugars

and the free amino groups of proteins-Early compounds. Schiff bases,

undergo gradual rearrangement to form more stable ketoamines called

Amadori products. Progression of the reaction occurs in proportion to

the level of glycaemia. Therefore, the level of Amadori products, of

which glycated haemoglobin (HbA C) is one example, is a clinically

useful measure of medium term glycaemic control. Subsequent to the

generation of Amadori products, a series of condensation reactions occur

that ultimately result in the formation of AGE.

The formation of AGEs is a ubiquitous reaction. Indeed, AGEs

•I

accumulate during normal ageing and may account for some

physiological age-dependent vascular changes. However, AGEs

accumulate to a greater extent in diabetes and it appears that they have a

pathogenic role in diabetic microvascular disease. AGEs are postulated

to exert their effects through

“BiociemUtnq<m«t’PaX/uyfUuttiolow ' 3
7
two main mcchanisms: direct cross linking of proteins and binding to specific

receptors. Proteins that have been modified by cross linking are stiffer, more

resistant to degradation and are able to quench the potent vasodilator nitric

oxide, with consequent detrimental effects upon tissue structure and function.

In addition, it is believed that AGEs interact with specific reccptors to cause

injury,and several AGE binding proteins have now been described on numerous

cell types with in the Kidney. The exact role of these receptors is still being

elucidated. The best characterized is the receptor for advanced glycation end

products (RAGE), with AGE - RAGE binding activating nuclear factor-kappa

beta (NF-k(3) (Intra cellular signaling molecules) and stimulating the

production of several cytokines, chemokines and vasoactive hormones. Other

receptors may be more important in the clearance of AGEs.

Inhibitors of AGE accumulation

Inhibition of AGE accumulation has demonstrated benefits in

experimental models of diabetic renal disease. Use of ammoguanidine, a

hydrazine derivative, was associated with a reduced accumulation of AGEs,

as well as retardation of the development of albuminuria and an amelioration of

mesangial expansion. Aminoguanidine not only inhibits AGE formation,

but also has other actions that include inhibition of inducible nitric oxide

Z>ioc4crHt6t’i<f and "P^

synthase (iNOS).

( ompound that disrupt AGE mediated protein cross links, such

as

developments

that offer the potential to reverse established diabetes related tissue damage. I

liese drugs have been demonstrated to restore vessel compliance; however, their

roles in diabetic nephropathy has not yet been

established.

Polyol pathway
;;
■ In the polyol pathway, the enzyme aldose reductase catalyses the
if

conversion ot glucose to sorbitol, using reduced nictoinamidc adenine dinucleotide

phosphate (NADPH) as a cofactor. In the context of hyperglycaemia, the

metabolism of high levels of glucose causes sorbitol accumulation and the

consumption of NADPH and is associated with depletion of intracellular

myoinositol and altered cellular redox potential. NADPH is required for nitric

oxide synthase to produce nitric oxide and in the regeneration of the free radical

scavenger glutathione. This pathway has been extensively studied in the

pathogenesis of diabetic nephropathy with more limited investigation in renal

disease. Renal polyol accumulation has been reported in diabetic nephropathy and

inhibition has been observed with various aldose reductase inhibitors. The

importance of this pathway in diabetic nephropathy is emphasized by the

demonstration that aldose reductase inhibition is associated with reduced protein

Kinase C (PKC) activation and transforming growth factor beta (TGF-p) production
in human mesangial cells in response to glucose.In experimental diabetes, several

studies have examined the effect of aldose reductase inhibition, on various

structural and functional

\ §,|| markers of diabetic nephropathy; however, results have been inconsistent. In ;

; diabetic nephropathy, the results of aldose reductase inhibition have

h also been conflicting and in general rather disappointing.

■ ■

. V; Protein kinase C activation

- A direct pathogenic role for glucose has been suggested by cell

r , - culture experiments demonstrating that glucose itself could induce cellular

hypertrophy, ECM synthesis and cytokine production.These effects have

been
^ f- .
■ ‘ --ir • ■
attributed to activation ofPKC, a ubiquitous family of serine/threonine kinase MM

that are increased in the retina, arota, heart and glomeruli of diabetic animals.
; --; -
' i Radiolabelling of glucose shows that glycolytic intermediates are
I .

. incorporated directly into the glycerol backbone of a compound called

■ ^i-acylglycerol (DAG). The major endogenous activator ofPKC. The

increase ®P ; ;in.rihese intermediates in the context of hyper glycaemia

—-
results in a de novo increase in DAG and PKC activation. A second

relevant pathway also exists, as ligands such as angiotensin II (All).

Vascular endothelial growth factor (VEGF) and endothelin are able to

generate DAG via interaction with a cell membrane G-protein coupled

receptor mediated phospholipid cascade.

The PKC family regulates a diverse range of vascular functions

including regulation of blood flow, cellular differentiation and cytokine

generation. The observation that there is preferential activation of the

beta isoform ofPKC in diabetes has led to the development and study of

isoform specific inhibitors such as LY 333531. Significant amelioration

of glomerular
hyperfiltration, albuminuria and an attenuation ofTGFp expression and tissue

g matrix accumulation have been demonstrated by use of this compound in :

experimental diabetes. The 16 week administration of LY 333531 to a rat ffe,

fflodel of type 2 diabetes reduced albuminuria and TGFp expression, as well

as fibronectin and collegen IV accumulation in addition to normalizing PKC

activity. Recent work has also demonstrated that the ACE inhibitor ramipril

and interestingly the AGE inhibitor aminogunanidine, were both associated

1: with prevention of diabetes associated increased in PKC activation.

V It is therefore possible that PKC activation represents a critical

v downstream event in the pathogenesis of diabetic nephropathy. Indeed a

number bf the cytokines implicated in diabetic complications, such as VEGF,

are PKC dependent and PKC has been shown to modulate the effects of

glucose upon VEGF and TGF-P expression.

Oxidative stress
-■ r-. Oxidative stress is widely recognized by some investigators as a

key component in the development of diabetic complications. It is unknown

however, if it is an important early link between hyper glycaemia and

Complication or a consequence of other pathogenic mechanisms. Recently it

was demonstrated that the inhibition of reactive oxygen species (ROS)

formation in vitro interfered with multiple independent pathways of hyper

glycaemic damage, namely PKC activation, NF-k|3 activation and the

formation ofAGEs. Links between ROS and several metabolic pathways seem
•V —- “* " ■ i—i— i.» 1.1, .i■.-.I..... ..■I,, i— ... ... ...... i, , - I,..— i i,
 ■ •**
41

to be bi-directional. For example, glucose-induced generation of lipid peroxides

was attenuated by PKC inhibition, yet ROS have been shown to activate PKC.

Haemodynamics
It has been suggested that the earliest predictor for the development

of diabetic nephropathy is hyperfiltration, although this is not a universal

finding. The mechanisms implicated in the increase of GFR in diabetes involve

glucose dependent effects on arteriolar dilation mediated by a range of

vasoactive factors including All, insulin-like growth factor I (1GF-I), nitric

oxide, and prostaglandins. Experimentally this hyper filtration is mediated by

afferent arteriolar dilatation with an increase in glomerular hydrostatic

pressure. Measure that reduce glomerular pressured such as systemic blood

pressure reduction, a low protein diet or ACE inhibitors that block A1J

-mediated efferent arteriolar constriction all reduce the development of

glomerular damage and proteinuria.

Renin - Angiotensin system

The studies by Brenner ’s group have emphasized the role of

haemodynamic factors in diabetic nephropathy; in particular the potential of

inhibitors of the RAS as renoprotective agents. Micro puncture studies in

diabetic rats have revealed a range of intrarenal haemodynamic abnormalities

that included increased intra glomerular pressure and preferential afferent

versus efferent arteriolar vasodilation, which are modulated by All.

‘SCocAemUPuf cutci ' 42

 It is considered that the RAS may be locally activated in the diabetic Kidney,

and evidence continues to accrue in animal models showing a .range of

effects of agents that interrupt the RAS. These include reduction

in PKC activation, amelioration of renal TGF-p expression and

-ron
\ /- » ..
restoration ;; • ofthe depletion in the slit pore protein,

nephrin.

Endothelin
V -

The role of endothelin in progressive renal disease remains

controversial. Several groups have reported reno-protective effects associated

with the use of endothelin inhibitors. Equally other groups, have failed to
I < -
cor.firm significant renoprotective actions of a range of these endothelin
:'r~ T 'rV
receptor antagonists. This may reflect differences in receptor selectivity of

the antagonists, or to the experimental context. It is also possible that the

.'
endothelin pathway does not play a central role in the pathogenesis of renal
.; T':’■
damage, but the context of RAS inhibition may offer additional benefits.
•• . ru

Other vaso-active pathways

:
h Although the focus has been on vasoconstrictor pathways,

glomerular vasomotor tone is also dependent on the status of vasodilators

such a bradykinin and atrial natriuretic peptide. A range of novel compounds

r
that act on various metallo proteinases, such as ACE and neutral

endopeptidase (NEP), have now been developed. These compounds, such as

-~r~v

the dual ACE/ JNEP inhibitors, inhibit formation of All and block the

enzyme NEP involved in the degradation of these vasodilators. The studies

suggests that these

<%H<1
compounds may confer renoproteclion in various forms of renal disease including

experimental diabetic nephropathy. I lie role of these compounds in human

diabetic nephropathy remains to be delineated.

Cytokines
An area of intense research has focused on the properties of soluble

growth factors called cytokines. Over-expression and inappropriate expression of

these powerful molecules is believed to play a central role in the development of

microvascular disease.

A. Transforming growth factor-bcta

TGF -(3 is considered a pivotal cytokine involved in a range of
j -

diverse fibrotic conditions that includes diabetic nephropathy. Il coordinates the

physiological process of normal tissue repair, potently stimulating matrix

protein production and inhibiting its degradation. In addition iGF-P has a

F -

number of powerful effects upon cellular differentiation and proliferation. The

pathogenic role of TGF-(3 is illustrated by gene-transfection experiments

where TGF-P is over expressed with the associated development of

\

glomerulosclerosis and mesangial matrix accumulation. Further evidence comes

from a mouse model of type 2 diabetes where the chronic administration of TGF-p

neutralizing antibodies prevented diabetes associated accumulation of glomerular

ECM. In addition,TGF-P neutralization prevented diabetes-related decrease in

GFR, though interestingly it failed to normalize pxoteinuria. This indicates that

one cannot assume that various reno protective

lai ‘SuKAewiAtui and W
BSP,: 'therapies will affect functional parameters such as albuminuria and

renal structural changes in a similar manner.

B. Connective tissue growth factor

Although TGF-p - appears to plays a central role in diabetic renal

disease, it is now dear that other prosclerotic cytokines, acting either

individually or in concert with TGF-P , play important biological roles in

diabetic nephropathy. Connective tissue growth factor (CTGF), a cysteine rich

peptide, is one such cytokine. CTGF is stimulated by TGFp and by mechanical

stretch and has been shown to be up-regulate 10 fold in the diabetic Kidney. In

vitro CTGF induces HCM production and inhibition of this growth factor

ameliorates collagen 1 secretion, indicating that TGF -{J alone does not fully

explain diabetes associated HCM accumulation. Of particular interest is the

recent finding, in a noil-renal context, that AGEs may induce HCM production

via a C l Gl dependent pathway .

U"

(.. Vascular endothelial growth factor

i A role for VHGF in the Kidney is highly attractive as certain

manifestations of the diabetic state, such as increased endothelial permeability

and cellular proliferation arc readily induced in vessels by VHGF. Renal VEGF.

mRNA and immuno reactivity is increased in the diabetic Kidney. Although All.

TGFp. hypoxia and glucose all stimulate VEGF, the role of VEGF in the Kidney

remains to be clarified, particularly in view of the observation that in non-

diabetic renal disease (NDRD), certain VBGL

isoforms may mediate glomerular repair. This suggests that increased VEGF

expression may represent an attempt at repair rather than as an inducer of

injury and this issue remains to be adequately resolved.

D. Other cytokines

A number of other cytokines have been explored in diabetic

nephropathy. These include epidermal growth factor (EGF), heparin-binding

epidermal growth factor-like growth factor (HB-EGF) and platelet derived

growth factor(PDGF) but their respective roles await further elucidation. This

is of particular interest with the advent of approaches involving specific

blockade of tyrosine receptor kinase, these agents have been shown to confer

reno protection in non-diabetic models of renal disease.

Conclusion |

The last decade has seen major advances in our understanding of a

number of the mechanism that underlie the development of diabetic

nephropathy. Modulations of these perturbations at a number of levels, by

influencing systemic vascular pressure, or at the level of intracellular signaling

pathways and downstream cytokine production offers exciting opportunities to

reduce the disease burden for the expanding diabetic population.

Chapter 4
 Renin-Angiotensin System in Diabetic
Nephropathy

Introduction
/ "Vhe"RAS is traditionally viewed as an endocrine system primarily involved in

the regulation of systemic blood pressure and salt and water homeostasis.

However, All, the effectors molecule of the RAS, has many actions beyond its

effects on blood pressure regulation and they can either be viewed as

homodynamic or non-hacmo dynamic. In many instances these actions are

complementary or synergistic in their capacity to regulate blood pressure and

volume haemostasis and can also conspire to promote tissue injury. As such, this

has prompted the hypothesis that therapeutic inhibition of the RAS may provide

protection of vulnerable target organs, beyond the expected benefit of blood

pressure reduction alone. This chapter will consider this concept by first over

viewing the key components of the RAS and then

TReii.1 - /fftqiotcKtU S<fd(ctfc in 'DutSetic 'HeiAwfrxtkt ~ 4

7 focusing on the may diverse actions ol All and their potential role in the

pathogenesis of diabetic nephropathy.

Overview of the renin angiotensin system

Classically, the RAS is viewed as an endocrine system in which

renin is secreted by the juxtaglomerular apparatus (JGA) with in the kidney

and acts on its specific substrate angiotensinogen, produced by the liver, to

generate Al. The A1 is then cleaved by ACE, predominantly with in the

pulmonary circulation, to yield All Al and 11 are subsequently degraded by

various proteolytic enzyme systems to generate A [1-7], AIII or A1V.

Renin

Renin is a 38kDa aspartyl protease that shows high substrate for

angiotensinogen. Renin is produced by specialized smooth muscle like cells

(the macula densa) with in a unique anatomical structure adjacent to renal

glomeruli, the JGA. With in the JGA, produced as a 45kDa Pre-prorenin and

released as active renin after a two stage cleavage of the signal sequence and

the pro segment. Renin release by the JGA is influenced by many factors such

as

♦ Renal nerve activation. Increased sympathetic nerve activity

increases rennin release.

♦ Renal perfusion pressure. Renal ischemia increases renin

release and vice versa; and

♦ Various'hormonal influences: All suppresses renin release.

Angiotensinogen

Angiotensinogen
TRCKU - Sis t;i 60kDa
f A tprotein
e m andCHthe only substrate from which All
Viatetic -48can be
generated. Angiotensinogen is predominantly produced by the liver but it is also expressed in

many other tissues, including the brain ,

(at, the heart, the kidney, lung and fibroblasts. Renin cleaves angiotensinogen

to yield Al. This molecule has little biological activity and function as the

precursor peptide for the generation of All via the actions of ACE.

Angiotensin converting enzyme

ACE is a zinc metalloproteinase that cleaves two peptides (histidine

and leucine) from the carboxy terminus of the decapepitide Al to yield the

biological active octapeptide, All. It is important to note, however , t h a t the

action of ACE is not specific for Al. ACE, also termed kininase 11, is involved

in the enzymatic cleavage and inactivation of other peptides such as bradykinin.

Therefore, inhibition of ACE not only decreases All production, it also inhibits

the degradation of bradykinins and other peptides normally subject to

degradation by leutinizing hormone, among others. The potentiation of kinins

by ACE inhibition is thought to be important in the development of ACE

inhibitor induced cough and they may have other actions, such as the potential

clinical benefits of bradykinins

potentiation However, to date, the benefits of Kinin potentiation Via
.1

System Ch 'DCaAetif. - 49
ACE inhabitation remain largely hypothetical in humans. The concept of a
‘tissue renin- angiotensin’ system
The classical RAS pathway implies that the majority of All is

generated systcmically within the vascular compartment. However there is

considerable evidence to suggest that A1J can also be generated locally

within tissues. This has given rise to the concept of a “circulating” and a

‘tissue’ RAS, subserving different functions but operating in tandem. Most

of the key elements of the RAS, such as renin and angiotensinogen, can be

detected and synthesized in many tissues and the expression and activity of

the RAS in these tissues appears to be locally and independently regulated.

This is of particular relevance and importance to tissues such as the kidney

in which
$

high levels of All are generated. Further more, this concept of local All

generation implies that inhibition of systemic or circulating All production

does not necessarily imply inhibition of tissue All production. This concept

may be of considerable importance, mindful of the potentially important role

of All in the pathogenesis of tissue injury in diabetes.

Multiple pathways of angiotensin II generation

It is clear that the aforementioned pathway of the RAS are not the

only mechanism where by All can be generated from its solitary substrate

angiotensinogen. Various non- renin angiotensinogenases are now known to

exist that are capable of generating both AI and All directly from

angiotensinogen, without involvement of renin of ACli. These enzymes

included. / -
♦ Chymostatin All generating enzyme (CAGE).
 ♦ Chymases (serine proteases),many forms of which have been identified in

various tissues including kidney.

Other enzymes not traditionally considered to be relevant to the RAS, for

example tissue plasminogen activator, cathepsin G and tonin.

♦ Recognition of these additional enzymes system may have considerable

therapeutic importance as total blockage of All production is unlikely to

be achieved by ACE inhibition alone.

Angiotensin II receptors
The emergence of highly specific and selective AllAs along with

molecular cloning of All receptor subtypes has led to a tremendous growth

inknowledge about the function and complexity of the All receptor system. A

full review of All receptor is outside the scope of this chapter. Suffice to say

that at least four subtypes of All receptors exist in humans (AT 1-4), the two

main subtypes being ATI and AT2 the ATI receptors have a classic G-protein

linked peptide receptor structure with seven transmembrane spanning helices,

coupled to G proteins (Gi and Gq) for intracellular signalling. The ATI

receptors are widely distributed and almost all of the recognized renal and

cardiovascul&actions of All cited below appear to be mediated, via the ATI

receptor. The function and biological relevance of the AT2 receptor is less

- s^KfiGteKdi* Sy&tem it 'Diabetic ~

well defined. I he AI2 receptor is not ;is ubiquitously expressed as the ATI receptor and is

often only transiently expressed during tissue development and repair, suggesting a

potentially important role in the regulation of cell growth and cell death.

Renin-angiotensin system in diabetes

Diabetes, particularly type 2, is a state of sodium retention and

volume expansion and one would expect this to suppress the RAS. It is

therefore not surprising that many studies in humans have reported suppression

of the circulating RAS in patients with diabetes, especially those with

nephropathy. Despite this, many studies in experimental animals have shown

that key components of the RAS are expressed at normal or increased level in

diabetic renal tissues. This supports the hypotheses that renal tissue RAS is

inappropriately activated in diabetes and that measurements of plasma renin

activity (PRA) are a poor surrogate for tissue RAS activity. In support of this,

a series of studies by Hollenberg in patients with type 2 diabetes have

demonstrated increased functional renal RAS activity. They reported that in

patients with type 2 diabetes receiving a high salt diet, PRA was suppressed

less well in diabetic patients when compared to non diabetic controls .The reno

vaso dilator response to the ACE-inhibitor enalapril was substantially greater

in type 2 diabetic patients on a high salt diet when compared to non-diabetic

controls on the same diet who exhibited a minimal response, together these

observations support the hypothesis that the renal

-v'

Keni* -/fuQiotcKAiK System in 'Dux&etic ' ^2

 1S
RAS inappropriately active in patients with type 2 diabetes, even prior to the

development of nephropathy.

Biological actions of angiotensin II and their relevance to the

development of diabetic nephropathy

AH has both haemodynamic and non-haemodynamic action on renal

tissues (see by table) these actions are relevant and important for the development

and progression of renal injury in diabetes and it must he emphasized that they act

in concert in the initiation and progression of diabetic nephropathy.

Haemoctynamic and non-hacmodynamic actions of angiotensin II in the

pathogenesis of diabetic nephropathy.

<=> Systemic hypertension O Glomerular hypertension >=> Increase glomerular

permeability <=> Increase oxidative stress/inflammation Increased growth factor.

Increased TGF-a fibroblast and fibrosis =>

Monocvte migration and activation

 ngiotensin II. systemic and glomerular haemodynamic action

The importance of systemic hypertension in the development and

progression of diabetic nephropathy cannot be overstated. The reduction in

systemic blood pressure when patients are treated with AlIAs implies an

important role for A II in the maintenance of systemic hypertension in

patients with diabetes. However, All also has haemodynamic action beyond

the systemic circulation and plays a key role in the fine regulation of

pressures with in the glomerular microcirculation.

The glomerular microcirculation is unusual in that it is supplied

and drained by arterioles , the afferent and efferent arterioles respectively.

This is because the pressure with in the microcirculation is a key

determinant of glomerular filtration and is actively and finely regulated by

the vascular tone with in the afferent and efferent arterioles. Diabetes is

associated with a generalized impairment of microvascular blood flow, auto

regulation, the consequence of which is an increase in glomerular pressure

.This is due to a deficiency in afferent arteriolar tone in the phase of an

increased perfusion pressure due to coexisting systemic hypertension. In

effect, the diabetic glomerular microcirculation is a ‘pressure passive

microvascular bed, at the mercy of systemic hypertension. This explains the

increased renal plasma flow and GFR that are frequently observed in patients

early alter the onset ot diabetes. This unforgiving state is compounded by the

aforementioned inappropriate activation of the renal RAS. All constricts the

efferent arteriole,
tJia - 54
 furl her elevating glomerular mierovasculatory pressure. This increase in

inicrovascular pressure promotes an increase in albumin leakage across the

glomerular capillary bed into I he urinary space and is key to the

development of microalbuminuria and proteinuria, the hallmark ofdiabctic

nephropathy. The increase in urinary albumin excretion (I / AI v) rale

observed in the early stage ofdiabctic nephropathy is an important bedside

surrogate masker of increased glomerular pressure.

Non-haemodynamic actions of angiotensin II and the pathogenesis

of diabetic nephropathy.

In the past 20yrs, there has been increasing recognition of the fact

that the spectrum of All activity extends well beyond its haemodynamic

actions.

■ All can directly modulate glomerular permeability to

macromolecules, independent of its effects on glomerular

pressure.

■ All has powerful growth promoting action and can induce the

phenotypic shift of many cell types towards a pro-

inflammatory, pro-synt hetic/fibrotic phenotype, more

commonly associated with tissue injury and repairs.

 All is powerfully pro-oxidant and prothrombotic and these

actions further augment tissue injury and fibrosis. The simplistic overview

reveals why, in the context of the diabetic kidney, where the RAS is inappropriately

active, All has been so strongly implicated in the pathogenesis of diabetic nephropathy

Angiotensin II and proteinuria

An increased UAE rate is the hallmark of diabetic

nephropathy.In early stages of nephropathy, this is functional and

reversible, driven by changes in glomerular pressure. The GBM is a

complex structure that play a fundamental role in the regulation of

macromolecular passage into the urinary space. The GBM separates the

glomerular capillary space from the urinary space of the Bowmans’s

capsule. The passage of macromolecules such as albumin across the GBM is

determined not only by the pressure differential across it, but also the

specific physical properties of the macromolecule, such as size and charge.

Therefore, the GBM exhibits size and charge selectivity and disturbances to

these properties can influence macromolecular trafficking across the GBM.

GBM perm selectivity for macromolecule can be measured

experimentally by intravenously infusing a solution of dextran molecules of

different radius into the circulation and measuring their urinary clearance. In

these circumstances the fractional clearance of dextrans is inversely

proportional to the functional integrity of the GBM. In animal models of

chronic renal disease, following unilateral nephrectomy glomerular perm

selectivity is reduced just as it is in early diabetes in humans. The treatment

tin System it "Diabetic - 56

of such experimental animals with losartan to selectively inhibit the action of All

via the ATI, receptor restores glomerular perm selectivity to normal. This

observation suggests that All directly and adversely influences the selectivity

characteristics of GBM and could thereby directly reduce the barrier to proteins

trafficking across the GBM.

These observation reveal too important All driven mechanisms for the

initiation of proteinuria in diabetes:-

1. Increased glomerular pressure due to distuibed microvascular

blood flow regulation and

2. reduced glomerular perm selectivity for macromolecules such as

proteins

These two early mechanisms are compounded at a later stage by

inflammation, structural disorganization and damage leading to heavier protein

loss. These later processes are also, at least in part, driven by A II.

Angiotension II as a growth factor

All is a growth factor for many cell types including vascular smooth

muscle cells, cardiac myocytes and glomerular mesangial cells. All also promotes

the synthesis and release of potent vascular and mesangial cell mitogens such as

PDGF, basic fibroblast growth factor (bFGF) and others that mediate some of its

cell specific growth promoting actions. It has been ■mplicated in the

development of glomerular mesangial expansion via

57mesangial cell proliferation and increased mesangial matrix deposition. This

is an early histological feature of diabctic nephropathy. Mesangial expansion

within the enclosed glomerular space occurs at the expense of the glomerular

capillary tuft that is compressed, thereby reducing surface area for glomerular

filtration. Consequently, mesangial expansion is inversely proportional to GFR

in patients with diabetic nephropathy. The expression of another growth factor,

VEGF, is also increased by All via the ATI receptor. VEGF potently induces

angiogenesis and has been strongly implicated in the pathogenesis of diabetic

neovascularization and capillary leakage, particularly in the retina.

It is not widely appreciated that similar microvascular proliferative changes and

microaneurysm formation also occur in the glomerulus. It is likely that such

structural disorganization driven by growth factors such as VEGF is of

unrecognized importance in the pathogenesis of diabetic nephropathy. In this

regard, the observation that Al increases VEGF production and that this action

maybe inhibited by ATI receptor antagonism may be important therapeutically to

limit diabetic revascularization in the kidney and beyond.

Angiotensin II, oxidant stress and renal inflammation

Diabetic nephropathy exhibits all the hallmarks of a chronic inflammatory

process culminating in glomerular and interstitial fibrosis. All has recently been

shown to induce oxidative stress with in the cardiovascular and renal systems.

All acting via the ATI receptor, activates a reduced nicotinamide adenine

dinucleotide (NAl)H) /NADPH oxidase within thevascular smooth muscle and

glomerular mesangial cells that leads to the pioduction of a highly reactive

oxygen radical, super oxide. Super oxide is pro-inflammatory and injurious to

local tissues. Super oxide also reacts with and quenches nitric oxide there by

forming another toxic radical peroxynitrite.

The resulting loss of nitric oxide has devastating consequences lor the kidney.

Nitric oxide is a potent vasodilator and plays a key role in the regulation of

glomerular haemodynamics. Nitric oxide is anti proliferative and its loss

enhances the cellular growth promoting action of All. Nitric oxide is also

antithrombotic.

There prooxidant action of All are likely to be particularly damaging

in patients with diabetes. Normally the reactive oxygen species are rapidly

removed by efficient anti-oxidant system to prevent tissue injury. There is

abundant evidence that oxidant stress is increased in patients with diabetes and

that antioxidant defenses are diminished, probably by excess consumption.

In this setting, the oxidant stress induced by All in the kidney and other vulnerable

tissues will be particularly devastating. This is potentially one of the most

important mechanisms where by All exhibits its direct toxic and pro-inflammatory

actions on renal and cardiovascular cells.

In addition to oxidant stress , All can directly augment renal

System U Vudetic - 59
inflammation via complementary mechanisms. All augments the production of

many pro-inflammatory cytokines. Moreover, All up regulates the expression of

cell surface proteins (eg.monocyte chemotractant protein-1) that promote

Stf&tem CH 'Dia&etic - 60
 illation ol inflammatory cells, such as monocytes and macrophages into the

kidney thereby complying the renal inflammatory cascade.

Angiotensin 11 induced renal matrix synthesis and fibrosis

A 11 strongly induces the production of TGF-p, apparent

multifunctional cytokine that plays a key role in the development of renal

fibrosis. TGF-(3 orchestrates the regulation of many key enzyme systems

involved in the regulation of extra cellular protein biosynthesis and

degradation. Specifically, TGF-p increases matrix synthesis and inhibits many

of the enzymes (metallo proteinases) that are involved in matrix protein

degradation. Therefore TGF-p shills the dynamic balance between matrix

synthesis and degradation in favour of matrix accumulation. In addition to All

other factors relevant to the pathogenesis of kidney disease in diabetes also

increase TGF-p and matrix production by glomerular mesangial cells. These

include elevated extra cellular glucose concentrations and mechanical strains

(haemodynamic stress).

Numerous studies have confirmed that TGF-P expression is increased

in renal tissues from experimental animals and patient with diabetes. Moreover,

in patients with types 2 diabetes and nephropathy, circulating levels of TGF-P

are increased, which suggests that diabetic is a pro-inflammatory state. In a

recent study of patients with type 2 diabetes and microalbhuminuria treatment

with losartam (losartan) reduced both microalbaninuria and TGF-P level. This is

strong evidence for a key role for All in the development of

I*
Microalbuminuria and activation of TGF-p in patients with type 2 diabetes.

Stf&tem CH 'Dia&etic - 60
 Implications beyond the kidney

There is stiong evidence implicating All in the pathogenesis of

diabetic nephropathy is haemodynamic and non-haemodynamic mcchanisms. The

pathogenesis ol diabetic renal disease involves the activation of an inflammatory

process culminating in progressive renal fibrosis. Many of the processes involved

are identical to those leading to the development of atherosclerosis. This explains

the well recognized association between the onset of nephropathy and the

accelerated development of atherosclerosis in people with diabetes. This being so,

one would expect that blockade of the AT p receptor might have the potential to

prevent the development of atherosclerosis. A recent study using a primate model

of atherosclerosis has confirmed that treatment with losartan can prevent the

formation of atherosclerotic plaques and reduce the circulating levels of

inflammatory cytokines known to be involved in the pathogenesis of

atherosclerosis.

In summary, the RAS is inappropriately activated in people with diabetes

and multiple strands of evidence suggest an important role for AH acting via the AT,

receptor in the pathogenesis of diabetic nephropathy. These findings provide a strong

rationale for selective and specific inhibition of the ATI receptor in an endeavour to

prevent the development and/or limit the progression of nephropathy in people with

type 2 diabetes. The benefit afforded by such a strategy is likely to be

complementary to that achieved by systemic blood pressure reduction. With early and

prolonged intervention ol this kind, it is likely that the benefit will extend beyond

the Kidney

“i^CKCH ~ S<f&te*K in