REVIEWS

Present and future drug treatments

for chronic kidney diseases:
evolving targets in renoprotection
Norberto Perico, Ariela Benigni and Giuseppe Remuzzi
Abstract | At present, there are no specific cures for most of the acquired chronic kidney
diseases, and renal transplantation is limited by organ shortage, therefore present efforts
are concentrated on the prevention of progression of renal diseases. There is robust
experimental and clinical evidence that progression of chronic nephropathies is
multifactorial; however, intraglomerular haemodynamic changes and proteinuria play a
key role in this process. With a focus on renoprotection, we first examine more established
therapies — such as those that modulate the renin–angiotensin–aldosterone system —
that can be used for the treatment of proteinuric renal diseases. We then discuss
examples of novel drugs and biologics that might be used to target the inflammatory and
profibrotic process, and glomerular injury, highlighting results from recent clinical trials.

Department of Medicine
and Transplantation,
Ospedali Riuniti di Bergamo -
Mario Negri Institute for
Pharmacological Research,
Via Gavazzeni 11-24125,
Bergamo, Italy.
Correspondence to G.R.
e-mail:
gremuzzi@marionegri.it
doi:10.1038/nrd2685
Published online
10 October 2008
Chronic kidney disease (CKD) is a worldwide threat to
public health, but the scale of the problem is probably not
fully appreciated. Estimates of the global burden of dis-
eases report that diseases of the kidney and urinary tract
contribute to approximately 830,000 deaths annually1.
CKD that requires renal-replacement therapy (RRT),
which consists primarily of kidney transplantation,
haemodialysis and peritoneal dialysis, are rising sharply
worldwide so that the global end-stage renal disease
(ESRD) population will exceed 2 million patients by the
year 2010 (REF.1). The aggregate cost for treatment during
the coming decade will be more than US$1 trillion1.
Patients on RRT can be regarded as the tip of an iceberg,
as the number of those with CKD not yet in need of RRT
is much greater. However, only rough estimates exist for
the prevalence of predialysis CKD.
It is increasingly being recognized that the burden of
CKD is not limited to its implications on demands for
RRT, but has a major impact on the health of the overall
population. Indeed, patients with reduced kidney function
represent a population not only at risk for progression of
kidney disease and for ESRD, but also at even greater risk
for cardiovascular diseases2. This suggests that patients
with CKD who are progressing towards ESRD carry the
heaviest burden of cardiovascular disease, and that this
frequently leads to death before ESRD is reached. So,
CKD, through their impact on cardiovascular morbidity,
may directly contribute to increasing mortalities, even in
emerging countries.
CKD encompasses different types of renal disease,
and the biggest group includes glomerular diseases of
which diabetic and hypertensive nephropathies are the
most common cause of CKD. They are considered to
be degenerative renal diseases. There are also inflam-
matory glomerular diseases, the prototype being lupus
nephritis. Other renal diseases affect primarily the
tubulointerstitium and they include polycystic kidney
disease.
At present, there is no specific cure for most of the
acquired CKDs, nor is there any reasonable expectation
that gene therapy will be available soon enough to treat
genetic forms of kidney diseases, such as polycystic
kidney disease. Renal transplantation is limited by organ
shortage3, a worldwide problem that is not likely to be
resolved in the near or immediate future. Therefore, the
best therapeutic option at the present time is to concen-
trate our efforts on preventing the progression of renal
diseases.
Progression of chronic nephropathies
Certain renal diseases, although rare, have a rapid course
that quickly leads to irreversible ESRD. More common
nephropathies progress less rapidly, but still evolve to
ESRD within one to four decades after diagnosis. During
the past 20 years, research in animals and humans have
provided significant insights on the mechanisms by
which CKD progresses and has indicated preventive
strategies. Many studies have established that progressive

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Mesangial cell
A key cell of the glomerulus
with enlarged cytoplasmic
processes that extend around
the capillary lumen and
insinuate themselves between
the glomerular basement
membrane and the overlying
endothelium.
Podocytes
The visceral epithelial cells
of the glomerulus with large
cytoplasmic processes that
extend from the main body.
Glomerulosclerosis
Mesangial accumulation
of hyaline material that
progressively encroaches
on capillary lumina, which
obliterates the Bowman’s
space and results in global
sclerosis of the glomerulus.
Slit diaphragm
A thin membrane that bridges
the gap between adjacent foot
processes of the podocytes
near the glomerular basement
membrane.
nATURE REvIEwS | drug discovery
deterioration of renal function is the result of compen-
satory glomerular haemodynamic changes in response
to nephron loss due to various original insults. In the
widely used experimental model of renal mass reduc-
tion by partial five-sixths nephrectomy, the remnant
glomeruli undergo hypertrophy and the tone of afferent
arterioles drops by a greater degree than that of efferent
ones. This increases glomerular capillary hydraulic pres-
sure, leading to more filtrate formed per nephron4. These
changes serve initially to minimize the functional conse-
quences of nephron loss, but ultimately are detrimental,
causing relentless injury of remaining intact nephrons5.
Enhancing intraglomerular capillary pressure and per-
fusion pressure result in stretching of the three major
cell types — endothelial cells, mesangial cells and podo-
cytes — in the glomerulus, leading to direct cell injury6
(FIG. 1). The mechanical cell damage together with con-
comitant induction of pro-inflammatory and profibrotic
phenotypic alterations in glomerular cells promote the
development of glomerulosclerosis7–15. Impairment of the
selective function of the glomerular capillary wall is a
further mechanism by which glomerular hypertension
contributes to glomerular injury, resulting in excessive
protein ultrafiltration in animal models16. An excessive
protein load in podocytes is a factor underlying pro-
gressive injury of these glomerular cells. These injured
glomerular cells release transforming growth factor-β
(TGF-β) in the microenvironment, which ultimately
promotes activation of adjacent mesangial cells and
deposition of extracellular matrix17 (FIG. 1).
A key player of these glomerular haemodynamic
changes that are central to progressive renal injury is
angiotensin II18. In vitro experimental studies have also
revealed several non-haemodynamic effects of angio-
tensin II that are relevant in renal disease progression.
They include stimulation of mesangial cell proliferation
and extracellular matrix deposition19, and production
of plasminogen activator inhibitor, type 1 (PAI1) by
endothelial cells20. Moreover, angiotensin II alters size-
selective properties of the glomerular capillary barrier by
binding to angiotensin II subtype 1 receptors on podo-
cyte foot processes. This leads to rearrangement of the
cell cytoskeleton and in the distribution or expression
of protein components of the slit diaphragm, which even-
tually allows excess plasma proteins to move into the
urinary space21 (FIG. 1).
Exposure of proximal tubuli to the excessive filtered
proteins leads to further intrinsic toxicity of this nephron
segment17. Thus, both in vitro and in vivo protein overload
causes increased production of vasoactive, inflammatory
mediators and growth factors such as endothelin 1 (ET1),
monocyte chemoattractant protein 1 (MCP1), RAnTES
and osteopontin. Transcription analysis by cDnA micro-
array of renal tubular cells from patients with proteinuric
nephropathies22 identified more than 160 genes as being
regulated differently compared with those of proximal
tubular cells from control subjects.
The activation of various molecules, including cyto-
kines, growth factors and vasoactive substances, results in
abnormal accumulation in the interstitium of extracell-
ular matrix collagen, fibronectin and other components
REVIEWS
that are responsible for interstitial fibrosis23. Tissue injury
promotes the generation of reactive oxygen species and
an endoplasmic reticulum stress response24. This leads
to the oxidative modification of membrane lipids, pro-
teins and DnA that initiates cell-death responses, which
results in tissue inflammation24. loss of peritubular capil-
laries also reduces oxygen availability. The danger signal
secondary to hypoxia links cell injury to the activation of
Toll-like receptors, which further enhances the produc-
tion of pro-inflammatory cytokines and chemokines and
contribute to the local recruitment of macrophages and
lymphocytes24,25. In turn, they are capable or stimulating
the transformation of tubular cells into myofibroblasts.
Modest derangement of these processes often escapes
clinical or laboratory detection because of compensa-
tory adaptations by the myriad of unimpaired nephrons.
beyond a certain level of injury, however, compensatory
adaptations no longer keep pace with nephron loss. As
a result, glomerular filtration rate declines and organic
nitrogenous wastes accumulate in plasma and other
body fluids. In line with these experimental observa-
tions is the evidence that in humans with nephropathy,
more severe and persistent proteinuria means more
rapid progression of disease26. The role of proteinuria as
a strong, independent predictor of ESRD has also been
documented in a mass-screening setting27.
In summary, there is robust experimental and clini-
cal evidence that progression of chronic nephropathies
is multifactorial, but intraglomerular haemodynamic
changes and proteinuria have a key role in this process.
Renin–angiotensin–aldosterone system blockade
Although traditionally the renin–angiotensin–aldosterone
system (RAAS) was considered a systemic endocrine
system, an intrarenal local RAAS has also been described
to operate independently. The overview of the RAAS
cascade is reported in Supplementary information S1
(box).
RAAS can be inhibited at various points of the cas-
cade. Three strategies have already been developed by
pharmaceutical companies: inhibition of angiotensin-
converting enzyme (ACE), competitive inhibition of
the binding of angiotensin II to cell-surface receptors
and inhibition of the enzymatic action of renin. Most
trials with these drugs have been performed or are being
performed on diabetic nephropathy. ACE inhibitors and,
increasingly, angiotensin II receptor antagonists are now
widely prescribed for the treatment of proteinuric renal
diseases, including diabetic nephropathy.
ACE inhibitors and angiotensin II receptor blockers
or their combination for renoprotection. In five-sixths
nephrectomized rats, ACE inhibitors or angiotensin II
receptor antagonists slowed the development of protein-
uria and limited renal structural damage compared with
conventional antihypertensive drugs16. As a consequence
of these effects, progressive loss of kidney function was
attenuated16. In this nephrectomy model, as in other
models of diabetic and non-diabetic renal disease, ACE
inhibitors and angiotensin II receptor antagonists have
the unique property of normalizing intraglomerular
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Degenerative/ Proliferation/
inflammatory extracellular Glomerulosclerosis
glomerular diseases matrix deposition

Nephron loss

Mesangial cells
Compensatory
Mechanical
glomerular capillary Endothelial cells
stretching
hypertension
Podocytes ↑Ang II synthesis

Glomerular Increased glomerular

hyperfiltration permeability Podocyte protein Gene
accumulation activation ↑TGF-β
to proteins

Cytoskeleton rearrangement/
Proteinuria loss of differentiated phenotype

Slit-diaphragm Foot-process
dysfunction effacement

Figure 1 | Mechanisms of progressive glomerular injury. A reduction in the number of nephrons as a consequence
of various glomerular diseases results in compensatory glomerular haemodynamic changes that are ultimately
detrimental. In particular, by mechanical stretching, the increased glomerular capillaryNature
pressure directly
Reviews | Druginjures
Discovery
glomerular cells — namely endothelial cells, mesangial cells and podocytes. Glomerular hypertension also impairs the
glomerular capillary size-permeable-selective function, which causes excessive protein ultrafiltration and eventually
proteinuria. The concomitant protein overload of podocytes leads to rearrangement of the cell cytoskeleton and loss
of differentiated phenotype. This eventually contributes to the structural and functional changes of foot processes and
slit diaphragm of podocytes, which further enhances protein traffic through the glomerular capillary wall. Injured
podocytes release transforming growth factor-β (TGF-β) in the microenvironment, which activates adjacent mesangial
cells to proliferate and produce excessive extracellular matrix components, and thus contributes to the development
of glomerulosclerosis. A key player in these haemodynamic and non-haemodynamic events of progressive glomerular
injury is angiotensin II (Ang II).

capillary pressure and size selectivity of the glomerular
capillary barrier, which limits protein traffic and conse-
quent nephrotoxicity28.
before 1995, several randomized trials evaluating the
effect of ACE inhibitors in humans had conflicting results,
possibly because of the heterogeneity of the patients and
treatments studied. More importantly, most studies
included patients with no or mild proteinuria, which was
associated with slow disease progression and negligible
treatment effect. This, combined with the small sample
size, reduced the power of the analyses and increased
the risk of random data fluctuations29. More convincing
evidence of a specific renoprotective and dialysis-saving
potential of ACE inhibitors and angiotensin II receptor
antagonists was then provided by a series of large clinical
trials in patients with diabetes and in patients without dia-
betes (TABLE 1). The REIn study30 showed that in patients
with non-diabetic nephropathy an ACE inhibitor (rami-
pril) slowed the rate of decline in glomerular filtration
rate and halved the risk of ESRD at equivalent levels of
blood-pressure control in comparison with conventional
antihypertensive therapy. Treatment effect was a func-
tion of baseline proteinuria, and the highest benefit was
observed in those with most severe proteinuria to start
with30,31. Subsequent meta-analysis confirmed that pro-
teinuria strongly correlated with renal disease progres-
sion, and treatments that fail to reduce proteinuria are
unlikely to exert significant renoprotection32,33. However,
proteinuria is currently not recognized by the US Food
and Drug Administration (FDA) as an outcome for CKD
treatment. nevertheless, in a recent meeting, discussion
has been initiated between leading nephrologists and
FDA representatives for qualification of proteinuria as
a surrogate marker in CKD (see Further information).
In chronic nephropathies, high blood pressure is also a
major determinant of disease progression, and blood-
pressure reduction is renoprotective34. However, it is
controversial whether ACE inhibition therapy in combi-
nation with efforts to reduce blood pressure — to lower
than usual targets — in patients with CKD will provide
additional renoprotection. This issue has been addressed
by the REIn-2 trial, which showed that in patients with
non-diabetic proteinuric nephropathies receiving back-
ground ACE-inhibitor therapy, no additional benefit from
further blood-pressure reduction by the dihydropyridine
calcium-channel blocker felodipine was achieved in terms
of delaying decline in glomerular filtration rate and pro-
gression to ESRD35.

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Albumnin/creatinine ratio
Ratio between the
concentrations of albumin
and creatinine measured in
urine. It is used to detect
early chronic nephropathy,
particularly diabetic
nephropathy.
nATURE REvIEwS | drug discovery
ACE inhibitors are also renoprotective in patients with
type 1 diabetes. They reduced the incidence of ESRD as
well as progression to overt nephropathy in those with
macroalbuminuria36. Relatively small studies have also
found that ACE inhibitors are renoprotective in type 2
diabetes37, but there has been no adequately powered
trial conducted yet that evaluates their effect on renal
function decline or progression to ESRD. nevertheless,
in the bEnEDICT trial38, evaluation of 1,204 patients with
type 2 diabetes and hypertension (but no evidence of renal
involvement) has shown that ACE inhibitors may prevent
the onset of nephropathy, which is the strongest risk fac-
tor for cardiovascular mortality in this population. More
recently, the IRMA-2 (REF. 39), REnAAl40 and IDnT41
trials have provided evidence that angiotensin II receptor
antagonists are also renoprotective, independently of their
antihypertensive effect, in patients with type 2 diabetes
and incipient or overt nephropathy (TABLE 1).
nevertheless, more effective intervention strategies
are needed to limit the still excessive renal and cardio-
vascular risk of people with diabetes. In this regard, it is
important to note that angiotensin II can be also gener-
ated by non-ACE pathways, such as chymase, that are
not inhibited by ACE inhibitors (see Supplementary
information S1 (box))42. The combination of an ACE
inhibitor and an angiotensin II receptor antagonist has
been suggested as a way to maximize RAAS blockade by
affecting both the bioavailability and activity of angio-
tensin II. Moreover, an ACE inhibitor might block the
compensatory increase in angiotensin II synthesis that
is observed during angiotensin II receptor antagonist
therapy, and these antagonists might block the activity of
angiotensin II produced by ACE-independent pathways.
So, dual RAAS blockade is probably a rational approach
to achieve a more effective inhibition of the system and,
conceivably, maximize renoprotection and cardiopro-
tection in patients at risk.
Animal studies and clinical studies have consistently
found that ACE inhibitors and angiotensin II receptor
antagonists in combination reduce proteinuria more
effectively than either agent alone43. However, in the
clinical studies, the combined therapy included an ACE
inhibitor and an angiotensin II receptor antagonist at full
recommended doses, and the dual RAAS blockade had a
more robust antihypertensive effect than monotherapy43.
The full dosages of ACE inhibitor and angiotensin II
receptor antagonist used in these studies did not allow
the assessment of whether the superior antiproteinuric
effect of combined therapy really reflected more effective
RAAS blockade or, rather, was just a function of a greater
reduction in blood pressure. This issue was addressed in
a cohort of patients with non-diabetic CKD given full
doses of the ACE inhibitor benazepril or of the angio-
tensin II receptor antagonist valsartan, or half doses of
the two drugs used in combination44. A similar reduc-
tion in blood pressure was seen in the three treatment
groups, whereas a more consistent proteinuria reduction
was seen in those on dual therapy. Thus, the superior
antiproteinuric effect of dual compared with single
drug RAAS blockade was not explained by a superior
antihypertensive effect, but in all likelihood by a more
REVIEWS
effective inhibition of the RAAS. Finding that combined
therapy also ameliorated concomitant cardiovascular
risk factors such as hypertension and dyslipidaemia
suggests that dual RAAS blockade could be the treatment
of choice to limit renal and cardiovascular morbidity and
mortality in this population. Along this line, post-hoc
analyses of the OnTARGET trial45 will be important to
assess whether dual RAAS blockade may reduce renal
and cardiovascular events more effectively than an ACE
inhibitor or an angiotensin II receptor antagonist alone
in patients with renal dysfunction at inclusion.
Renin inhibitors. Monotherapy with ACE inhibitors or
angiotensin II receptor antagonists provides incomplete
inhibition of the RAAS, particularly after long-term use
in which a reactive rise in plasma renin activity may
limit efficacy. So, targeting renin, the rate-limiting step
in angiotensin II synthesis, may afford better blockade
of the RAAS. The first renin inhibitors were peptide ana-
logues of the prosegment of renin or substrate inhibitory
peptides of the renin cleavage site of angiotensinogen46.
They effectively inhibited renin activity in animals and
humans46, but their limitation was that they had to be
administered parenterally. This pitfall was overcome by
novel orally active renin inhibitors (see Supplementary
information S1 (box)). The first generation of orally
active renin inhibitors — with a molecular mass of a
tetrapeptide — were never used clinically because of the
short half-life, bioavailability of less than 2% and mild
blood-pressure-lowering effect47. The discovery of the
new orally active renin inhibitor aliskiren (Tekturna;
novartis), which has a prolonged half-life and high bio-
availability, in addition to the high specificity for human
renin48, renewed interest in this class of antihypertensive
drugs. Aliskiren directly blocks the function of renin by
occupying the enzymatic pocket of the molecule, thereby
providing greater inactivation of the RAAS and possibly
more renoprotective benefit than other RAAS blockers.
This possibility is supported by findings in hyper-
tensive double transgenic rats harbouring the human
renin and angiotensinogen genes: aliskiren lowered
blood pressure and albuminuria and normalized serum
creatinine49. Clinical trials in hypertensive patients
have demonstrated the efficacy of aliskiren in lower-
ing arterial blood pressure at comparable or at an even
higher extent than angiotensin II receptor antagonists50.
Clinical effects of oral renin inhibitors on kidney dam-
age remain to be determined. The only large clinical trial
of aliskiren in patients with CKD (the AvOID study)
has just been completed51. This study was performed in
patients with type 2 diabetes who also have hyperten-
sion and overt nephropathy. Participants were assigned
to receive aliskiren or placebo in addition to the maxi-
mum recommended renoprotective dose of losartan
(Cozaar; Merck). Aliskiren reduced the albumin/creatinine
ratio by 20% compared with placebo during 24 weeks of
follow-up51. These promising findings, however, should
be tempered by the fact that the AvOID study has been
relatively short-term and lacks relevant information
regarding long-term outcomes such as the progression
of kidney disease to ESRD.
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Table 1 | Renoprotection by angiotensin II blockers in large cohorts of patients with nephropathy

Trial Patients Treatment (median end point/outcome Proteinuria
follow-up) changes
Non-diabetic nephropathies
AIPRI 583 normotensive Double-blind placebo controlled Doubling of serum Benazepril was
(REF. 207) or hypertensive trial with benazepril (3 years) creatinine or need for –29%, placebo
patients with renal dialysis: benazepril was +9%
insufficiency due to was 10.3%, placebo was
various disorders 20.1%
REIN 352 normotensive or Double-blind placebo controlled Doubling of serum Ramipril was
(REF. 30) hypertensive patients trial with ramipril; both plus creatinine or end-stage –50%, placebo
with non-diabetic conventional antihypertensive renal disease: ramipril had no change
proteinuric therapy (3.4 years) was 23.1%, placebo was
nephropathies 45.4%
REIN-2 338 patients Randomized controlled trial Time to end-stage renal Similar in both
(REF. 35) (non-diabetic) comparing conventional BP disease: conventional BP groups
with proteinuric control plus ramipril with control was 20%, intensive
nephropathies intensified BP control (ramipril BP control was 23%
plus felodipine) (3 years)
Diabetic nephropathies
RENAAL 1,513 patients with Double-blind placebo controlled End-stage renal disease: Losartan was
(REF. 40) type 2 diabetes and trial with losartan; both plus losartan was 19.6%, –35%, placebo
overt nephropathy conventional antihypertensive placebo was 25.5% there was
therapy (3.4 years) a trend to
increase
IDNT 1,715 hypertensive Double-blind placebo controlled Doubling of serum Irbesartan
(REF. 41) patients with type 2 trial comparing irbesartan with creatinine, onset of was –33%,
diabetes and overt amlodipine; all plus conventional end-stage renal disease amlodipine was
nephropathy antihypertensive therapy or death: irbesartan was –6%, placebo
(2.6 years) 32.6%, amlodipine was was –10%
41.1%, placebo was 39%
IRMA-2 590 hypertensive Double-blind placebo controlled Time to onset of Not measured
(REF. 39) patients with type trial comparing irbesartan macroalbuminuria:
2 diabetes and 300 mg with irbesartan 150 irbesartan 300 mg was
microalbuminuria mg; both plus conventional 5.2%, irbesartan 150 mg
antihypertensive therapy was 9.7%, placebo was
(3.4 years) 14.9%
BENEDICT 1,204 hypertensive Double-blind placebo controlled Development of persistent Not measured
(REF. 38) patients with type trial comparing trandolapril with microalbuminuria:
2 diabetes and trandolapril/verapamil, and with trandolapril was 6.0%,
normoalbuminuria verapamil; all plus conventional trandolapril/verapamil
antihypertensive therapy was 5.7%, verapamil was
(3.6 years) 11.9%, placebo was 10.0%
Type 1 409 patients with Double-blind placebo Doubling of serum Significantly
diabetes type 1 diabetes and controlled trial with captopril creatinine: captopril less proteinuria
mellitus nephropathy 300 mg; both plus conventional was 12.1%, placebo was in the captopril
(REF. 36) antihypertensive therapy 21.2% group than in
(2.7 years) the placebo
group
ACE, angiotensin converting enzyme; AIPRI, ACE inhibition in progressive renal insufficiency; BENEDICT, Bergamo nephrologic
diabetes complications trial; BP, blood pressure; IDNT, irbesartan diabetic nephropathy trial; IRMA-2, irbesartan in patients with
type 2 diabetes and microalbuminuria; REIN, ramipril efficacy in nephropathy; REIN-2, BP control for renoprotection in patients
with non-diabetic chronic renal disease; RENAAL, reduction of end points in non-insulin-dependent diabetes mellitus with the
angiotensin II antagonist losartan.

Aldosterone antagonists. An additional key component of
the RAAS is aldosterone — a mineralocorticoid hormone
that is produced in the adrenal cortex, and in endothelial
and vascular smooth muscle cells in the heart, blood
vessels and brain. Spironolactone and eplerenone are
steroid analogues that strongly resemble aldosterone
structurally, thereby functioning as competitive antago-
nists of the mineralocorticoid receptor52. Eplerenone is
a 9α,11α epoxy derivative of spironolactone and is less
potent in blocking mineralocorticoid receptors than
spironolactone. However, eplerenone is substantially
more selective than spironolactone and has little ago-
nist activity for the oestrogen receptor and the proges-
terone receptor52. Therefore, eplerenone is associated
with a lower incidence of side effects. At variance with
spironolactone, eplerenone has no active metabolites,
which results in a shorter effective half-life and therefore
a more rapid time to peak response. Selective aldosterone

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Dahl salt-sensitive rat
An experimental model of
hypertension and renal injury
(glomerulosclerosis and
tubulointerstitial damage)
induced in rats sensitive to
a high-salt diet.
Idiopathic membranous
nephropathy
The most common cause for
nephrotic syndrome in adults,
which occurs as idiopathic
(primary) disease of the kidney.
Nephrotic proteinuria
Urinary protein excretion of
more than 3 g per day in the
setting of glomerular disease.
nATURE REvIEwS | drug discovery
receptor antagonism may have benefit for the kidney
independent of its effects on blood pressure. Indeed,
in recent years there has been a striking paradigm shift
with respect to our understanding of the widespread
effects of aldosterone that are in addition to the classical
effects on sodium and potassium transport in the renal
tubules. This points to a possible contribution of this
RAAS component to the development and progression
of chronic injury. Recent evidence indicates that in rats,
aldosterone can be produced in the renal cortex53, and
its mineralocorticoid receptor has been localized to pre-
glomerular vasculature, mesangial cells and fibroblasts,
as well as distal tubular cells of the nephron54. Thus, the
intrarenal sites of aldosterone production and expression
pattern of its receptor, together with the observation
that mediators of renal disease progression promote the
local synthesis of aldosterone, suggest a possible direct
role of this mineralocorticoid hormone in kidney dam-
age. Although the molecular pathways of aldosterone-
mediated renal injury have not been fully elucidated, they
all seem to contribute to the final common pathway of
renal fibrosis55 (FIG. 2).
As aldosterone acts through production of growth
factors and reactive oxygen species, and inhibition of
extracellular matrix degradation — important mediators
of renal fibrosis — blockade of the mineralocorticoid
receptor should prevent or limit renal injury. Indeed, it
has been shown that spironolactone limits glomerulo-
sclerosis and tubulointerstitial fibrosis in rats with
streptozotocin-induced diabetic nephropathy56, in a
genetic model of type 2 diabetes57, and in spontan-
eously hypertensive rats of the stroke-prone substrain58.
Similarly, in Dahl salt-sensitive rats, eplerenone prevented
podocyte damage, proteinuria and glomerulosclerosis59.
More importantly, inhibition of the aldosterone recep-
tor showed renoprotection in animals that have estab-
lished renal injury. In rats with five-sixths nephrectomy,
spironolactone induced regression of existing proteinuria
and glomerular damage60. Tubulointerstitial fibrosis and
vascular lesions improved in parallel with glomerulo-
sclerosis. The renoprotective effects of spironolactone
were potentiated when rats were given the aldosterone
receptor antagonist in combination with the angiotensin
II receptor antagonist losartan. These experimental
findings may have important clinical implications, as
they indicate that blockade of the aldosterone receptor
is instrumental to protect the kidney from further dam-
age even in the presence of an angiotensin II receptor
antagonist. whether this novel renoprotective approach
would translate into clinical benefits for patients with
chronic nephropathies remains unclear, as available data
are scarce and are mainly from non-controlled or small,
short-term controlled studies that are focused only on
the effect on urinary protein excretion. The results show
consistent renoprotection (reduction of albuminuria or
of proteinuria) in patients with type 1 or type 2 diabetes
and overt nephropathy when aldosterone antagonists are
given alone or as add-on therapy to RAAS blockade61,62.
Even in patients with non-diabetic chronic disease
already treated with ACE inhibitors and/or angiotensin
II receptor antagonists, spironolactone effectively reduced
REVIEWS
proteinuria63. by contrast, in patients with idiopathic
membranous nephropathy and nephrotic proteinuria ,
spironolactone given with full-dose ACE inhibitor
therapy did not provide additional antiproteinuric
effects (G. Remuzzi and P. Ruggenenti, unpublished
observations). However, a systematic review of 15 studies
that give an aldosterone blocker in addition to an ACE
inhibitor and/or angiotensin II receptor antagonist for
treatment of patients with proteinuric CKD showed
decreases in urinary protein excretion, but highly vari-
able changes in renal function64. The clinically significant
heterogeneity of these studies precludes any recommen-
dation about using sequential blockade of the RAAS with
an ACE inhibitor/angiotensin II receptor antagonist and
aldosterone antagonist to provide additional long-term
benefits to patients with chronic nephropathies. As there
is also concern with regards to the risk for developing
hyperkalaemia, future randomized trials are necessary
to confirm the efficacy and safety of aldosterone receptor
antagonism on the progression of CKD.
Anti-inflammatory and antifibrotic therapeutics
In addition to components of the RAAS, a large variety
of inflammatory cells are recruited in the kidney tissues
and are activated to produce mediators that trigger pro-
gression of renal disease towards ESRD. Inflammatory
cells — apart from being involved in the development of
glomerular injury — are key players of the tubulointer-
stitial damage, which eventually leads to interstitial fibro-
sis (FIG. 3). Mediators of tubulointerstitial injury would
represent novel potential targets for therapeutics that
eventually could help to complement the renoprotection
afforded by RAAS inhibitors, and in the future could
be part of a multidrug approach to provide the ultimate
treatment for progressive renal disease. In the following,
we will examine these new therapeutic options; most of
them are still in the preclinical development while a few
are now reaching the clinical arena.
Endothelin receptor antagonists. The endothelin system
comprises a family of three isopeptides that have power-
ful vasoconstrictor and profibrotic properties of which
ET1 is the predominant and biologically more relevant
isoform in human kidneys65. It is produced by glomerular
endothelial, epithelial and mesangial cells, and by renal
tubular and medullary collecting duct cells. The renal
medulla contains the highest concentration of ET1 of
any organ66. The effects of endothelin are mediated by
two cell-surface receptors belonging to the superfamily
of rhodopsin-like G-protein-coupled receptors, namely
the type-A (ETA) and the type-b (ETb) receptors67,68. ETA
receptors reside in vascular smooth-muscle cells and
mediate vasoconstriction and cell proliferation69, whereas
ETb receptors mainly reside on endothelial cells and
their activation results in vasodilation via prostacyclin
and nitric oxide70. However, ETb receptors on smooth-
muscle cells can elicit vessel contraction71. besides its
vasodilatory effect, the ETb receptor acts as a clearance
receptor for circulating ET1, and regulates water and
sodium handling. Selective and non-selective endothe-
lin-receptor antagonists have been developed and have
vOlUME 7 | nOvEMbER 2008 | 941
REVIEWS

showed promising results in animal models of hyperten-
sion, heart failure and renal disease72. In particular, the
renoprotective effect of endothelin-receptor blockade
has been documented in several models of progressive
nephropathies, including renal mass ablation73,74, acceler-
ated passive Heymann nephritis75, streptozotocin-induced
diabetes76 and lupus nephritis77. Animal data suggest
that concomitant blockade of the endothelin system and
the RAAS produces greater haemodynamic changes78
and more renoprotective effects75 than those seen with
blockade of either system alone. This synergism is based
on the interaction of endothelin and angiotensin II at
the molecular level79, and would represent the basis for a
OH
combination therapy in patients who do not fully respond
to ACE inhibitors. The question of whether selective ETA
or non-selective ETA/ETb receptor antagonists should
be used is still a matter of investigation. Although both
antagonists reduce blood pressure, preliminary evi-
dence in patients with CKD showed that selective ETA
receptor antagonists have additional desirable effects on
renal haemodynamics80. Concomitant blockade of ETb
receptors seems to offer no advantage over selective ETA
antagonism in terms of preserving natriuresis, diuresis
and glomerular haemodynamics, but studies are needed
to discern the theoretical benefit. Despite the successful
use of endothelin-receptor antagonists in pulmonary

O O
HO

Chaperone
O
Aldosterone
MR MR

Transcription
MR Na+/K+
ATPase
MR Target gene
Non-genomic K+
transactivation Na+
ATP

EGFR ADP
P ERK1/2 SGK1
phosphorylation

NADPH ENaC
oxidase
Na+
↑ICAM1 ↑Collagen
↑MCP1 ↑TGF-β
↑IL6 ↑ROS ↑PAI1

Inflammation Extracellular ↑Blood

matrix pressure
accumulation

Renal damage

Figure 2 | Mechanisms of aldosterone-induced kidney damage. Activation of the mineralocorticoid receptor (MR) by
aldosterone results in its dissociation from molecular chaperones, translocation into the nucleus and binding to hormone-
Nature Reviews | Drug Discovery
response elements in the regulatory region of target gene promoters to enhance expression. Stimulatory action of
aldosterone on Na+ reabsorption in renal tubular cells depends mostly on transcriptional regulation of Na+/K+-ATPase and
early induction of serum/glucocorticoid regulated kinase 1 (SGK1), which de-represses the surface expression and activity
of the epithelial Na+ channel (ENaC). Aldosterone enhances the gene expression of profibrotic molecules, including
collagen, transforming growth factor-β (TGF-β) and plasminogen activator inhibitor, type 1 (PAI1). Moreover, aldosterone
stimulates inflammation through the generation of reactive oxygen species (ROS) by increasing the expression of
Heymann nephritis
NADPH oxidase. Aldosterone can also exert rapid, non-genomic effects, which involves transactivation of the epithelial
An experimental model of growth-factor receptor (EGFR) and the phosphorylation of extracellular-signal-regulated kinase 1 (ERK1)/ERK2, thereby
glomerular disease that upregulating the expression of pro-inflammatory transcription factors and related molecules. In the kidney, the net effect
mimics membranous of aldosterone-induced inflammation and extracellular matrix accumulation is tissue fibrosis and ultimately renal
nephropathy in humans. scarring. ICAM1, intracellular adhesion molecule 1; IL6, interleukin 6; MCP1, monocyte chemoattractant protein 1.

942 | nOvEMbER 2008 | vOlUME 7 www.nature.com/reviews/drugdisc


Crescentic
glomerulonephritis
An aggressive form of
glomerulonephritis that
is characterized by intensive
glomerular inflammation,
which induces epithelial cell
proliferation and macrophage
maturation (cellular crescents).
ANCA-associated vasculitis
An inflammatory disease
involving blood vessels, also of
the kidney, characterized by
the concomitant presence of
antineutrophil cytoplasmic
antibodies (ANCA).
nATURE REvIEwS | drug discovery
hypertension81, their possible antiproteinuric and reno-
protective effects in patients with CKD remain to be eval-
uated. This could be due to the fact that pharmaceutical
companies are in favour of performing acute/short-term
clinical studies, such as those in pulmonary hyperten-
sion, but are reluctant to embark in trials with long-term
outcomes, such as those required to define the potential
renoprotective effects of endothelin-receptor antagonists.
Moreover, major side effects of hepatotoxicity and fluid
retention documented with this drug class raise concern
for their long-term clinical use72.
Statins. Statins are inhibitors of the 3-hydroxy-3-methyl-
glutaryl coenzyme A (HMG-CoA) reductase, the rate-
limiting step in cholesterol synthesis — namely the
conversion of HMG-CoA into mevalonate (see Supple-
mentary information S2 (box)). Statins therefore effec-
tively reduce serum levels of low-density lipoprotein
cholesterol and, to a lesser extent, they reduce triglycer-
ide levels. However, statins also inhibit the synthesis of a
range of compounds including the so-called isoprenoids
(farnesyl-PP, geranyl-geranyl-PP) that are derived from
the mevalonate pathway, which are involved in numer-
ous biological processes in all types of cells, including
cell proliferation and generation of pro-inflammatory
cytokines82 (see Supplementary information S2 (box)).
Studies in rat models of cyclosporine-induced nephro-
pathy or unilateral ureteral obstruction have shown that
statins do protect from renal scarring83,84. Although the
renoprotective effect can be attributed to the lipid-lowering
property of statins, their anti-inflammatory and antipro-
liferative actions could also have a role in attenuating renal
inflammation, tubular transdifferentiation and interstitial
fibrosis85. Furthermore, in a severe model of progressive
nephropathy resistant to ACE inhibition, combining
ACE inhibitors and angiotensin II receptor antagonists
with a statin arrested proteinuria and protected against
impairment of renal function and structure86.
Preliminary clinical data support the renoprotective
effects of statins. A meta-analysis of 15 small, randomized,
placebo-controlled studies involving 1,384 participants
with CKD found that statins reduced albuminuria and
proteinuria within a median of 6 months after initia-
tion of therapy87. Individually, these studies, however,
showed heterogeneous effects and were often of poor
quality. More recently, another meta-analysis on 18,176
patients with low glomerular filtration rate documented
that statins modestly reduced proteinuria but did not
significantly change the rate of renal function decline
in comparison with placebo88. This conclusion chal-
lenges a previous meta-analysis study that showed a
small benefit of statins in slowing the rate of decline in
glomerular filtration rate, but a similar effect on pro-
teinuria89. Moreover, there are observations that raise
concern about the safety of statins (particularly rosuva-
statin) owing to the risk of increasing urinary protein
excretion90. So, only the results of ongoing large trials in
patients with CKD will help to elucidate the real thera-
peutic potential of this class of drugs to kidney disease.
with this in mind, the PlAnET 1 and PlAnET 2 studies
(ClinicalTrials.gov database identifier nCT00296374
REVIEWS
and nCT00296400, respectively; see Further infor-
mation) are currently underway to assess the effect of
rosuvastatin on proteinuria and renal function during
1 year of follow-up. These trials may provide definitive
evidence to the renoprotection and safety of statins.
nevertheless, more relevant would be trials with
clinically important outcomes of progression to ESRD.
Hopefully, the results of the SHARP trial (ClinicalTrials.
gov database identifier nCT00125593; see Further
information) that is currently being conducted in 6,000
patients with CKD will help to provide this information
in the near future91.
Cytokine and chemokine receptor antagonists and
chemokine inhibitors. Members of the tumour necrosis
factor (TnF) superfamily of cytokines regulate several
cell responses including proliferation, differentiation,
inflammation and cell death92. Some of these cytokines
such as TnF-α have been extensively studied in kidney
disease and have been shown to mediate renal damage.
Antibody blockade of TnF-α reduces inflammation and
scarring in experimental crescentic glomerulonephritis93 and
ameliorates obstruction-induced renal fibrosis and dys-
function in rodents94. The recombinant TnF-α receptor
etanercept (Enbrel; Amgen/wyeth) has been used in the
clinic and is highly effective in patients with rheumatoid
arthritis95. However, clinical trials that use TnF-α block-
ers in patients with ANCA-associated vasculitis gave mixed
results96. Of note, in a large-scale, randomized trial, treat-
ment with etanercept was found to be ineffective and
resulted in an excess of treatment-related mortality96.
Moreover, autoimmunity that is induced by anti-TnF-α
therapy remains an issue of concern in patients with
AnCA-associated vasculitis as it is in patients with rheu-
matoid arthritis96. In addition, the recent observation of
serious infections and malignancies in randomized clini-
cal trials95, as well as the increased mortality in patients
with chronic heart failure97, give doubt over the possibility
of designing clinical trials for CKD and renal fibrosis.
Several chemokines and their receptors act as pro-
moters of renal fibrosis98. The profibrotic activity of
MCP1 and its receptor CCR2 (REFs 99,100), as well as of
RAnTES and its receptor CCR1 (REF. 101), is mediated by
the recruitment of inflammatory cells into the glomeruli
and the tubulointerstitium. Evidence is available dem-
onstrating that blockade of CCR2 (REF. 99) or CCR1
chemokine receptors ameliorated progressive fibrosis in
the kidney as shown in rats with unilateral ureter liga-
tion101 and in mice with glomerulosclerosis and nephrotic
syndrome102. However, the attempt to block chemokines
and their receptors in humans is more complex. Indeed
translation of experimental findings into clinical trials is
difficult owing to the large species differences in the tissue
expression of chemokine receptors, the heterogeneity of
the response to block them in animal models103 and the
limited specificity in humans of the chemokine recep-
tor antagonists (CCR2 and CCR5 antagonists) that have
been developed for clinical use so far. A possible prom-
ising alternative is the inhibition of MCP1 production
with bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-in-
dazol-3-yl]methoxy]propanoic acid)104. In murine lupus
vOlUME 7 | nOvEMbER 2008 | 943
REVIEWS

Increased
potent pro-inflammatory mediator that induces cell
Excessive migration, enhances cell adhesion and stimulates the
glomerular
protein tubular
permeability
reabsorption oxidative burst108. So, the prevention of C5 cleavage
to proteins
Proximal tubular cell would have substantial anti-inflammatory consequences
in a relatively late stage of the complement activation
cascade without affecting the activation and function
of the early components. Amelioration of the course of
Nuclear signals for
Cell injury vasoactive and lupus nephritis was documented in mice after treatment
inflammatory genes with a monoclonal antibody that is specific for the C5
component109. The specific blockade of C5 cleavage can
be achieved either by a humanized monoclonal antibody
mRNA: ET1,
(eculizumab) or its 25-kDa single-chain version lack-
RANTES,MCP1, ing the whole constant region including the Fc part of
TGF-β the antibody (pexelizumab). Eculizumab was recently
approved by the FDA and the European Commission —
under the trade name Solaris (Alexion Pharmaceuticals)
Corresponding — for the treatment of patients with paroxysmal nocturnal
proteins
haemoglobinuria based on the demonstration that this
monoclonal antibody inhibited complement-mediated
Interstitium
Lymphocyte/ intravascular haemolysis110. with regards to kidney dis-
macrophage ease, after favourable Phase I safety results in patients
EMT infiltration with systemic lupus erythematosus (SlE), a Phase II trial
Cell
proliferation using eculizumab was initiated in patients with prolifer-
ative lupus nephritis in the United States, but unfor-
Interstitial ↑ECM protein Interstitial TGF-β, EGF, Cytokines/
fibrosis synthesis fibroblasts bFGF, PDGF chemokines tunately was then stopped owing to difficulties in the
recruitment of such patients110. On the other hand, a ran-
↓ECM
degrdation domized controlled trial in 200 patients with idiopathic
Figure 3 | Mechanisms of renal interstitial fibrosis. The injury of tubular membranous nephropathy — a disease in which C5b-9
epithelium as well as the protein overload of proximal tubular cells as a consequence activation of the podocytes have a pathogenetic role, at
of the increased glomerular permeability to proteins in the setting of glomerular least in experimental animals111 — found no difference
Nature
disease, activates intracellular signals that cause increased Reviewsof| Drug
production Discovery
vasoactive, between eculizumab and placebo in the primary out-
inflammatory mediators and growth factors, such as endothelin 1 (ET1), RANTES, come variable of urinary protein excretion112. However,
monocyte chemoattractant protein 1 (MCP1) and transforming growth factor-β since complement activity was not fully inhibited in the
(TGF-β). These substances are released into the interstitium and promote local majority of patients, and because of the short-term treat-
recruitment of inflammatory cells, which in turn are stimulated to release cytokines/ ment, the study may have been insufficient to document
chemokines and growth factors. In particular, TGF-β induces epithelial to a true therapeutic effect of eculizumab.
mesenchymal transition (EMT) and ultimately fibroblast generation. Furthermore,
growth factors stimulate interstitial fibroblasts to proliferate and increase the
production of extracellular matrix (ECM) proteins. These events, combined with the Anti‑TGF‑β antibodies, inhibitors of TGF‑β production
reduction in ECM degradation, eventually lead to interstitial fibrosis and renal and receptor kinases. Transforming growth factor-β
scarring. EGF, epidermal growth factor; bFGF, basic fibroblast growth factor (also (TGF-β) encompasses a family of ligands, the prototype
known as FGF2); PDGF, platelet-derived growth factor. of which is TGF-β1. Although most research focuses on
the TGF-β1 isoform, the β2 and β3 isoforms also have
profibrotic effects113. They bind to specific transmem-
autoimmune disease, bindarit prevented renal MCP1 brane type I (also termed activin receptor-like kinase,
upregulation, limited glomerular injury and prolonged AlK5) and type II serine/threonine kinase receptors,
animal survival105. Similarly, anecdotal observation in which subsequently propagate the signal by phosphory-
patients with active lupus nephritis showed that bindarit lation of several SMAD transcription factors that
administration significantly reduced urinary albumin accumulate in the nucleus. Eventually they control the
and MCP1 excretion, and was well tolerated106. Therefore, expression of target genes, including those encoding for
targeting MCP1 could be a new way to provide renopro- extracellular matrix components114 (FIG. 4). So, targeting
tection. A clinical trial aimed to reduce albuminuria and of the TGF-β pathway may have therapeutic potential
renal disease progression with bindarit added on to RAAS for tackling profibrotic processes, as suggested by studies
blockade therapy is ongoing in patients with type 2 dia- using an anti-TGF-β monoclonal antibody, inhibitors of
betes and with microalbuminuria or macroalbuminuria TGF-β production, and inhibitors of TGF-β type I and
(national Monitoring Centre for Clinical Trials database type II receptor kinases. Although the mechanism of
identifier 2006-006191-38; see Further information). action is not fully understood, it has been shown that
pirfenidone — which is known to be a collagen syn-
Complement inhibition with anti‑C5 antibodies. thesis inhibitor and an antifibrotic p38 MAP kinase
Cleavage of the C5 component, which results in the inhibitor — suppresses both TGF-β gene expression
fragments C5a and C5b, is the critical event during the at the transcription level and protein expression115, in
activation of the complement cascade107. C5a is a highly addition to antagonizing TnF-α signalling116 and being

944 | nOvEMbER 2008 | vOlUME 7 www.nature.com/reviews/drugdisc

 REVIEWS

O The functional role of TGF-β in vivo is emphasized

O by evidence showing that abrogation of TGF-β signalling
O
N
O
by long-term treatment with an anti-TGF-β antibody
H
in db/db mice with type 2 diabetes-like disease almost
NH
completely prevented the increase in collagen and
N N O
N
fibronectin expression and mesangial matrix expan-
N N NH2 sion121. Moreover, pirfenidone reduced the expression
N H
of glomerular TGF-β, attenuated the accumulation of
GW788388 SB431542 glomerular extracellular matrix components122 in the
five-sixths nephrectomy rat model, and improved renal
fibrosis and function in animals with ureteral obstruc-
TGF-β tion123. In addition, in a rat model of puromycin-induced
TGFBRII TGFBRI
(ALK5) nephritis the TGF-β type 1 receptor kinase inhibitor
Sb525334 reduced procollagen 1α(I) deposition in the
GW788388 P P SB431542, renal tissue124. Recently, when the inhibitor Gw788388
GW788388 was given orally for 5 weeks to db/db mice, it reduced
P the mRnA expression of critical factors in extracellular
matrix remodelling, namely COl-I, COl-III and PAI1,
R-SMAD2/3 and significantly lowered renal fibrosis119.
SMAD4 Overall, these observations provide the rationale
for adding agents that are capable of reducing TGF-β
activity or production in combination with inhibitors of
angiotensin II activity as a multimodal intervention to
implement renoprotection. Indeed, treatment with an
SMAD4 anti-TGF-β antibody, when added on the background
Fibronectin of chronic ACE inhibition, normalized proteinuria
Collagen I and abrogated glomerulosclerosis and tubular damage
PAI1
as compared with ACE inhibitor alone in diabetic rats
with overt nephropathy125. Despite encouraging results
in experimental animals by the multimodal intervention
that includes anti-TGF-β antibody, this regimen is not
so easily adaptable to patients with CKD. Anti-TGF-β
antibodies are just at the beginning of their clinical
Figure 4 | structures of TgF-β receptor inhibitors and their inhibitory actions. development in proteinuric chronic nephropathies, such
Nature Reviews
Transforming growth factor-β (TGF-β) binds to the TGF-β-receptor | Drug Discovery
II (TGFBRII), as focal segmental glomerulosclerosis. Certainly, however,
which forms a heteromeric receptor complex with TGF-β-receptor I (TGFBRI or activin more data are needed about their renoprotective effects
receptor-like kinase, ALK5)114. The resulting kinase activation leads to the recruitment before anti-TGF-β antibodies will be part of the thera-
and phosphorylation of specific-receptor regulated (R)-SMADs, which are the peutic options for renoprotection. Of note, in a small
intracellular effectors of TGF-β family members. TGF-β induces SMAD2/3 study in patients with advanced focal segmental glomer-
phosphorylation. This phospho-SMAD forms a heteromeric complex with SMAD4, ulosclerosis, pirfenidone showed a beneficial effect on
which is transported into the nucleus where it regulates the expression of target genes
slowing the loss of glomerular filtration rate, but had no
such as fibronectin, collagen I and plasminogen activator inhibitor, type 1 (PAI1).
effect on proteinuria during 13 months of treatment126.
Apart from stimulating extracellular matrix protein synthesis204, TGF-β induces fibrosis
by inhibiting matrix degradation through the suppression of the activity of matrix However, the safety profile of both anti-TGF-β antibodies,
metalloproteinases205 and activating epithelial to mesenchymal transition, a process pirfenidone and TGF-β receptor kinase inhibitors should
whereby epithelial cells are transformed into migratory myofibroblasts206. TGF-β be carefully explored in view of the fact that complete
receptor inhibitors block activation of TGFBRI (SB431542 and GW788388) and of inhibition of the potent immunosuppressive activity of
TGFBRII (GW788388). TGF-β could have serious adverse consequences.

Thiazolidinediones. Thiazolidinediones (TZDs) represent

Db/db mouse
The db/db mouse is a
hyperinsulinaemic model of
genetic diabetes that develops
abnormalities in renal
morphology and function
that mimic those in human
diabetic nephropathy.
a reactive oxygen species scavenger117. The inhibition of
TGF-β type I and type II receptor kinases is achieved by
small-molecule competitive antagonists of the receptor
kinase activity that interact with the ATP-binding site,
which prevents phosphorylation of SMAD proteins118,119
(FIG. 4). The AlK5 inhibitor Sb431542 is commonly used
in experimental in vitro and in vivo studies. Recently, a
new TGF-β receptor kinase inhibitor, Gw788388, has
been developed that has an improved pharmacokinetic
profile in comparison with Sb431542 (FIG. 4). In vitro,
Gw788388 inhibits both TGF-β type I and type II receptor
kinase activities120.
a class of compounds that are currently in use for the
treatment of type 2 diabetes; they exert their hypogly-
caemic properties through reduction of insulin resist-
ance127. These compounds act by stimulating perixosome
proliferator-activated receptor-γ (PPAR-γ), which are
members of the nuclear hormone receptor superfamily
(FIG. 5). Synthetic TZDs, such as pioglitazone (Actos;
Takeda) and rosiglitazone (Avandia; GlaxoSmithKline),
are extensively used as antidiabetic or insulin-sensitizing
agents (FIG. 5). Troglitazone, the first PPAR-γ agonist, is
no longer available for clinical use owing to its hepato-
toxicity128. Apart from ameliorating insulin resistance,

nATURE REvIEwS | drug discovery vOlUME 7 | nOvEMbER 2008 | 945

REVIEWS

O O

NH NH
N N S S
O N O
O O
Rosiglitazone Pioglitazone

PPAR RXR-α Target genes Leptin

PPAR RXR-α TNF-α
aP2

PPRE

Figure 5 | structures and agonistic effects of thiazolidinediones. Thiazolidinediones, for example, rosiglitazone and
pioglitazone, bind to peroxisome proliferator-activated receptors (PPARs). After ligand binding, PPARs form heterodimers
Nature Reviews
with retinoid X receptor-α (RXR-α), thereby undergoing to conformational changes, which facilitate | Drug Discovery
their binding to the
peroxisome proliferative response element (PPRE) in the enhancer regions of target genes such as leptin, tumour-necrosis
factor-α (TNF-α) and adipocyte fatty acid-binding protein (aP2).

Puromycin-induced
nephrotic syndrome
An experimental model of
nephrotic syndrome that
is characterized by heavy
proteinuria and progressive
glomerular and
tubulointerstitial injury
induced by single or repeated
injections of the puromycin
aminonucleoside.
Focal segmental
glomerulosclerosis
A diagnostic term for a clinical
pathological syndrome that
has multiple aetiologies and
pathogenic mechanisms
characterized by proteinuria
and focal segmental glomerular
consolidation or scarring.
TZDs also decrease the expression of TGF-β in glomeruli
independently of their effect on glucose129, as well as
suppress the glucose-induced production of TGF-β
from tubular cells130. So, TZDs may offer a clinically
promising approach to target the profibrotic TGF-β
activity specifically for diabetic nephropathy.
In streptozotocin-induced experimental diabetes,
pioglitazone suppressed the renal expression of TGF-β,
reduced urinary albumin excretion and ameliorated
glomerular injury131. Treatment with rosiglitazone also
reduced albuminuria in patients with type 2 diabetes
through the inhibition of TnF-α and increase of adipo-
nectin132. Preliminary data are already available that
demonstrate the favourable effects of combining angio-
tensin II receptor antagonists and pioglitazone to target
proteinuria and to achieve better renoprotection as com-
pared with angiotensin II receptor antagonists alone in
patients with type 2 diabetic nephropathy133. Although
TZDs seem promising for the implementation of reno-
protection in diabetic nephropathy, caution should be
taken given the recent signal of concern of increased risk
of myocardial infarction and, albeit non-significant, of
death from cardiovascular causes134.
Vitamin D analogues. Calcium-phosphate deposition is
a frequent histological finding in end-stage kidney biop-
sies, irrespective of the underlying cause of renal failure.
Calcium deposition in the renal parenchyma is asso-
ciated with ultrastructural evidence of mitochondrial
disorganization and calcium accumulation135. It there-
fore may contribute to renal injury via uncoupling of
mitochondrial respiration and generation of reactive
oxygen species136. It is well established that the effects of
vitamin D on endocrine system functions extend beyond
the regulation of calcium and phosphate metabolism.
Several important activities of vitamin D may contrib-
ute to its renoprotective property, which include inhi-
bition of profibrotic growth factors and inflammatory
cytokines and suppression of the RAAS137–139. Moreover,
the synthetic vitamin D analogue paricalcitol (Zemplar;
Abbott) inhibits renal inflammation by promoting
vitamin D receptor-mediated sequestration of nuclear
factor-κb (nF-κb) signalling in a human proximal
tubular cell line140.
Animal studies have revealed pivotal protective roles
of vitamin D in the renal and cardiovascular systems141.
Evidence is also mounting showing that a vitamin D
analogue is renoprotective in different experimental
nephropathies142–144. A recent pilot clinical trial shows
that paricalcitol reduces albuminuria and inflammation
in patients with CKD145,146. nevertheless, long-term ran-
domized, controlled trials are required to confirm these
benefits of vitamin D analogues.
Growth‑factor inhibitors. Connective tissue growth
factor (CTGF) is a downstream mediator of TGF-β
with a potent profibrotic activity that operates directly
by binding to integrins and extracellular matrix or by

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Table 2 | Efficacy of CDK inhibitors in models of chronic renal diseases
Model
Mesangial proliferative glomerulonephritis
Anti-Thy 1.1 antibody
(rat)
Crescentic glomerulonephritis
Nephrotoxic form (rat)
Anti-total glomerular
protein form (mice)
HIV-induced collapsing glomerulopathy
Tg26 (mice)
Tg26 (mice)
Membranous nephropathy
Passive Heymann
nephritis (rat)
Proliferative lupus nephritis
NZB × NZW (mice)
NZB × NZW (mice)
Diabetic nephropathy
Streptozotocin
-induced diabetes (rat)
Polycystic kidney disease
jck (mice)
cpk (mice)
BIO, (2′2,3′E)-6-bromoindirubin-3-oxime; CDK,cyclin-dependent kinase; cpk, congenital
polycystic kidney (also known as Cys1); jck, juvenile cystic kidney (also known as Nek8).
Collapsing glomerulopathy
Collapsing glomerulopathy is a
morphological variant of focal
segmental glomerulosclerosis
that is characterized by
segmental and global collapse
of the glomerular capillaries,
marked hypertrophy and
hyperplasia of podocytes, and
severe tubulointerstitial disease.
Intestinal crypts
Mucosal epithelium that is
extensively invaginated.
nATURE REvIEwS | drug discovery
REVIEWS
Therapeutics to target immunological processes
Cyclin‑dependent kinase/glycogen synthase kinase‑3
drug Therapeutic response refs inhibitors. Cyclin-dependent kinase and glycogen syn-
thase kinase-3 (GSK3) are families of serine/threonine
Roscovitine Decreases mesangial cell 162 kinases that share high structural similarity at their
proliferation and mesangial matrix ATP-binding and catalytic domains158. They have been
production; ameliorates renal recognized to participate in the pathogenesis and repair
insufficiency of renal tissue injury through their ability to promote
engagement (G0 to G1) and progression (G1 to M) of the
Roscovitine Decreases crescent formation 208 cell cycle; to influence proliferation, apoptosis and inflam-
mation in part by direct control of gene expression; and
Roscovitine Decreases glomerular matrix 163
deposition; ameliorates renal to modulate tissue morphogenesis159. Moreover, GSK3
insufficiency is known to control the nOTCH pathway160, which has
recently been shown to participate in podocyte injury
and possibly to the development of glomerulosclerosis161.
Flavopiridol Prevents and regresses existing 209
Small-molecule cyclin-dependent kinase/GSK3 inhibi-
nephropathy
tors have been proposed as therapeutic molecules for
Roscovitine Prevents and regresses existing 164 CKD (TABLE 2). In addition, some cyclin-dependent
nephropathy
kinase/GSK3 inhibitors have recently been shown to
inhibit the nOTCH signalling pathway160.
Roscovitine Decreases renal-cell proliferation; 210 Cumulative evidence suggests the preclinical efficacy
stabilizes podocyte injury of cyclin-dependent kinase/GKS3 inhibitors as renopro-
tective agents. This has been documented mainly with
Roscovitine Decreases glomerular 165
roscovitine in experimental models of mesangial prolifer-
hypercellularity ative glomerulonephritis162, crescentic glomerulonephritis
and collapsing glomerulopathy163,164, and proliferative lupus
Roscovitine Decreases tubulointerstitial 166
damage, immunoglobulin and nephritis165,166. However, clinical trials of roscovitine in
complement deposition, patients with immunoglobulin A (IgA) nephropathy
and proteinuria were halted over concerns of potential toxicity with its
prolonged use167.
BIO Inhibits mesangial cell apoptosis; 211
decreases proteinuria Gamma‑secretase inhibitors. nOTCH genes encode
large, single-transmembrane receptors that regulate
a broad range of cell-fate decisions 168. Recently, the
Roscovitine Long-lasting arrest of renal 212 transmembrane receptor nOTCH has been shown to
cystogenesis be expressed on the podocyte surface in the setting of
Roscovitine Long-lasting arrest of renal 212 animal and human glomerulopathies. Moreover, the
cystogenesis nOTCH pathway is involved in the development of
glomerular disease161. binding of the ligand jagged 1
(JAG1) to nOTCH renders the transmembrane receptor
susceptible to γ-secretase-mediated proteolytic cleavage,
activating the intracellular nOTCH pathway, which
enhancing the signalling of other growth factors including eventually results in the upregulation of target genes
epidermal growth factor (EGF), platelet-derived growth and cell apoptosis161. Thus, γ-secretase could represent a
factor (PDGF) and basic fibroblast growth factor (bFGF; new therapeutic target for glomerulopathies. Inhibitors
also known as FGF2)147. Inhibition of CTGF markedly of γ-secretase have been developed and are already in
reduced renal fibrosis in experimental models of dia- Phase II and III clinical trials for Alzheimer’s disease169
betic nephropathy, renal mass ablation and unilateral (FIG. 6). Among them, l-685458, an aspartyl protease
ureteral obstruction147–150. Therapy with an anti-CTGF transition-state mimic, is a potent inhibitor of amyloid-β-
antibody is now in clinical trials in diabetic nephropathy protein precursor γ-secretase activity170. Dibenzazepine
(FibroGen Clinical Trials; see Further information). inhibits the proliferation of cells in intestinal crypts
Similarly, PDGF is a key mediator of fibrosis in different and adenomas, thereby suggesting benefit in colorec-
organs including the kidney151. The multikinase inhibi- tal neoplastic disease171. DAPT (N-[N-(3,5-difluoro-
tor imatinib (Gleevec/Glivec; novartis), which is widely phenyl)-l-alanyl]-S-phenylglycine-1,1-dimethylester)
used in cancer therapy, also blocks PDGF-receptor- blocks the nOTCH pathway and inhibits chondro-
activated tyrosine kinases and attenuates renal damage genesis in human mesenchymal stem cells172. Of note, all
in experimental unilateral ureteral obstruction152, lupus these γ-secretase inhibitors not only prevented the devel-
nephritis153 and diabetic nephropathy154,155. However, it is opment of albuminuria, but also lowered established albu-
uncertain whether imatinib could be a useful therapeutic minuria in experimental puromycin-induced nephrotic
approach in patients with CKD given its side effects on syndrome161. The anti-albuminuric effect was associated
haematopoiesis, heart and bone156,157. with preservation of podocytes and protection against
vOlUME 7 | nOvEMbER 2008 | 947
REVIEWS

O F
O N
O
H
F N O NH
N F
H N
O O O
O
DAPT H H Dibenzazepine
F O N N NH2
N
H
O O O

L-685458

γ-secretase inhibitors

TGF-β

Podocyte Podocyte
γ-secretase
JAG1 NOTCH–ICD
Apoptosis
NOTCH

JAG1 p300
RBPJ Target genes
MAML ↑TGF-β

Figure 6 | structure and function of γ-secretase inhibitors on podocytes. In podocytes, transforming growth
factor-β (TGF-β) activates the transcription of the NOTCH ligand jagged 1 (JAG1). NOTCH is a transmembrane receptor
to γ-secretase-mediated
that is expressed on the surface of podocytes. Binding of JAG1 renders NOTCH susceptibleNature Reviews | Drug Discovery
proteolytic cleavage, thereby resulting in the release of the NOTCH intracellular domain (ICD) from the plasma
membrane and its translocation into the nucleus. Here, NOTCH–ICD binds to the DNA-binding protein recombinant
signal-binding protein J (RBPJ) and forms a nuclear complex with the cofactors MAML and p300. This complex blocks
transcriptional inhibition of RBPJ and leads to upregulation of NOTCH target gene transcripts, including those that
encode TGF-β. In turn, TGF-β may activate intracellular pathways that promote cell apoptosis. Inhibitors of γ-secretase
prevent NOTCH cleavage and eventually podocyte apoptosis. DAPT, N-[N-(3,5-difluorophenyl)-l-alanyl]-S-phenylglycine-
1,1-dimethylester.

Glomerular basement
membrane
This is one of the components
of filtration barrier between
the blood and urinary space,
which is composed of a central
dense layer, the lamina
densa, and two thinner,
more electron-lucent layers,
the lamina rara externa
and the lamina rara interna.
glomerulosclerosis161. These findings provide proof of
principle that inhibition of γ-secretase could be a new
modality for treatment of chronic glomerulopathies.
Rituximab. Rituximab (MabThera; Roche) is an engi-
neered chimeric monoclonal antibody that is directed
against the CD20 molecule found on pre-b and mature
b cells173. It was first approved in 1997 for the treat-
ment of relapsed or refractory b-cell non-Hodgkin’s
lymphoma174. Although the exact mechanism by which
rituximab exerts its effect remains unclear, it is likely
that b-cell depletion occurs through antibody-mediated
apoptosis, antibody-dependent cellular cytotoxicity and
complement-mediated toxicity175.
Antibody production by b cells is characteristic of
numerous primary and secondary glomerular diseases
of the kidney. Antibody binding to glomerular cells and/
or basement membranes promotes an inflammatory
response that alters the size-selective properties of the
glomerular capillary barrier to macromolecules, which
leads to proteinuria and eventually glomerulosclero-
sis and tubulointerstitial fibrosis. Corticosteroids and
cytotoxic drugs are currently used as induction and/or
maintenance therapy176, but attempts to modulate this
antibody-mediated response have not been consistently
successful, in addition to exposing patients to a number
of serious side effects. More specific and less toxic thera-
pies that target antibody-producing b cells are therefore
needed. based on its property of selectively depleting
CD20+ b cells, rituximab has been recently proposed as
a therapeutic option for antibody-mediated glomerulo-
pathies, namely membranous nephropathy, cryoglobuli-
naemic glomerulonephritis and SlE.
Indeed, the hallmark of idiopathic membranous
nephropathy (IMn) is the subepithelial deposition of
IgG on the glomerular basement membrane, in some cases
associated with CD20+ infiltrates177. A study in eight
patients with persistent IMn and nephrotic syndrome

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first showed that four infusions of 375 mg per m2 rituxi-
mab at weekly intervals significantly reduced proteinuria,
with two patients achieving full remission (proteinuria
<1 g per day)178. Rituximab treatment was well tolerated.
Recently, similar results have been achieved with only a
Box 1 | New therapies for genetic renal diseases
Insights into hereditary diseases of the kidney are constantly evolving as new
information about their molecular and cellular mechanisms become available at an
increasingly rapid rate. A better understanding of the genetic and pathophysiology of
these diseases and the availability of animal models that are orthologous to the human
disease has enabled the development of preclinical studies and the identification
of promising candidate drugs for clinical trials in some renal diseases for which, until
recently, only genetic counselling and symptomatic therapies were available.
recombinant α-galactosidase A for Fabry’s disease
Fabry’s disease, also known as angiokeratoma corporis diffusum universalis,
is an X-chromosome-linked inborn error of metabolism caused by a deficiency of
α-galactosidase A, an enzyme that is necessary for the catabolism of ceramide
trihexoside (a glycosphingolipid)194. This enzymatic defect interferes with
intralysosomal degradation of glycosphingolipids and results in their accumulation
in various tissues of the body outside the central nervous system with various
manifestations. Renal involvement is common in male hemizygotes and is occasional in
female heterozygotes. The disease presents with haematuria and proteinuria, which
often progresses to nephrotic levels. Recently, recombinant human α-galactosidase A
replacement therapy has become available, and two randomized controller trials have
demonstrated its safety and efficacy. In one short-term study, α-galactosidase A
treatment improved neuropathic pain, decreased mesangial widening and increased
renal function195. In another study repeated renal biopsies showed decreased
microvascular endothelial deposits of globotriaosylceramide196. On this basis, clinical
guidelines for the management of Fabry’s disease have recently been published197.
They encourage enzyme-replacement therapy as early as possible in all males with
Fabry’s disease and in female carriers with substantial disease manifestations.
Novel disease-targeted treatments for AdPKd
Autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive
polycystic kidney disease (ARPKD) are the most well known of a large family of
inherited diseases that are characterized by the development of renal cysts of tubular
epithelial cell origin198. Identification of the causative mutated genes and elucidation
of the function of their encoded proteins is shedding insights on the mechanisms that
underlie tubular epithelial cell differentiation198. These advances in the understanding
of ADPKD pathogenesis open the possibility for novel disease-specific treatment
strategies that overcome the current therapies directed towards limiting the
morbidity and mortality from the complications of the disease, which include
hypertension, pain, cyst haemorrhage and infection, and nephrolithiasis. Two major
approaches as potential treatment are targeting cell proliferation and lowering
intracellular cyclic AMP199. They have been shown to be effective in preventing
cyst formation and renal-disease progression in several models of PKD (see
Supplementary information S3 (table)).
Caution, however, is necessary when directly extrapolating results form rodent
models of renal cysts for human PKD. Apart from differences in genotype, cysts may
originate from different nephron segments and this could influence the effectiveness
of different drugs. Nevertheless, clinical studies with some novel therapeutics are
ongoing. Phase II clinical trials with the vasopressin 2 receptor antagonist tolvaptan
have been completed in patients with ADPKD, and a Phase III trial with this molecule
is under way worldwide198. In a pilot study the synthetic metabolically stable
somatostatin analogue octreotide inhibited renal growth and stabilized renal function
in patients with ADPKD200. Given the safety and efficacy of the treatment, a long-term
Phase III study with octreotide has been started. Similarly, based on encouraging
results in three rodent models of PKD demonstrating that mTOR (mammalian target
of rapamycin) inhibitors significantly retard cyst expansion and protect renal
function201,202, Phase II clinical trials of rapamycin and everolimus, two mTOR inhibitors,
are being implemented203. Given the complexity of the cystic disease process, it seems
likely that a combination strategy will be required for maximal therapeutic benefit.
Nevertheless in the next decade, much promise is expected of an effective therapy
for a so far untreatable disease.
nATURE REvIEwS | drug discovery
REVIEWS
single dose of the anti-CD20 antibody179. However, ritux-
imab did not lower proteinuria at the same extent in all
patients179,180. This was not explained by different effects
of the depleting antibody on b lymphocytes, but to the
severity of the accompanying chronic renal lesions. It
has been suggested that the immune pathways of IMn
can be effectively affected by b-cell depletion therapy,
whereas non-immune mechanisms could be control-
led by treatments, such as RAAS blockade179. Therefore,
renal biopsy findings may help to tailor treatment to
individual patients with IMn.
Type II mixed essential cryoglobulinaemia is often associ-
ated with hepatitis C virus (HCv) infection, and leads
to membranoproliferative glomerulonephritis and usu-
ally systemic vasculitis181. very few studies, however, are
so far available of rituximab therapy in this disorder. In
six patients with HCv-associated cryoglobulinaemia
and renal involvement, a prolonged regimen of rituxi-
mab resulted in reduced proteinuria and improved renal
function at 12–18 months of follow-up182.
SlE is the prototypical immune complex disorder,
and involves a wide range of autoantibodies. nephritis
is common in SlE, perhaps invariably. In a small study
of ten patients with proliferative nephritis the standard
therapeutic protocol of rituximab and oral steroids suc-
cessfully achieved complete remission in four patients
after 1 year of follow-up183. One patient attained tem-
porary complete remission and the other three attained
partial remission. In other studies of SlE patients treated
with rituximab, only a small proportion also had renal
disease184,185. Overall, rituximab provided some clinical
and biochemical benefits in the majority of patients.
nevertheless, rituximab use should be confined to
patients who are resistant to standard induction therapy
or to patients with contraindications to corticosteroids
and immunosuppressive agents until more data are avail-
able from ongoing clinical trials.
Conclusions
One of the milestones in nephrology has been the char-
acterization — during the middle of the 1980s — of
the intraglomerular mechanisms underlying the pro-
gressive nature of proteinuric chronic nephropathies
towards ESRD4 and the identification of specific treat-
ments targeting the deleterious effect of excessive traffic
of proteins through the glomerular capillary barrier16.
The evidence in animal models that ACE inhibitors,
besides lowering blood pressure, had selective antipro-
teinuric and renoprotective properties opened a new era
for the treatment of patients with CKD. This dictated
the current and consolidated therapeutic approach for
proteinuric chronic nephropathies based on blockade
of the RAAS with ACE inhibitors and/or angiotensin
II receptor antagonists, which limit proteinuria and
reduce renal function decline and risk of ESRD more
effectively than other antihypertensive treatments. Even
more intriguing are the more recent experimental186 and
clinical187,188 observations that regression of glomerular
structural changes and remodelling of the glomeru-
lar architecture is achievable with RAAS blockade.
However, precisely which cells are involved in the process
vOlUME 7 | nOvEMbER 2008 | 949
REVIEWS

Type II mixed essential
cryoglobulinaemia
Cryoglobulinaemia refers to a
pathogenic condition caused
by the production of
circulating immunoglobulins
that precipitate upon cooling.
Type II cryoglobulinaemia
is defined as mixed
cryoglobulins containing at
least two immunoglobulins,
and has no clear aetiology.
Systemic vasculitis
systemic vasculitis comprises
a large group of inflammatory
diseases with a suggestive or
proven immunopathogenesis
involving blood vessels of
various sizes and affecting
various organs including
the kidney.
of scar tissue removal and regeneration of endothelial
and mesangial cells and podocytes is not yet known.
Regression of glomerulosclerosis and neoformation of
glomerular tissue has been linked to progenitor or stem
cells of renal or extrarenal origin189,190, and ACE inhibi-
tors or angiotensin II receptor antagonists may promote
their mobilization and/or activation at the site of renal
injury191. For non-responders or partial responders, treat-
ment procedure to remission and/or regression must
include a multi-pharmacological strategy to promote
renoprotection through novel anti-inflammatory and
antifibrotic molecules. Added on to RAAS inhibitors,
they might theoretically potentiate the tissue reparative
effects of resident or extrarenal stem cells. The possibility,
however, exists of patients with CKD who are resistant
to the multimodal therapeutic options that have regen-
erative potential, particularly when pharmacological
intervention is started late. This could reflect the very
low number of remaining stem cell niches in the adult
injured kidney. In this setting, cell therapy with exogenous
mesenchymal stem cells — as recently documented in
experimental models of acute renal injury192,193 — would
be a promising future alternative to repair chronic renal
injury and promote renoprotection.
Moreover, there is a clear need to find further thera-
pies to better control antibody production in immune-
related nephropathies, and rituximab is a promising
option. Certainly, it seems likely that biological agents
that are directed at b-cell depletion or inhibition
will form part of the immunotherapeutic armamen-
tarium in the future treatment of immune-mediated
glomerulopathies.
Of note, recent insights into the genetic and patho-
physiology of some hereditary diseases of the kidney
allowed the identification of promising candidate drugs
for diseases for which only symptomatic treatments so
far are available (BOX 1).
Overall, as existing drugs for the treatment of chronic
nephropathies have a favourable risk/benefit ratio, new
therapies to induce renoprotection will require a similar
or better safety profile if they are to be widely used and
accepted.

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Acknowledgements
We are deeply grateful to P. Ruggenenti, M. Abbate and
B. Bikbov for their suggestions and criticisms. We also
thank F. Gaspari for technical assistance in preparing the
manuscript.
FURTHER INFORMATION
ClinicalTrials.gov: http://clinicaltrials.gov
FibroGen Clinical Trials: http://www.fibrogen.com/trials
National Monitoring Centre for Clinical Trials:
http://ossc-svil.test.cineca.it/index_ingl.htm
NKF, FDA Host Conference to Assess Proteinuria as
a Surrogate Outcome in Chronic Kidney Disease:
http://www.kidney.org/professionals/physicians/
proteinuria.cfm
SUPPLEMENTARY INFORMATION
See online article: S1 (box) | S2 (box) | S3 (table)
All liNKs Are AcTive iN The oNliNe PdF
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