# Peroxisome Proliferator-Activated Receptors in Diabetic Nephropathy

Authors: Shinji Kume; Takashi Uzu; Keiji Isshiki; Daisuke Koya
Journal: PPAR Research
 (2008)

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## Abstract

Diabetic nephropathy is a leading cause of end-stage renal disease, which is increasing in incidence worldwide, despite intensive treatment approaches such as glycemic and blood pressure control in patients with diabetes mellitus. New therapeutic strategies are needed to prevent the onset of diabetic nephropathy. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that play important roles in lipid and glucose homeostases. These agents might prevent the progression of diabetic nephropathy, since PPAR agonists improve dyslipidemia and insulin resistance. Furthermore, data from murine models suggest that PPAR agonists also have independent renoprotective effects by suppressing inflammation, oxidative stress, lipotoxicity, and activation of the renin-angiotensin system. This review summarizes data from clinical and experimental studies regarding the relationship between PPARs and diabetic nephropathy. The therapeutic potential of PPAR agonists in the treatment of diabetic nephropathy is also discussed.


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## Body

## 1. Introduction

The incidence and prevalence of type 2 diabetes mellitus
(DM) have been
increasing worldwide since the 1980s, and this rise is estimated to continue in
the future [1, 2]. Diabetic nephropathy is a common complication of DM and
represents one of the major challenges for modern nephrology as the most common
cause of end-stage renal disease, accounting for about 40% of new cases [3, 4].
The increasing prevalence of DM and its complications including diabetic
nephropathy have
therefore become a major health problem worldwide, and new therapeutic
strategies to prevent diabetic nephropathy are urgently needed.Peroxisome
proliferator-activated receptors (PPARs) are ligand-activated transcription
factors belonging to the nuclear hormone receptor superfamily. They were
originally cloned from rodent liver while screening for molecular mediators of
peroxisome proliferation [5, 6]. Three isoforms have been cloned (PPARα, PPARβ/δ, and PPARγ) and characterized. Each has a unique
expression pattern and ligand-binding specificity, as well as distinct
metabolic functions [7]. PPARs regulate diverse cell functions, including fatty
acid metabolism, adipocyte differentiation, inflammation, atherosclerosis, and
cell cycle [8–11]. PPARα plays an important role in lipid metabolism in
several tissues including liver and kidney [12]. PPARβ/δ is associated with cell survival and colon carcinogenesis
[13] and was recently implicated as an important regulator of mitochondrial
biogenesis and subsequent lipid metabolism in skeletal muscle [14]. PPARγ plays a pivotal role in adipogenesis, and its
activation by thiazolidinediones (TZDs) improves insulin sensitivity via this
role in adipocyte differentiation [15]. Accordingly, TZDs are widely used as
oral antidiabetic agents in patients with type 2 diabetes [15, 16]. It is clear
that substantial experimental and clinical research is still needed to clarify
the role of PPARγ in the whole body physiology and the
pathophysiology of various diseases such as diabetes, obesity, hypertension,
atherosclerosis, and cancer.In
addition to the demonstrated physiological roles, several clinical and
experimental studies have implicated PPARs in the pathogenesis of diabetic
nephropathy. This review summarizes these clinical and experimental data with a
particular focus on the therapeutic potential of PPAR modulators in diabetic
nephropathy.


## 2. Structure of PPARs

PPAR was initially identified in a mouse cDNA library in
1990 [6], and since then three PPARs have been cloned: PPARα, PPARβ/δ, and PPARγ (Figure 1(a)) [7]. PPARγ mRNA has three splicing forms derived from a
single gene in human [17]. There are no splicing variants of PPARα or PPARβ/δ mRNA. Two PPARγ protein isoforms result from the translation
of each of the three PPARγ mRNAs to produce PPARγ1 and γ2 [18], with both PPARγ1 and PPARγ3 mRNAs giving rise to the same protein, PPARγ1. PPARγ2 is the larger of the two isoforms, with 30
additional N-terminal amino acids. Due to different promoter usage, PPARγ1 and PPARγ2 have different expression patterns [19].Structure and action of PPARs. (a) Domain structure of human PPARs. (b)
Molecular mechanism of PPARs. After ligand binding, PPARs undergo conformational change with RXR and cofactors.
(a)(b)All
PPARs possess four domains similar to those found in other nuclear hormone
receptors [5, 20]: an NH2-terminal ligand-independent transactivation domain
(activation function-1 (AF-1)), which regulates PPAR activity (A/B domain) [21, 22]; a DNA-binding domain of 70 amino acids (two zinc fingers) (DBD, C domain);
a docking domain for cofactors (D domain); a COOH-terminal region containing
the ligand-binding domain (LBD) and AF-2 domain (E/F domain). DBD and LBD are
approximately 70% homologous among the three PPARs.


## 3. PPAR Ligands

PPARs are ligand-activated transcriptional factors
belonging to the nuclear hormone receptor superfamily, whereby modulation of
target gene transcription depends on the binding of ligands to the receptor.
PPARs form heterodimers with the 9-cis retinoic acid receptor, retinoid X
receptor (RXRα). Activation of the PPAR:RXRα heterodimers by PPAR ligands and/or RXR
ligands triggers a conformational change in the receptors. This in turn allows
the heterodimers to bind to PPAR responsible element
containing the sequence AGGTCANAGGTCA in the promoter region of the target
genes, and thus modulate gene transcription (Figure 1(b)).Many
ligands including natural and synthetic compounds have been identified for each
PPAR isoform in both functional (cell-based transactivation efficiency) and in
vitro interaction assays [8, 23]. The different amino acids sequences in the
LBD of each PPAR provide the molecular basis for ligand specificity. Each PPAR
can accommodate several structurally diverse ligands due to a large
ligand-binding pocket [24]. PPARα binds unsaturated fatty acids with the highest
affinity of the three isoforms [25–28]. Natural
ligands for PPARγ also include several unsaturated fatty acids
such as oleate, linoleate, eicosapentaenori and arachidonic acids, and 15dPGJ2
[8, 23, 29, 30]. TZD compounds such as troglitazone (was the first agent of
this class on the market, but withdrawn due to liver toxicity), ciglitazone,
pioglitazone, and rosiglitazone act as synthetic PPARγ ligands and promote adipocyte differentiation
via activation of the receptor [23, 31–35]. Termisaltan, an angiotensin II
type 1 receptor blocker (ARB), was recently shown to bind PPARγ and reduce blood glucose levels [36, 37].


## 4. Distribution of PPARs in Kidney

Expression of the three PPAR isoforms has been examined
in many species including Xenopus, rat, mouse, rabbit, and human. PPARα is mainly expressed in tissues exhibiting high
catabolic rates of fatty acids such as adipose tissue, liver, heart, and
skeletal muscle [38, 39]. PPARβ/δ is ubiquitously expressed, while PPARγ is highly expressed in white and brown adipose
tissues that store large amounts of fatty acids, and in other selected tissues at low levels such
as heart, liver, immune cells (monocytes and macrophages), placenta, and colon
[40–42].All
three PPARs are expressed in the kidney [38, 41–43]. PPARγ mRNA has been demonstrated in the medullary
collecting ducts and pelvic urothelium of kidney [44], as well as in isolated glomeruli
and cultured mesangial cells [45, 46]. PPARα and γ1, but not γ2, protein was detected in kidney tissue by
immunoblot analysis, while immunohistochemical analysis revealed PPARα and γ1 proteins in the nuclei of mesangial cells and
epithelial cells in glomeruli, proximal and distal tubules, the loop of Henle,
medullary collecting ducts, and the intima/media of renal vasculatures [47].
Large amounts of PPARα have also been detected in proximal tubular
cells, and renal lipid metabolism is highly regulated by PPARα [48]. In contrast to PPARα, PPARγ protein is highly expressed in the nephron
segment, predominantly in collecting ducts, implicating PPARγ in systemic water and sodium retention [49, 50].


## 5. Experimental (Animal) Studies

PPARγ is the best characterized of the PPAR isoforms
in diabetic animal models. The first evidence for a possible renoprotective
effect of PPARγ agonists came 15 years ago, with the TZD
compound troglitazone decreasing urinary albumin excretion and reducing blood
pressure in obese Zucker rats [51]. Further studies since then also showed the beneficial
effects of TZD compounds on renal injury in type 1 and type 2 diabetic animal
models, as summarized in Table 1 [50, 52–60]. Several
experimental studies also showed similar or superior protection against
diabetic nephropathy for PPARγ agonists such as TZD, with results comparable
to other renoprotective agents such as renin-angiotensin system blockers.Table 1
Animal studies.
AuthorsTZDAnimal modelDurationEffect on UAEEffect on BPOther effectsModel of type 1 diabetesFujii et al. [54]TroSTZ-induced diabetic rats12 weeks↓NSNDIsshiki et al. [56]TroSTZ-induced diabetic rats12 weeks↓NDHyperfiltration↓Nicholas et al. [58]TroSTZ-induced diabetic rats12 weeks↓NSNDYamashita et al. [60]Tro, pioSTZ-induced diabetic SHR rats12 weeks↓NSLoss of glomerular basement
membranes↓Model of type 2 diabetesYoshioka et al. [51]TroObese Zucker rats4 and 8 weeks↓↓NDFujiwara et al. [55]TroWistar fatty rats24 weeks↓↓NDYoshimoto et al. [50]PioDiabetic Wistar fatty rats13 weeks↓↓Glomerulosclerosis↓ intrarenal
arteriolosclerosis ↓Tanimoto et al. [59]PioDiabetic KK/Ta mice4 and 8 weeks↓NSGlomerular enlargement↓Buckingham et al. [53]RosiObese Zucker rats4 and 9 months↓↓Glomerulosclerosis↓
tubulointerstitial fibrosis ↓Baylis et al. [52]RosiObese Zucker rats6 months↓NSGlomerulosclerosis↓
tubulointerstitial fibrosis ↓Khan et al. [57]RosiObese Zucker rats12 weeks↓↓NDTZD, thiazolidinedione; Tro, troglitazone; Pio, pioglitazone; Rosi, rosiglitazone;
STZ, streptozotocin; SHR, spontaneously hypertensive rats; UAE, urinary albumin
excretion; BP, blood pressure; NS, no significant effects; ND: not determined;↓, significant reductions.PPARα is highly expressed in renal proximal tubules
and helps to maintain a sustained balance of energy production and expenditure
in the kidney [61]. The role of PPARα in renal cortex lipid metabolism was
demonstrated when the activation of PPARα by clofibrates induced expression of β-oxidation enzymes [62]. In db/db type 2 diabetic mice [63] and Zucker diabetic rats [64], treatment with PPARα activator, fenofibrate, improved urinary
albumin excretion rates and glomerular mesangial expansion. These experimental
studies suggest PPARα agonists as potentially useful therapeutic
agents for diabetic nephropathy.


## 6. Human Clinical Trials

Several clinical
trials of PPARγ agonists have been conducted over the past
decade that together confirm the renoprotective action of PPARγ (Table 2) [65–79]. PPARγ agonist, TZD, is an approved therapeutic agent
for glycemic control in patients with type 2 DM, and thus is effective in
preventing type 2 diabetic nephropathy. The beneficial effect of pioglitazone
on urinary albumin excretion was also demonstrated in large, multicenter
intervention studies, which compared the general efficacy and safety of TZD
agents to other oral antidiabetic agents in patients with type 2 DM over 1
year. Either pioglitazone or the antidiabetic, metformin, was given to 639-randomized
patients already receiving a sulfonylurea [72]. Although the two regimens had
comparable effects on glycemic control, urinary albumin excretion was reduced
by 15% in the group receiving pioglitazone and increased by 2% in the metformin
group. In another study from the same group on drug-naive patients with type 2
DM, pioglitazone significantly reduced urinary albumin excretion, whereas
metformin had no effect. A similar follow-up study showed that administration
of pioglitazone in those patients who had previously received metformin therapy
was associated with a decreased urinary albumin excretion of 10%, whereas
another TZD compound, gliclazide, caused an increase of 6% [74]. Taken
together, these data from both large and small clinical studies showed that
PPARγ agonists have a beneficial effect on diabetic
nephropathy compared to other antidiabetic agents.Table 2
Human clinical studies.
Authorssubjects
(Type 2 DM)nregimensDurationEffect on UAE (%)Effect on BP (mmHg)Sironi et al. [65]hyp40200 mg toroglitazone versus plb8 weeks+11%−4/−3Imano et al. [66]mA, hyp30400 mg toroglitazone versus 500 mg metformin12 weeks−39%a−3/0Nakamura et al. [67]mA or MA32400 mg toroglitazone versus 5 mg glibenclamide12 months−67%ain mA 0% in MA−6cNakamura et al. [68]mA4530 mg Pio versus 5 mg glibenclamide versus
0.6 mg Vog3 months−66%a−6/−4Nakamura et al. [69]mA2830 mg Pio versus plb6 months−59%a−4cAljabri et al. [70]mA, hyp6230–45 mg Pio
versus isophane insulin16 weeks−44%−8/−5Yanagawa et al. [71]mA, hyp40Pio versus Met or glibenclamide12 weeks−45%aNAHanefeld et al. [72]mA, hyp63915–45 mg Pio
versus 850–2550 mg metformin12 months−15%aNASchernthaner et al. [73]hyp119915–45 mg Pio
versus 850–2550 mg metformin12 months−19%aNAMatthews et al. [74]hyp63015–45 mg Pio
versus 80–320 mg glibenclamide12 months−10%aNAAgarwal et al. [75]MA, hyp44Pio versus Glip4 months−7%+3.7/+2.2Lebovitz et al. [76]mA, hyp4934 or 8 mg Rosi versus plb26 weeks4 mg group: −14% 8 mg group: −22%aNASarafidis et al. [77]hyp, mA204 mg Rosi6 months−35%a−5.4a/−4.1aPistrosch et al. [78]mA, hyp19non-mA patients: Rosi versus Nat, mA patients: Rosi versus plb12 weeksnon-mA patients: +18%b, mA patients:
−66%a,b,NASignaificant changes from
baseline levels or other groups;bchange versus the group compared;cmean change for systolic BP versus baseline in
patients treated with the TZD.DM, diabetes mellitus; hyp,
hypertension; mA, microalbuminuria; MA, macroalbuminuria; Glip, glipizide; Nat,
nateglinide; plb, placebo; Pio, pioglitazone; Vog, voglibose; UAE, urine
albumin excretion; NA, changes in blood pressure levels not applicable.It should be noted that PPARγ agonists could potentially cause heart failure
due to the associated water retention. Recent clinical trials in patients with
impaired glucose tolerance (IGT) and/or impaired fasting glucose (IFG) showed
that rosiglitazone, which reduces the onset of diabetes, also reduced the
development of renal disease; however, it increased the adverse risk of heart
failure, compared to ramipril [79]. Therefore, PPARγ agonists should be used only with intensive
monitoring of volume retention in patients with cardiac risk factors.Clinical evidence also suggests the
beneficial effect of PPARα ligands on diabetic nephropathy. Treatment of
type 2 diabetes-associated dyslipidemia with gemfibrozil, an antidyslipidemic
agent and PPARα activator, stabilized urinary albumin
excretion rates [80, 81]. In addition, a large randomized controlled trial in 2005 determined that long-term fenofibrate therapy significantly
reduced the rate of progression to albuminuria in patients with type 2 DM [82].
Although not extensive, these clinical data suggest the therapeutic efficacy of
PPARγ agonists in preventing diabetic nephropathy.


### 6.1. Effects of PPARγ Ligands on Diabetic Nephropathy

#### 6.1.1. Improving Hyperglycemia

The Diabetes
Control and Complications Trial (DCCT) and the United Kingdom Prospective
Diabetes Study (UKPDS) suggested that the adverse effects of hyperglycemia on
metabolic pathways are the main causes of long-term complications such as kidney
disease in diabetes [83, 84]. TZDs are a new class of oral antidiabetic agents
used widely to improve insulin resistance, hyperinsulinemia, and hyperglycemia
in patients with type 2 diabetes [85–87]. Since the improvement
of hyperglycemia in such patients can prevent the development and progression
of diabetic nephropathy, TZDs are potential protective agents for nephropathy
in type 2 diabetes patients and animal models by virtue of their
insulin-sensitizing action [66].


#### 6.1.2. Lowering Blood Pressure with or without Improved Insulin Resistance

Hypertension is
commonly linked to obesity and insulin resistance [88]. TZDs have a possible
antihypertensive effect through improvement of insulin resistance because
insulin sensitivity is related to blood pressure levels both in diabetic
animals and patients [50, 89–92]. On the other
hand, PPARγ ligands could directly affect vascular
function because of their expression in endothelial cells and vascular smooth
muscle cells (VSMCs) [93–95]. Indeed,
pioglitazone lowered the blood pressure in 5/6 nephrectomized hypertensive
rats, and the effect was not associated with insulin resistance [96, 97]. The
demonstrated antihypertensive effects of TZDs could involve the release of
vasodilators such as nitric oxide and prostaglandins [98], the decrease in
fatty acid levels, and/or modification of vasoactive peptide synthesis
including endothelin-1 [47]. Recently, PPARγ downregulated the expression of angiotensin II
type 1 receptor and in turn decreased vascular smooth muscle tone, thereby
reducing vascular contractility [99]. Although the underlying functional
mechanisms remain unclear, PPARγ expression probably contributes to blood
pressure regulation through multiple mechanisms.


### 6.2. Renoprotective Effects of PPARγ Ligands Due to Mechanisms Other Than Changes in Blood Glucose Levels

TZD treatment
ameliorated renal abnormalities in streptozotocin- (STZ-) induced diabetic rats,
a type 1 diabetic model, without changing blood glucose levels [54, 56]. These
findings suggest that the protective effects of PPARγ ligands on diabetes-induced renal dysfunction
are independent of its insulin-sensitizing property. Multiple biochemical
mechanisms have been proposed to explain the adverse effects of hyperglycemia
in diabetes, and the effects of PPARγ ligands on each of these mechanisms is
discussed below.


#### 6.2.1. Amelioration of DGK-DAG-PKC Pathway Activation

The diacylglycerol- (DAG-) protein kinase C- (PKC-) extracellular signal-regulated kinase (ERK)
pathway is enhanced in mesangial cells cultured under high-glucose conditions
and in glomeruli isolated from streptozotocin- (STZ-) induced diabetic rats [100–103].
In these animals, troglitazone ameliorated the diabetes-associated increases in
glomerular filtration rate, urinary albumin excretion, and mRNA expressions of
extracellular matrix (ECM) proteins (fibronectin and type IV collagen) and
transforming growth factor-β (TGF-β) without changing the blood glucose levels
[56]. These findings provided the first evidence that PPARγ ligands can protect glomerular function
independent of their insulin-sensitizing action. In mesangial cells cultured
under high-glucose conditions and in isolated glomeruli from diabetic rats, it
was confirmed that TZDs inhibited the accumulation of DAG and its subsequent activation
of the PKC-ERK pathway. Furthermore, another TZD, pioglitazone, also prevented
DAG-PKC-ERK pathway upregulation in mesangial cells exposed to high glucose
[56]. Finally, TZDs and potent PPARγ ligand, 15dPGJ2, increased the protein
expression of DGK to block DAG-PKC signaling in endothelial cells [103].


#### 6.2.2. Attenuation of Oxidative Stress

Increased
oxidative stress is observed in renal glomeruli and a variety of vascular and
nonvascular tissues exposed to hyperglycemia [104–106].
Troglitazone has potent antioxidant effects, evident by it suppressing
phosphoenolpyruvate gene expression in vitro and scavenging reactive oxygen
species in vivo [107]. It also normalizes the decrease in plasma lipid
hydroperoxide concentration and increase of superoxide dismutase activity in Otsuka
Long-Evans Tokushima Fatty rats, a type 2 diabetic animal model, and improves
the decreased skin blood flow in STZ-induced diabetic rats [98, 108, 109].
Pioglitazone also reduces oxidative stress in the kidney of alloxan-induced
diabetic rabbits [110, 111] and reduces renal lipid peroxides, urinary
isoprostane excretion, and expression of p47 phox and gp91 phox in
high-fat diet-induced obese rats [112].


#### 6.2.3. Suppression of Inflammation

Hyperglycemia and
the diabetic state can induce cytokine production in some tissues. In diabetic
nephropathy, macrophages infiltrates appear in glomeruli and the interstitial
spaces between tubules [113, 114]. Both PPARα and γ  have
potent anti-inflammatory effects in macrophages [115, 116]. The endogenous and
potent PPARγ ligand, 15dPGJ2, is a natural metabolite
derived from prostaglandin (PG)D2, the most abundant prostaglandin in normal
tissues with the highest binding affinity to PPARγ of the J-series prostaglandins [117]. Several
studies demonstrated that the anti-inflammatory effect of 15dPGJ2 or TZDs seems
to be regulated through transcriptional inhibition by both PPARγ-dependent [115, 116, 118] and PPARγ-independent mechanisms [119–121]. Nuclear
factor-κB (NF-κB), a well-known inflammatory transcription
factor, is repressed by 15dPGJ2 in a PPARγ-independent manner [122]. It was also reported
that 15dPGJ2 inhibits interleukin-1β- (IL-1β-) induced cyclooxygenase-2 expression and PGE2
production independently of PPARγ activation in mesangial cells, by suppressing
ERK and c-Jun NH2-terminal kinase (JNK) pathways and AP-1 activation [123].
Another TZD agent, ciglitazone, inhibited platelet-derived growth
factor-induced mesangial cell proliferation without changing ERK activation,
through inhibiting the activation of serum response element directly [124].


#### 6.2.4. Modification of Atherosclerotic Changes

Renal atherosclerotic
changes such as renovascular stenosis and atheroemboli are common findings in
elderly diabetic patients and are known to accelerate renal dysfunction [125, 126]. PPARγ activation also may modify the progression of
atherosclerosis through multiple mechanisms including foam cell
differentiation, inflammatory reactions, and cell proliferation [127]. The
infiltrating monocytes take up oxidized low-density lipoprotein (OxLDL) via
scavenger receptors, resulting in the accumulation of intracellular lipids and
generation of foam cells [127]. The OxLDL scavenger receptor, CD36, is under
direct control of PPARγ [29, 30]. OxLDLs include natural PPARγ agonists such as 9-hydroxyoctadecadienoic acid
(HODE) and 13-HODE. Furthermore, OxLDL induces the expression of PPARγ [115], which has an anti-inflammatory effect
in monocytes by reducing proinflammatory cytokine production [115] via
inhibition of proinflammatory transcription factors such as NFκB, AP-1, and STATs [116]. PPARγ has other effects on atherosclerosis including
induction of apoptosis in monocytes [128], inhibition of VSMC proliferation
[94, 129], and suppression of matrix metalloproteinase-9 expression [130].


### 6.3. Effects of PPARγ Ligands in Tubular Tissue

Patients with
diabetic nephropathy frequently show a nephrotic state, whereby large
quantities of albumin enter the renal tubular system and carry with it a heavy
load of fatty acids. Albumin-bound fatty acids can activate PPARγ and induce apoptosis of proximal tubular
cells. PPARγ agonists might inhibit tubular cell
proliferation, whereas activation of albumin-bound fatty acids is accompanied
by increased proliferation [131]. In particular, pioglitazone increases the
tubular cell albumin uptake and reverses the expression of inflammatory and
profibrotic markers, monocyte chemoattractant protein-1 (MCP-1) and TGF-β [132].


## 6.1. Effects of PPARγ Ligands on Diabetic Nephropathy

### 6.1.1. Improving Hyperglycemia

The Diabetes
Control and Complications Trial (DCCT) and the United Kingdom Prospective
Diabetes Study (UKPDS) suggested that the adverse effects of hyperglycemia on
metabolic pathways are the main causes of long-term complications such as kidney
disease in diabetes [83, 84]. TZDs are a new class of oral antidiabetic agents
used widely to improve insulin resistance, hyperinsulinemia, and hyperglycemia
in patients with type 2 diabetes [85–87]. Since the improvement
of hyperglycemia in such patients can prevent the development and progression
of diabetic nephropathy, TZDs are potential protective agents for nephropathy
in type 2 diabetes patients and animal models by virtue of their
insulin-sensitizing action [66].


### 6.1.2. Lowering Blood Pressure with or without Improved Insulin Resistance

Hypertension is
commonly linked to obesity and insulin resistance [88]. TZDs have a possible
antihypertensive effect through improvement of insulin resistance because
insulin sensitivity is related to blood pressure levels both in diabetic
animals and patients [50, 89–92]. On the other
hand, PPARγ ligands could directly affect vascular
function because of their expression in endothelial cells and vascular smooth
muscle cells (VSMCs) [93–95]. Indeed,
pioglitazone lowered the blood pressure in 5/6 nephrectomized hypertensive
rats, and the effect was not associated with insulin resistance [96, 97]. The
demonstrated antihypertensive effects of TZDs could involve the release of
vasodilators such as nitric oxide and prostaglandins [98], the decrease in
fatty acid levels, and/or modification of vasoactive peptide synthesis
including endothelin-1 [47]. Recently, PPARγ downregulated the expression of angiotensin II
type 1 receptor and in turn decreased vascular smooth muscle tone, thereby
reducing vascular contractility [99]. Although the underlying functional
mechanisms remain unclear, PPARγ expression probably contributes to blood
pressure regulation through multiple mechanisms.


## 6.1.1. Improving Hyperglycemia

The Diabetes
Control and Complications Trial (DCCT) and the United Kingdom Prospective
Diabetes Study (UKPDS) suggested that the adverse effects of hyperglycemia on
metabolic pathways are the main causes of long-term complications such as kidney
disease in diabetes [83, 84]. TZDs are a new class of oral antidiabetic agents
used widely to improve insulin resistance, hyperinsulinemia, and hyperglycemia
in patients with type 2 diabetes [85–87]. Since the improvement
of hyperglycemia in such patients can prevent the development and progression
of diabetic nephropathy, TZDs are potential protective agents for nephropathy
in type 2 diabetes patients and animal models by virtue of their
insulin-sensitizing action [66].


## 6.1.2. Lowering Blood Pressure with or without Improved Insulin Resistance

Hypertension is
commonly linked to obesity and insulin resistance [88]. TZDs have a possible
antihypertensive effect through improvement of insulin resistance because
insulin sensitivity is related to blood pressure levels both in diabetic
animals and patients [50, 89–92]. On the other
hand, PPARγ ligands could directly affect vascular
function because of their expression in endothelial cells and vascular smooth
muscle cells (VSMCs) [93–95]. Indeed,
pioglitazone lowered the blood pressure in 5/6 nephrectomized hypertensive
rats, and the effect was not associated with insulin resistance [96, 97]. The
demonstrated antihypertensive effects of TZDs could involve the release of
vasodilators such as nitric oxide and prostaglandins [98], the decrease in
fatty acid levels, and/or modification of vasoactive peptide synthesis
including endothelin-1 [47]. Recently, PPARγ downregulated the expression of angiotensin II
type 1 receptor and in turn decreased vascular smooth muscle tone, thereby
reducing vascular contractility [99]. Although the underlying functional
mechanisms remain unclear, PPARγ expression probably contributes to blood
pressure regulation through multiple mechanisms.


## 6.2. Renoprotective Effects of PPARγ Ligands Due to Mechanisms Other Than Changes in Blood Glucose Levels

TZD treatment
ameliorated renal abnormalities in streptozotocin- (STZ-) induced diabetic rats,
a type 1 diabetic model, without changing blood glucose levels [54, 56]. These
findings suggest that the protective effects of PPARγ ligands on diabetes-induced renal dysfunction
are independent of its insulin-sensitizing property. Multiple biochemical
mechanisms have been proposed to explain the adverse effects of hyperglycemia
in diabetes, and the effects of PPARγ ligands on each of these mechanisms is
discussed below.


### 6.2.1. Amelioration of DGK-DAG-PKC Pathway Activation

The diacylglycerol- (DAG-) protein kinase C- (PKC-) extracellular signal-regulated kinase (ERK)
pathway is enhanced in mesangial cells cultured under high-glucose conditions
and in glomeruli isolated from streptozotocin- (STZ-) induced diabetic rats [100–103].
In these animals, troglitazone ameliorated the diabetes-associated increases in
glomerular filtration rate, urinary albumin excretion, and mRNA expressions of
extracellular matrix (ECM) proteins (fibronectin and type IV collagen) and
transforming growth factor-β (TGF-β) without changing the blood glucose levels
[56]. These findings provided the first evidence that PPARγ ligands can protect glomerular function
independent of their insulin-sensitizing action. In mesangial cells cultured
under high-glucose conditions and in isolated glomeruli from diabetic rats, it
was confirmed that TZDs inhibited the accumulation of DAG and its subsequent activation
of the PKC-ERK pathway. Furthermore, another TZD, pioglitazone, also prevented
DAG-PKC-ERK pathway upregulation in mesangial cells exposed to high glucose
[56]. Finally, TZDs and potent PPARγ ligand, 15dPGJ2, increased the protein
expression of DGK to block DAG-PKC signaling in endothelial cells [103].


### 6.2.2. Attenuation of Oxidative Stress

Increased
oxidative stress is observed in renal glomeruli and a variety of vascular and
nonvascular tissues exposed to hyperglycemia [104–106].
Troglitazone has potent antioxidant effects, evident by it suppressing
phosphoenolpyruvate gene expression in vitro and scavenging reactive oxygen
species in vivo [107]. It also normalizes the decrease in plasma lipid
hydroperoxide concentration and increase of superoxide dismutase activity in Otsuka
Long-Evans Tokushima Fatty rats, a type 2 diabetic animal model, and improves
the decreased skin blood flow in STZ-induced diabetic rats [98, 108, 109].
Pioglitazone also reduces oxidative stress in the kidney of alloxan-induced
diabetic rabbits [110, 111] and reduces renal lipid peroxides, urinary
isoprostane excretion, and expression of p47 phox and gp91 phox in
high-fat diet-induced obese rats [112].


### 6.2.3. Suppression of Inflammation

Hyperglycemia and
the diabetic state can induce cytokine production in some tissues. In diabetic
nephropathy, macrophages infiltrates appear in glomeruli and the interstitial
spaces between tubules [113, 114]. Both PPARα and γ  have
potent anti-inflammatory effects in macrophages [115, 116]. The endogenous and
potent PPARγ ligand, 15dPGJ2, is a natural metabolite
derived from prostaglandin (PG)D2, the most abundant prostaglandin in normal
tissues with the highest binding affinity to PPARγ of the J-series prostaglandins [117]. Several
studies demonstrated that the anti-inflammatory effect of 15dPGJ2 or TZDs seems
to be regulated through transcriptional inhibition by both PPARγ-dependent [115, 116, 118] and PPARγ-independent mechanisms [119–121]. Nuclear
factor-κB (NF-κB), a well-known inflammatory transcription
factor, is repressed by 15dPGJ2 in a PPARγ-independent manner [122]. It was also reported
that 15dPGJ2 inhibits interleukin-1β- (IL-1β-) induced cyclooxygenase-2 expression and PGE2
production independently of PPARγ activation in mesangial cells, by suppressing
ERK and c-Jun NH2-terminal kinase (JNK) pathways and AP-1 activation [123].
Another TZD agent, ciglitazone, inhibited platelet-derived growth
factor-induced mesangial cell proliferation without changing ERK activation,
through inhibiting the activation of serum response element directly [124].


### 6.2.4. Modification of Atherosclerotic Changes

Renal atherosclerotic
changes such as renovascular stenosis and atheroemboli are common findings in
elderly diabetic patients and are known to accelerate renal dysfunction [125, 126]. PPARγ activation also may modify the progression of
atherosclerosis through multiple mechanisms including foam cell
differentiation, inflammatory reactions, and cell proliferation [127]. The
infiltrating monocytes take up oxidized low-density lipoprotein (OxLDL) via
scavenger receptors, resulting in the accumulation of intracellular lipids and
generation of foam cells [127]. The OxLDL scavenger receptor, CD36, is under
direct control of PPARγ [29, 30]. OxLDLs include natural PPARγ agonists such as 9-hydroxyoctadecadienoic acid
(HODE) and 13-HODE. Furthermore, OxLDL induces the expression of PPARγ [115], which has an anti-inflammatory effect
in monocytes by reducing proinflammatory cytokine production [115] via
inhibition of proinflammatory transcription factors such as NFκB, AP-1, and STATs [116]. PPARγ has other effects on atherosclerosis including
induction of apoptosis in monocytes [128], inhibition of VSMC proliferation
[94, 129], and suppression of matrix metalloproteinase-9 expression [130].


## 6.2.1. Amelioration of DGK-DAG-PKC Pathway Activation

The diacylglycerol- (DAG-) protein kinase C- (PKC-) extracellular signal-regulated kinase (ERK)
pathway is enhanced in mesangial cells cultured under high-glucose conditions
and in glomeruli isolated from streptozotocin- (STZ-) induced diabetic rats [100–103].
In these animals, troglitazone ameliorated the diabetes-associated increases in
glomerular filtration rate, urinary albumin excretion, and mRNA expressions of
extracellular matrix (ECM) proteins (fibronectin and type IV collagen) and
transforming growth factor-β (TGF-β) without changing the blood glucose levels
[56]. These findings provided the first evidence that PPARγ ligands can protect glomerular function
independent of their insulin-sensitizing action. In mesangial cells cultured
under high-glucose conditions and in isolated glomeruli from diabetic rats, it
was confirmed that TZDs inhibited the accumulation of DAG and its subsequent activation
of the PKC-ERK pathway. Furthermore, another TZD, pioglitazone, also prevented
DAG-PKC-ERK pathway upregulation in mesangial cells exposed to high glucose
[56]. Finally, TZDs and potent PPARγ ligand, 15dPGJ2, increased the protein
expression of DGK to block DAG-PKC signaling in endothelial cells [103].


## 6.2.2. Attenuation of Oxidative Stress

Increased
oxidative stress is observed in renal glomeruli and a variety of vascular and
nonvascular tissues exposed to hyperglycemia [104–106].
Troglitazone has potent antioxidant effects, evident by it suppressing
phosphoenolpyruvate gene expression in vitro and scavenging reactive oxygen
species in vivo [107]. It also normalizes the decrease in plasma lipid
hydroperoxide concentration and increase of superoxide dismutase activity in Otsuka
Long-Evans Tokushima Fatty rats, a type 2 diabetic animal model, and improves
the decreased skin blood flow in STZ-induced diabetic rats [98, 108, 109].
Pioglitazone also reduces oxidative stress in the kidney of alloxan-induced
diabetic rabbits [110, 111] and reduces renal lipid peroxides, urinary
isoprostane excretion, and expression of p47 phox and gp91 phox in
high-fat diet-induced obese rats [112].


## 6.2.3. Suppression of Inflammation

Hyperglycemia and
the diabetic state can induce cytokine production in some tissues. In diabetic
nephropathy, macrophages infiltrates appear in glomeruli and the interstitial
spaces between tubules [113, 114]. Both PPARα and γ  have
potent anti-inflammatory effects in macrophages [115, 116]. The endogenous and
potent PPARγ ligand, 15dPGJ2, is a natural metabolite
derived from prostaglandin (PG)D2, the most abundant prostaglandin in normal
tissues with the highest binding affinity to PPARγ of the J-series prostaglandins [117]. Several
studies demonstrated that the anti-inflammatory effect of 15dPGJ2 or TZDs seems
to be regulated through transcriptional inhibition by both PPARγ-dependent [115, 116, 118] and PPARγ-independent mechanisms [119–121]. Nuclear
factor-κB (NF-κB), a well-known inflammatory transcription
factor, is repressed by 15dPGJ2 in a PPARγ-independent manner [122]. It was also reported
that 15dPGJ2 inhibits interleukin-1β- (IL-1β-) induced cyclooxygenase-2 expression and PGE2
production independently of PPARγ activation in mesangial cells, by suppressing
ERK and c-Jun NH2-terminal kinase (JNK) pathways and AP-1 activation [123].
Another TZD agent, ciglitazone, inhibited platelet-derived growth
factor-induced mesangial cell proliferation without changing ERK activation,
through inhibiting the activation of serum response element directly [124].


## 6.2.4. Modification of Atherosclerotic Changes

Renal atherosclerotic
changes such as renovascular stenosis and atheroemboli are common findings in
elderly diabetic patients and are known to accelerate renal dysfunction [125, 126]. PPARγ activation also may modify the progression of
atherosclerosis through multiple mechanisms including foam cell
differentiation, inflammatory reactions, and cell proliferation [127]. The
infiltrating monocytes take up oxidized low-density lipoprotein (OxLDL) via
scavenger receptors, resulting in the accumulation of intracellular lipids and
generation of foam cells [127]. The OxLDL scavenger receptor, CD36, is under
direct control of PPARγ [29, 30]. OxLDLs include natural PPARγ agonists such as 9-hydroxyoctadecadienoic acid
(HODE) and 13-HODE. Furthermore, OxLDL induces the expression of PPARγ [115], which has an anti-inflammatory effect
in monocytes by reducing proinflammatory cytokine production [115] via
inhibition of proinflammatory transcription factors such as NFκB, AP-1, and STATs [116]. PPARγ has other effects on atherosclerosis including
induction of apoptosis in monocytes [128], inhibition of VSMC proliferation
[94, 129], and suppression of matrix metalloproteinase-9 expression [130].


## 6.3. Effects of PPARγ Ligands in Tubular Tissue

Patients with
diabetic nephropathy frequently show a nephrotic state, whereby large
quantities of albumin enter the renal tubular system and carry with it a heavy
load of fatty acids. Albumin-bound fatty acids can activate PPARγ and induce apoptosis of proximal tubular
cells. PPARγ agonists might inhibit tubular cell
proliferation, whereas activation of albumin-bound fatty acids is accompanied
by increased proliferation [131]. In particular, pioglitazone increases the
tubular cell albumin uptake and reverses the expression of inflammatory and
profibrotic markers, monocyte chemoattractant protein-1 (MCP-1) and TGF-β [132].


## 7. Involvement of PPARα and PPARβ/δ in Diabetic Nephropathy

PPARα agonists have renoprotective effects as
mentioned above. One possible mechanism underlying PPARα action on mesangial matrix production may be
related to hyperglycemia or TGFβ signaling [133]. Clofibrate directly inhibited
oxidative stress-induced TGFβ expression in mesangial cells [133], while
fenofibrate downregulated TGFβ and TGFβ receptors type II expression and decreased type IV collagen
accumulation in diabetic glomeruli, and inhibited the production of PAI-1 in
diabetic animals [63, 64].PPARβ/δ is expressed equally in the renal cortex and
medulla, although the role of PPARβ/δ in the kidney remains poorly understood [41].
Overexpression of this isoform protected cultured medullary interstitial cells
from hypertonicity-induced cell death, suggesting that PPARβ/δ is an important survival factor under
hypertonic conditions in renal medulla [134]. However, there are no reports
regarding the effect of PPARβ/δ on diabetic nephropathy. Further evidence from
both clinical and experimental studies is necessary to clarify the therapeutic potential
of PPARβ/δ and PPARα agonists in diabetic nephropathy.Several
recent studies suggested lipotoxicity from renal lipid accumulation as a
possible pathogenic mechanism underlying certain forms of renal injury
including diabetic nephropathy [135–137]. PPARα regulates lipid metabolism in the kidney [48],
and PPARα knockout mice develop severe interstitial
lesions induced by fatty acid overload [138]. PPARα agonists may, therefore, decrease lipotoxicity
and, consequently, inhibit the progression of diabetic nephropathy. PPARβ/δ also regulates lipid metabolism and
particularly lipid oxidation in several tissues, although its exact roles in
the kidney remain
unclear. Thus, both PPARβ/δ and PPARα agonists could be implemented in new
therapeutic strategies designed to prevent diabetic nephropathy by reducing
renal lipotoxicity. Further studies are required to prove this possibility.


## 8. Conclusion and Perspectives

The increased
incidence of diabetic nephropathy has become a major health problem worldwide.
As discussed in this review, PPARs comprise a subfamily of nuclear receptors
and transcription factors that play critical roles in modulating insulin
resistance, hypertension, dyslipidemia, obesity, hypertension, and
inflammation. Given the close relationship between PPAR activity and these
metabolic alterations, PPAR agonists are promising therapeutic agents for
diseases including type 2 diabetes, obesity, hypertension, hyperlipidemia, and
atherosclerosis. Fibrate PPARα agonists and TZD PPARγ agonists are already used successfully as
clinically effective hypolipidemic drugs and insulin sensitizers. PPARβ/δ agonists may provide additional insulin and
lipid modulators via their effects
on skeletal muscle. In addition, there is an increasing evidence suggesting that all three
PPARs contribute to the metabolic control of renal function and are involved in
the pathogenesis of diabetic nephropathy. PPARγ agonists are available as optional therapeutic
agents for nephropathy in type 2 diabetes. In the near future, both PPARα and PPARβ/δ agonists might be added to that strategy with
further evidence that these agents have a proven renoprotective effect in
diabetic animals and patients.



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*Source: 879523-2009-03-04.xml*