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. 2009 Oct 23;150(12):5273–5283. doi: 10.1210/en.2009-0628

Poly(Adenosine 5′-Diphosphate-Ribose) Polymerase Inhibition Counteracts Multiple Manifestations of Experimental Type 1 Diabetic Nephropathy

Viktor R Drel 1, Weizheng Xu 1, Jie Zhang 1, Ivan A Pavlov 1, Hanna Shevalye 1, Barbara Slusher 1, Irina G Obrosova 1
PMCID: PMC2795707  PMID: 19854869

Abstract

This study was aimed at evaluating the role for poly(ADP-ribose) polymerase (PARP) in early nephropathy associated with type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with one of two structurally unrelated PARP inhibitors, 1,5-isoquinolinediol (ISO) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427), at 3 mg/kg−1 · d−1 ip and 30 mg/kg−1 · d−1, respectively, for 10 wk after the first 2 wk without treatment. PARP activity in the renal cortex was assessed by immunohistochemistry and Western blot analysis of poly(ADP-ribosyl)ated proteins. Variables of diabetic nephropathy in urine and renal cortex were evaluated by ELISA, Western blot analysis, immunohistochemistry, and colorimetry. Urinary albumin excretion was increased about 4-fold in diabetic rats, and this increase was prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-associated increase in poly(ADP-ribose) immunoreactivities in renal glomeruli and tubuli and poly(ADP-ribosyl)ated protein level. Renal concentrations of TGF-β1, vascular endothelial growth factor, endothelin-1, TNF-α, monocyte chemoattractant protein-1, lipid peroxidation products, and nitrotyrosine were increased in diabetic rats, and all these changes as well as an increase in urinary TNF-α excretion were completely or partially prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-induced up-regulation of endothelin (B) receptor, podocyte loss, accumulation of collagen-α1 (IY), periodic acid-Schiff-positive substances, fibronectin, and advanced glycation end-products in the renal cortex. In conclusion, PARP activation is implicated in multiple changes characteristic for early nephropathy associated with type 1 diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies.

Evidence is provided of the important role for poly(ADP-ribose) polymerase activation in the pathogenesis of type 1 diabetic nephropathy, thus justifying the rationale for development and further studies of poly(ADP-ribose) polymerase inhibitors.

Despite a significant breakthrough in prevention and treatment of diabetic kidney disease in the last decade due to development of the renin angiotensin system blockers, there is still a vital need to identify and target novel pathophysiologic pathways (1). Activation of poly(ADP-ribose) polymerase (PARP), the enzyme that cleaves nicotinamide adenine dinucleotide (NAD+) with formation of nicotinamide and poly(ADP-ribose) polymer, is emerging as an important mechanism in the development of cardiovascular disease, cancer, and diabetes mellitus (2). PARP activation contributes to NAD+ depletion and energy failure (2,3), changes in transcriptional regulation and gene expression (2,4), impaired signal transduction (5), and, in extreme cases, induction of necrosis and apoptosis (2). In the last several years, PARP activation has been implicated in diabetes-associated endothelial and myocardial dysfunction (2,6,7), peripheral neuropathy (3,8), cataract (9), and retinopathy (9,10,11).

The role for PARP activation in diabetic nephropathy deserves a thorough evaluation considering that PARP-1 is abundantly expressed in both glomeruli and tubuli of the renal cortex and all types of endothelial cells examined so far (2,12) and that PARP activation is involved in diabetes-induced renal overexpression of endothelin-1 and endothelin receptors (12). A study in Leprdb/db (BKsJ) mice identified an important role of PARP activation in the pathogenesis of glomerulopathy associated with type 2 diabetes (13). Whereas growing evidence suggests that mechanisms underlying development of chronic complications in two types of diabetes are not necessarily similar (e.g. compare Refs. 14,15,16 identifying different roles of vascular endothelial growth factor in albuminuria and glomerular hypertrophy in animal models of type 1 and lean and obese type 2 diabetes), the role PARP in type 1 diabetic nephropathy, a devastating complication that often culminates in end-stage renal disease, remains unexplored. The nonspecific PARP inhibitor 3-aminobenzamide has been reported to reduce fibronectin mRNA expression and prevent mesangial expansion in streptozotocin (STZ)-diabetic rats (17). However, the study does not contain quantitative evaluation of the mesangial expansion data [periodic acid-Schiff (PAS) stainings] and furthermore evidence of a robust inhibition of renal poly(ADP-ribosyl)ation by 3-aminobenzamide. Thus, it is unclear whether reduced fibronectin mRNA abundance and other changes in 3-aminobenzamide-treated diabetic rats are due to PARP inhibition or other properties of the compound (1). The present study using streptozotocin-diabetic rat model and a pharmacological approach with two potent, specific, and structurally unrelated PARP inhibitors, 1,5-isoquinolinediol (ISO) and 10-(4-me-thyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one [GPI-15427 (8,9)], evaluated the role for PARP activation in early kidney disease associated with type 1 diabetes.

Materials and Methods

Reagents

Unless otherwise stated, all chemicals were of reagent-grade quality and were purchased from Sigma Chemical Co. (St. Louis, MO). GPI-15427 was obtained from Eisai Inc (Baltimore, MD). Mouse monoclonal anti-poly(ADP-ribose) antibody was purchased from Trevigen, Inc. (Gaithersburg, MD), rabbit polyclonal antinitrotyrosine (NT) antibody and mouse monoclonal anti-NT antibody from Upstate (Lake Placid, NY), goat polyclonal antimethylglyoxal-derived advanced glycation end product (AGE) antibody from Chemicon International Inc. (Billerica, MA), sheep polyclonal antiendothelin (ET) B receptor antibody from Alexis Biochemicals (San Diego, CA), rabbit polyclonal anticollagen-α1(IV) antibody from Chemicon International, and rabbit polyclonal anti-WT-1 (Wilms tumor gene product) antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Other reagents for immunohistochemistry have been purchased from Dako Laboratories, Inc. (Santa Barbara, CA).

Animals

The experiments were performed in accordance with regulations specified by the Guide for the Care and Handling of Laboratory Animals (National Institutes of Health publication 85-23) and Pennington Biomedical Research Center Protocol for Animal Studies. Male Wistar rats (Charles River, Wilmington, MA), body weight 250–300 g, aged 3–3.2 months, were fed a standard rat chow (PMI Nutrition International, Brentwood, MO) and had access to water ad libitum. STZ diabetes was induced by a single ip injection of STZ (50 mg/kg−1). Blood samples for glucose measurements were taken from the tail vein about 48 h after the STZ injection and the day before the study termination. All rats with blood glucose levels 13.8 mm or greater were considered diabetic. Diabetic rats were maintained on suboptimal doses of insulin (∼1–2 U every second day) to prevent ketoacidosis and weight loss. The experimental groups comprised control and diabetic rats treated with or without the PARP inhibitors, ISO (3 mg/kg−1 · d−1ip) or GPI-15427 (formulated as mesylate salt, 30 mg/kg−1 · d−1, in the drinking water), for 10 wk after the first 2 wk without treatment (n = 12/group). An initial 2-wk period without treatment was introduced to avoid β-cell regeneration and alleviation of hyperglycemia, which is known to occur when PARP inhibitors are administered together with streptozotocin or shortly after induction of diabetes (2). At the end of the study, rats were placed in individual metabolic cages (Lab Products Inc., Seaford, DE) and urine collected for 24 h. Urine specimen were centrifuged at 12,000 × g (4 C, 10 min) and frozen for subsequent assessment of albumin, creatinine, and TNF-α by ELISA.

Anesthesia, euthanasia, and tissue sampling

The animals were sedated by CO2 and immediately killed by cervical dislocation. One kidney from each rat was fixed in normal buffered 4% formalin for further assessment of NT, poly(ADP-ribose), and collagen-α1(IV) immunoreactivities and PAS-positive substance accumulation by conventional immunohistochemistry. The second kidney was immediately frozen in liquid nitrogen for subsequent Western blot analyses of poly(ADP-ribosyl)ated proteins, methylglyoxal-derived AGE protein adducts, and ET(B), assessment of podocyte counts by immunohistochemistry, lipid peroxidation product, i.e. malondialdehyde plus 4-hydroxylakenals (MDA+4-HA) concentration by a colorimetric assay, and TGF-β, ET-1, vascular endothelial growth factor (VEGF), fibronectin, NT, TNF-α, and monocyte chemoattractant protein (MCP)-1 concentrations by ELISA.

Specific methods

Urinary albumin, creatinine, and TNF-α

Urinary albumin, creatinine, and TNF-α excretions were assessed by ELISA. The Nephrat kit (Exocell, Philadelphia, PA), the creatinine parameter assay kit (R&D Systems, Minneapolis, MN), and the rat TNF ELISA kit II (BD Biosciences, San Diego, CA) were used for measurements of albumin, creatinine, and TNF-α, respectively. The assays were performed in accordance with the manufacturer’s instructions.

TGF-β, ET-1, VEGF, fibronectin, NT, TNF-α, and MCP-1 ELISA measurements in the renal cortex

For assessment of TGF-β, NT, and TNF-α, renal cortex samples were homogenized on ice in radioimmunoprecipitation assay buffer [1:10 (wt/vol)] containing 50 mm Tris-HCl (pH 7.2); 150 mm NaCl; 0.1% sodium dodecyl sulfate; 1% Nonidet P-40; 5 mm EDTA; 1 mm EGTA; 1% sodium deoxycholate; and the protease/phosphatase inhibitors leupeptin (10 μg/ml), aprotinin (20 μg/ml), benzamidine (10 mm), phenylmethylsulfonyl fluoride (1 mm), and sodium orthovanadate (1 mm). Homogenates were sonicated (3 × 5 sec) and centrifuged at 14,000 × g (4 C, 20 min). TGF-β, NT, and TNF-α concentrations were measured with the Quantikine mouse/rat/porcine/canine TGF-β1 kit (R&D Systems), the OxiSelect nitrotyrosine ELISA kit (Cell Biolabs, San Diego, CA), and the rat TNF ELISA kit II (BD Biosciences), respectively.

For VEGF measurements, renal cortex samples were homogenized in 20 mm PBS (pH 7.4) [1:5 (wt/vol)], on ice. Homogenate was used for VEGF measurements with the Quantikine rat VEGF ELISA kit (R&D Systems).

For fibronectin and MDA+4-HA measurements, renal cortex samples were homogenized in 20 mm PBS (pH 7.4) [1:10 (wt/vol)] on ice. Homogenate was centrifuged at 14,000 × g (4 C, 20 min). Supernatant fibronectin concentrations were measured with the AssayMax rat fibronectin ELISA kit (AssayPro, St. Charles, MO). Supernatant MDA+4-HA concentrations were measured with the Bioxytech LPO-586 kit (Oxis International, Inc., Foster City, CA).

For MCP-1 measurements, renal cortex samples were homogenized in 10 mm Tris-HCI buffer (pH 7.4), containing 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm EDTA, and 0.01% Triton X-100 [1:2 (wt/vol)]. Homogenates were centrifuged at 12,000 × g (4 C, 15 min). Supernatant MCP-1 concentrations were measured with the rat MCP-1 ELISA kit (BD Biosciences).

Renal ET-1 concentrations were measured with the ET-1 Biotrak assay kit (Amersham Biosciences, Pittsburgh, PA) as we described previously (12).

Immunohistochemical studies

All renal sections were processed by a single investigator and evaluated blindly. NT, poly(ADP-ribose), and collagen-α1(IV) immunoreactivities and PAS-positive substance accumulation were assessed by conventional immunohistochemistry. At least 10 fields of each section (∼50 glomeruli) were examined to select one representative image. Color intensity of renal sections stained for collagen-α1(IV) and PAS-positive substances was calculated using the ImageJ 1.32 software (National Institutes of Health, Bethesda, MD). Twenty-five to 40 glomeruli were evaluated in a blinded fashion for each animal. For assessment of podocyte counts, 4-μm frozen sections were brought to room temperature and air dried. Then they were fixed with prechilled (−20 C) acetone for 5 min and washed twice with PBS. Podocyte nuclei were detected with an anti-WT1 antibody and the ABC staining kit and visualized with the diaminobenzidine detection kit (both kits from Vector Laboratory Inc., Dedham, MA). Low-power observations of renal sections stained for NT, poly(ADP-ribose), collagen-α1(IV), and PAS-positive substances were made using an Axioskop microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY). Color images were captured with a Zeiss Axiocam HRc charge-coupled device camera at 1300 × 1030 resolution. Low-power images were generated with a ×40 acroplan objective using the automatic capturing feature of the Zeiss Axiovision software (version 3.1.2.1). Podocyte numbers were counted per glomerulus, and 15–20 glomeruli were examined for each animal.

Western blot analyses

Western blot analyses of poly(ADP-ribosyl)ated proteins, methylglyoxal-derived AGE protein adducts, and ET(B) were performed as described previously (18). Protein bands were visualized with the BM chemiluminescence blotting substrate (POD) (Roche, Indianapolis, IN). Membranes were then stripped and reprobed with β-actin antibody to verify equal protein loading. The data were quantified by densitometry (Quantity One 4.5.0 software; Bio-Rad Laboratories, Richmond, CA).

Statistical analysis

The results are expressed as mean ± sem. Data were subjected to equality of variance F test and then to log transformation, if necessary, before one-way ANOVA. Where overall significance (P < 0.05) was attained, individual between-group comparisons were made using the Student-Newman-Keuls multiple range test. Significance was defined at P ≤ 0.05. When between-group variance differences could not be normalized by log transformation (data sets for body weights and plasma glucose), the data were analyzed by the nonparametric Kruskal-Wallis one-way ANOVA, followed by the Bonferroni/Dunn or Fisher’s protected least significant difference tests for multiple comparisons.

Results

The initial (before STZ administration) body weights were similar in control and diabetic rats treated with or without ISO or GPI-15427. The final body weights were similarly reduced in untreated and PARP inhibitor-treated diabetic rats compared with the control group (Table 1). Initial blood glucose concentrations were increased 4.5-, 4.6-, and 4.5-fold in untreated and ISO- and GPI-15427-treated diabetic rats, respectively, compared with nondiabetic controls. In a similar fashion, final blood glucose concentrations were 4.7-, 4.5-, and 4.6-fold higher in untreated and ISO- and GPI-15427-treated diabetic rats than in nondiabetic controls. PARP inhibition did not affect either weight gain or blood glucose concentrations in nondiabetic rats.

Table 1.

Initial and final body weights and blood glucose concentrations in control and diabetic rats maintained with and without PARP inhibitor treatment

Body weight (g)
Blood glucose (mmol/liter)
Initial Final Initial Final
Control 291 ± 2.3 565 ± 22 5.7 ± 0.16 5.5 ± 0.4
Control + GPI-15427 299 ± 6.6 557 ± 19 5.4 ± 0.11 5.1 ± 0.3
Control + ISO 296 ± 3.2 537 ± 16 6.0 ± 0.2 5.2 ± 0.2
Diabetic 288 ± 3.8 353 ± 13a 25.4 ± 1.23a 26.1 ± 1.3a
Diabetic + GPI-15427 297 ± 4.5 359 ± 16a 26.2 ± 1.1a 24.5 ± 0.9a
Diabetic + ISO 298 ± 3.7 339 ± 18a 25.9 ± 1.0a 25.3 ± 1.1a
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Data are means ± sem (n = 12/group). 

a

P < 0.01 vs. controls. 

Renal poly(ADP-ribose) immunoreactivities were increased in glomeruli and tubuli of diabetic rats compared with nondiabetic controls, and this increase was essentially prevented by both ISO and GPI-15427 (Fig. 1, A and B). Poly(ADP-ribosyl)ated protein level, quantified by Western blot analysis, was increased by 48% in untreated diabetic rats compared with nondiabetic controls but remained essentially unchanged from the control level in diabetic rats treated with ISO or GPI-15427 (Fig. 1, C and D).

Figure 1.

Figure 1

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Panels A and B, Representative microphotographs of poly(ADP-ribose) immunostaining in renal glomeruli and tubuli. Panels C and D, Representative Western blot analysis of renal poly(ADP-ribosyl)ated proteins and poly(ADP-ribosyl)ated protein content (densitometry) in control and diabetic rats maintained with or without PARP inhibitor treatment. C, Control; D, diabetic. Magnification, ×100. Mean ± sem (n = 6–10/group). **, P < 0.01 vs. control group; ##, P < 0.01 vs. untreated diabetic group.

Urinary albumin excretion was increased 3.4-fold in untreated diabetic rats compared with nondiabetic controls, and this increase was essentially prevented by both ISO and GPI-15427 (Fig. 2A). In a similar fashion, urinary albumin/creatinine ratio was increased in untreated diabetic group, and this increase was blunted by both PARP inhibitors (Fig. 2B).

Figure 2.

Figure 2

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Panels A and B, Urinary albumin excretion and urinary albumin to creatinine ratio. Panels C–F, Renal TGF-β, VEGF, fibronectin, and ET-1 concentrations. Panels G and H, Representative Western blot analysis of endothelin (B) receptor protein and ET(B) receptor protein content (densitometry) in control and diabetic rats maintained with or without PARP inhibitor treatment. C, Control; D, diabetic. Mean ± sem (n = 6–10/group). *, **, P < 0.05 and < 0.01 vs. controls; #, ##, P < 0.05 and <0.01 vs. untreated diabetic group.

Diabetic rats displayed 66, 67, 74, and 152% increases in renal TGF-β1, VEGF, fibronectin, and ET-1 concentrations compared with nondiabetic controls (Fig. 2, C–F). Both ISO and GPI-15427 completely prevented diabetes-induced increase in TGF-β1 concentration in diabetic rats, without affecting this variable in nondiabetic control group. ISO reduced, but did not completely normalize, renal VEGF concentration in the diabetic group, whereas the effect of GPI-15427 was not significant. None of the two PARP inhibitors decreased renal VEGF concentrations in nondiabetic control rats. Both ISO and GPI-15427 prevented renal fibronectin and ET-1 accumulation in diabetic rats. Renal ET(B) expression was increased by 85% in diabetic rats compared with nondiabetic controls, and this increase was blunted, although not completely abolished, by ISO or GPI-15427 (Fig. 2, G and H).

One of the characteristic features of diabetic kidney disease was glomerular podocyte loss, which achieved 41% in untreated diabetic rats (Fig. 3, A and B). PARP inhibition reduced, although did not completely prevent, a reduction in glomerular podocyte counts in diabetic rats without affecting podocyte numbers in nondiabetic groups.

Figure 3.

Figure 3

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A, Representative microphotographs of glomerular podocyte immunostaining. B, Podocyte counts in control and diabetic rats maintained with or without PARP inhibitor treatment. C, Control; D, diabetic. Magnification, ×100. Mean ± sem (n = 8–10 rats/group); 15–20 glomeruli were examined for each rat. **, P < 0.01 vs. controls; ##, P < 0.01 vs. untreated diabetic group.

Collagen-α1 (IY) and PAS-positive substances immunoreactivities were increased in the renal cortex of untreated diabetic rats compared with nondiabetic controls (Fig. 4, A–D). This increase was completely or essentially prevented by ISO or GPI-15427. PARP inhibitors did not reduce collagen-α1 (IY) and PAS-positive substances immunoreactivities in nondiabetic rats.

Figure 4.

Figure 4

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Representative microphotographs and color intensities of collagen-α1(IV) (Panels A and B) and PAS-positive substances (Panels C and D) immunostainings in the renal cortex of control and diabetic rats maintained with or without PARP inhibitor treatment. C, Control; D, diabetic. Magnification, ×100. Mean ± sem (n = 6–10/group); 25–40 glomeruli were evaluated during quantitation for each rat. *, **, P < 0.05 and <0.01 vs. controls; ##, P < 0.01 vs. untreated diabetic group.

Methyglyoxal-derived AGE protein adduct expression was increased by 68% in the renal cortex of diabetic rats compared with nondiabetic controls, and this increase was prevented by both ISO and GPI-15427 (Fig. 5, A and B). PARP inhibition did not affect renal methylglyoxal-derived AGE protein adduct expression in nondiabetic control rats.

Figure 5.

Figure 5

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Representative Western blot analysis (A) and content (densitometry, B) of renal methylglyoxal-derived AGE protein adducts in control and diabetic rats maintained with and without PARP inhibitor treatment. C, Control; D, diabetic. Mean ± sem (n = 8–10/group). *, **, P < 0.05 and <0.01 vs. controls; ##, P < 0.01 vs. untreated diabetic group.

NT concentration was increased by 48% in the renal cortex of diabetic rats, and this increase was blunted by both PARP inhibitors (Fig. 6A). MDA+4-HA concentration was increased in the renal cortex of diabetic rats compared with controls (Fig. 6B). GPI-15427 treatment reduced MDA+4-HA concentration in diabetic rats, whereas the effect of ISO was not statistically significant. Neither ISO nor GPI-15427 significantly affected renal NT or MDA+4-HA concentrations in the nondiabetic rats.

Figure 6.

Figure 6

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Renal nitrotyrosine (A) and MDA+4-HA (B) concentrations in control and diabetic rats maintained with and without PARP inhibitor treatment. C, Control; D, diabetic. Mean ± sem (n = 6–10/group). **, P < 0.01 vs. controls; #, ##, P < 0.05 and <0.01 vs. untreated diabetic group.

Renal concentrations of the most potent proinflammatory cytokine, TNF-α, were increased by 143% in diabetic rats compared with controls, and this increase was essentially or partially prevented by a PARP inhibitor treatment (Fig. 7A). Urinary TNF-α excretion was increased 13.8-fold in diabetic rats compared with controls, and this increase was partially prevented by PARP inhibitors (Fig. 7B). Urinary TNF-α excretion remained elevated 7.7- and 9.1-fold in ISO- or GPI-15427-treated diabetic rats, respectively, compared with nondiabetic controls. MCP-1 concentration was increased by 182% in the renal cortex of diabetic rats compared with controls (Fig. 7C). MCP-1 concentrations were reduced in diabetic rats treated with ISO or GPI-15427, compared with untreated diabetic group, but remained significantly elevated compared with nondiabetic controls.

Figure 7.

Figure 7

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Renal TNF-α concentration (panel A), urinary TNF-α excretion, (panel B), and renal MCP-1 concentration (panel C) in control and diabetic rats maintained with and without PARP inhibitor treatment. C, Control; D, diabetic. Mean ± sem (n = 5–8/group). *,**, P < 0.05 and <0.01 vs. controls; #, ##, P < 0.05 and <0.01 vs. untreated diabetic group.

Discussion

The findings described herein provide the first evidence of the important role for PARP activation in albuminuria, mesangial expansion, and podocyte loss in experimental type 1 diabetic nephropathy. Accumulation of poly(ADP-ribosyl)ated proteins, an indicator of increased PARP activity, was clearly manifest in glomeruli and tubuli of the renal cortex of STZ-diabetic rats, consistent with previous findings of our group (18) and others (13) for high glucose-exposed human mesangial cells and mouse podocytes. Note that PARP-1 expression remains unchanged in the renal cortex of diabetic rats or high glucose-exposed mesangial cells, consistent with the current knowledge on PARP as an abundant, constitutively expressed enzyme (2).

PARP activation triggers multiple mechanisms participating in diabetic kidney disease. Our study implicates this mechanism in diabetes-induced renal accumulation of the fibrogenic cytokine TGF-β (19,20), which, through its phosphorylated mothers against decapentaplegic-3 (Smad3) signaling pathway, plays a key role in accumulation of mesangial extracellular matrix components and profibrotic changes in diabetic nephropathy (19,20). Angiotensin-converting enzyme, aldose reductase, and protein kinase C inhibitors, AGE cross-link breaker, and, recently, cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase counteracted renal TGF-β accumulation in animal models of type 1 diabetes (21,22,23,24,25,26). Taking into consideration that all these mechanisms contribute to oxidative stress in the diabetic kidney (1,15,25,26,27) and that antioxidant treatment also prevents diabetes-induced renal TGF-β accumulation (28,29), it is reasonable to suggest that oxidative damage plays a key role in TGF-β overexpression in type 1 diabetic nephropathy. This is in line with findings of a blunted renal TGF-β accumulation and less severe nephropathy in STZ-diabetic mice overexpressing Cu2+/Zn2+ superoxide dismutase (30) and, alternatively, a more profound TGF-β accumulation and accelerated kidney disease in diabetic Cu2+/Zn2+ superoxide dismutase-deficient mice (31). Therefore, the abrogatory effect of PARP inhibition on TGF-β accumulation may at least partially be mediated via alleviation of oxidative-nitrosative stress as evident from reduced renal NT and MDA+4-HA concentrations in diabetic rats treated with PARP inhibitors. Also note that PARP-1 has been demonstrated to directly control expression of TGF-β1, TGF-β2, and TGF-β3 genes in glia (4).

VEGF, a mesangial cell and podocyte-derived permeability and angiogenic factor, is another important player in diabetic kidney disease (reviewed in Ref. 20). A neutralizing VEGF antibody was found effective against renal hyperfiltration, albuminuria, and glomerular hypertrophy in STZ-diabetic rats (14). It also prevented glomerular hypertrophy in Zucker diabetic fatty rats (16). PARP inhibition counteracted diabetes-associated renal VEGF accumulation, consistent with previous findings of our group and others implicating PARP activation in diabetes- and hypoxia-induced retinal VEGF formation (10) as well as angiogenesis (32,33).

PARP plays an important role in transcriptional regulation (2,4). The list of transcription factors controlled by PARP through a direct binding, poly(ADP-ribosyl)ation, or both mechanisms includes nuclear factor-κB, activator protein 1, p53, fos, and others (4). Through these factors, PARP activation triggers gene expression of inducible nitric oxide synthase, cyclooxygenase-2, MCP-1, a variety of proinflammatory cytokines as well as P- and E-selectins (4), thus contributing to inflammatory disease (1). Evidence for the important role of low-grade inflammation in the progression of diabetic nephropathy is emerging (34,35,36,37,38). Inflammatory response manifest by increased expression of intercellular adhesion molecule-1, E-selectin, P-selectin, and proinflammatory cytokines in the kidney as well as macrophage infiltration in the glomeruli and interstitium is well documented in diabetic animal models and human subjects (34,35,36,37,38). Consistent with systemic and renal proinflammatory response, diabetic rats in our study displayed increased urinary TNF-α excretion and renal TNF-α and MCP-1 concentrations. PARP inhibition reduced, although did not completely prevent, urinary TNF-α excretion and renal TNF-α and MCP-1 accumulation, consistent with amelioration rather than disappearance of diabetes-associated systemic and renal proinflammatory response.

Our previous findings in short-term (4 wk) STZ-diabetic rat model added ET-1 and ET(A) and ET(B) receptors to the list of PARP-regulated genes (12). PARP inhibitors reduced diabetes-induced up-regulation of ET-1, ET(A), and ET(B) mRNA as well as ET-1 concentration in the renal cortex (12). A causal relation between renal PARP activation and ET-1 increase has also been identified in the present study in the STZ-diabetic rat model of longer duration. The effects of ISO and GPI-15427 on renal ET-1 accumulation could at least partially be due to blunting of ET(B) receptor protein expression. ET-1 is a potent vasoconstrictor peptide that has multiple signal transduction, metabolic, and pathophysiological effects (39). ET-1 overexpression has been reported in glomeruli, tubuli, and vasculature of diabetic rats, in which it has been implicated in albuminuria, tubulointerstitial inflammation, and mesangial expansion (40,41,42). ET-1 mRNA expression increases with the progression of diabetic nephropathy (43). Therefore, beneficial effects of PARP inhibitors on nephropathic changes in the present study may at least partially be mediated by arrest of diabetes-associated increase in renal ET-1 concentration.

Our findings are the first to implicate PARP activation in diabetes-induced renal accumulation of collagen-α1(IV), a podocyte-produced matrix molecule to the glomerular basement membrane. In the diabetic kidney, podocyte type IV collagen synthesis is increased by the diabetic milieu per se, hemodynamic changes, as well as by TGF-β and angiotensin II (44,45). Accumulation of extracellular matrix proteins, including type IV collagen, is an important mechanism contributing to glomerulosclerosis and tubulointerstitial fibrosis associated with advanced diabetic nephropathy (45). Several treatments, including, but not limited by, AGE inhibitors, antioxidants, omega-3 fatty acids, angiotensin-converting enzyme inhibitor, and angiotensin type 1 receptor antagonist, were found to reduce renal collagen-α1(IV) deposition and prevent glomerulosclerosis in animal models of diabetes (1,27,46,47,48,49,50). Our observation of a blunted effect of PARP inhibition on diabetes-related renal collagen-α1(IV) accumulation provides rationale for evaluating the efficacy of PARP inhibitors against glomerulosclerosis and tubulointerstitial fibrosis associated with advanced type 1 diabetic nephropathy.

All beneficial effects of PARP inhibition on early type 1 diabetic nephropathy in the present study are likely to be mediated by suppression of two most important and interrelated mechanisms, i.e. nonenzymatic glycation/glycooxidation and oxidative-nitrosative stress (1,22,28,29,30,31,51,52,53,54). Evidence for the important role of AGE in diabetic complications, including diabetic nephropathy, is emerging (1,8,28,51,52). AGE inhibitors and cross-link breakers have been reported to counteract all major biochemical and morphological changes of experimental type 1 diabetic kidney disease (1,8,28,51,52). PARP activation promotes formation of methylglyoxal-derived AGE through several mechanisms including depletion of the PARP substrate and glyceraldehyde 3-phopshate dehydrogenase (GAPDH) cofactor, NAD (2), as well as GAPDH poly(ADP-ribosyl)ation and nitrosylation (55,56). All these mechanisms lead to inhibition or insufficient (compared with the upper part of glycolysis) activation of GAPDH and concomitant diversion of the excessive glycolytic flux toward the formation of glycolytic by-product and AGE precursor, methylglyoxal, and accumulation of methylglyoxal-derived AGE protein adducts. Note, however, that the relation between PARP and AGE is bidirectional. AGEs generate oxidative stress (1,28) and therefore contribute to diabetes-associated increase in PARP activity.

Oxidative-nitrosative stress in tissue sites for diabetic complications is caused by free radicals and oxidants and, in the first place, peroxynitrite, a product of reaction of superoxide anion radicals with nitric oxide. Accumulation of NT, a footprint of peroxynitrite injury, is clearly manifest in the kidney of diabetic rats and, as we demonstrated previously, is localized to both glomeruli and tubuli of the renal cortex (13). Superoxide, required for peroxynitrite formation, is largely produced by nicotinamide adenine dinucleotide phosphate oxidase (NAD(P)H oxidase), playing an important role in mesangial expansion and podocyte injury in diabetic kidney disease (54,57). Evidence for overexpression of inducible nitric oxide synthase, a potentially most important source of nitric oxide, in the kidney of STZ-diabetic rats is also emerging (58,59). Note, however, that a recent study of renal biopsies of human subjects with diabetic nephropathy and nondiabetic individuals revealed diabetes-associated overexpression of endothelial, rather than inducible, nitric oxide synthase (60).

Until recently, PARP activation was regarded as a phenomenon arising exclusively from free radical- and peroxynitrite-induced DNA single-strand breakage (2). However, new studies revealed that in some tissues of diabetic animals PARP activation may lead to rather than result from oxidative-nitrosative stress (9,13,61) and that PARP activation does not necessarily require DNA single-strand breakage and can be caused by enzyme phosphorylation by ERK (62). In the diabetic rat retina, poly(ADP-ribosyl)ated proteins accumulated both in the cells containing DNA breaks and in those with preserved DNA integrity (11). Furthermore, poly(ADP-ribosyl)ated protein accumulation is clearly manifest in diabetic rat nerve (63), which does not display any DNA breakage in the absence of a superimposed ischemia-reperfusion injury (64).

The current findings suggest that PARP activation leads to oxidative-nitrosative stress associated with early type 1 diabetic nephropathy. Similar relations between the two phenomena have been identified by our group for other tissues (9,61). We also demonstrated that PARP inhibition counteracted superoxide formation, inducible nitric oxide overexpression, and nitrated protein accumulation in high glucose-exposed cultured human Schwann cells (61), whereas aldose reductase inhibition prevented both poly(ADP-ribosyl)ation and nitrosative stress in high glucose-exposed cultured human mesangial cells (18). Others found that PARP inhibition prevented high glucose-exposed ROS production and apoptosis in cultured mouse podocytes (13).

In conclusion, PARP activation contributes to AGE formation, oxidative-nitrosative stress, and proinflammatory response in the diabetic kidney and triggers multiple mechanisms leading to albuminuria, mesangial expansion, and podocyte loss in early type 1 diabetic nephropathy. These findings, consistent with a previous report on PARP contribution to the development of albuminuria, mesangial expansion, and podocyte loss associated with kidney disease in type 2 diabetes, provide rationale for further studies of a therapeutic potential of PARP inhibitors and PARP inhibitor-containing combination therapies.

Acknowledgments

We thank Jeho Shin and Nazar Mashtalir for expert technical assistance.

Footnotes

This work was supported by National Institutes of Health (NIH) Grant R21DK070720 and the Juvenile Diabetes Research Foundation International Grant 1-2005-223 (both to I.G.O.). The Cell Biology and Bio-Imaging Core used in this work is supported in part by Center of Biomedical Research Excellence (NIH Grant P20 RR021945) and Clinical Nutrition Research Unit (NIH 1P30-DK072476) center grants from the NIH.

Disclosure Summary: V.R.D., I.A.P., H.S., and I.G.O. have nothing to declare. W.X. and J.Z. were previously employed by MGI Pharma, which is now a part of Eisai Inc., a company developing PARP inhibitors, and B.S. is a current employee of Eisai Inc.

First Published Online October 23, 2009

Abbreviations: AGE, Advanced glycation end product; ET, endothelin; GAPDH, glyceraldehyde 3-phopshate dehydrogenase; GPI-15427, 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one; ISO, 1,5-isoquinolinediol; MCP, monocyte chemoattractant protein; MDA+4-HA, malondialdehyde plus 4-hydroxylakenals; NAD+, nicotinamide adenine dinucleotide; NT, nitrotyrosine; PARP, poly(ADP-ribose) polymerase; PAS, periodic acid-Schiff; STZ, streptozotocin; VEGF, vascular endothelial growth factor.

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