Yoshisuke Haruna

Oxidative Stress in Diabetic Nephropathy

Diabetic nephropathy is a leading cause of end-stage renal failure worldwide. Its morphologic characteristics include glomerular hypertrophy, basement membrane thickening, mesangial expansion, tubular atrophy, interstitial fibrosis and arteriolar thickening. All of these are part and parcel of microvascular complications of diabetes. A large body of evidence indicates that oxidative stress is the common denominator link for the major pathways involved in the development and progression of diabetic micro- as well as macro-vascular complications of diabetes. There are a number of macromolecules that have been implicated for increased generation of reactive oxygen species (ROS), such as, NAD(P)H oxidase, advanced glycation end products (AGE), defects in polyol pathway, uncoupled nitric oxide synthase (NOS) and mitochondrial respiratory chain via oxidative phosphorylation. Excess amounts of ROS modulate activation of protein kinase C, mitogen-activated protein kinases, and various cytokines and transcription factors which eventually cause increased expression of extracellular matrix (ECM) genes with progression to fibrosis and end stage renal disease. Activation of renin-angiotensin system (RAS) further worsens the renal injury induced by ROS in diabetic nephropathy. Buffering the generation of ROS may sound a promising therapeutic to ameliorate renal damage from diabetic nephropathy, however, various studies have demonstrated minimal reno-protection by these agents. Interruption in the RAS has yielded much better results in terms of reno-protection and progression of diabetic nephropathy. In this review various aspects of oxidative stress coupled with the damage induced by RAS are discussed with the anticipation to yield an impetus for designing new generation of specific antioxidants that are potentially more effective to reduce reno-vascular complications of diabetes.

Amelioration of progressive renal injury by genetic manipulation of Klotho gene

Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was ≈30% in ICGN mice, and ≈70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in β-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.

Angiotensin II type 1 receptor blocker ameliorates uncoupled endothelial nitric oxide synthase in rats with experimental diabetic nephropathy

Background Recent studies showed that angiotensin II type 1 receptor blocker (ARB) slows progression of chronic renal disease in patients with type 2 diabetes, regardless of changes in blood pressure. We showed that the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) due to endothelial NO synthase (eNOS) uncoupling contributed to renal dysfunction in the diabetic nephropathy. The aim of this study was to determine the effects of ARB on uncoupled eNOS in rat diabetic nephropathy. Methods. Diabetes was induced in Sprague-Dawley rats with streptozotocin (65 mg/ kg body weight). After 6 weeks, rats were divided into saline (DM; n = 11) and ARB, losartan groups (DM+Los; n = 11). After 2-week treatment, glomerular ROS production was assessed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA)-derived chemiluminescence. Renal NO and ROS production were imaged by confocal laser microscopy after renal perfusion with DCFH-DA and diaminorhodamine-4M acetoxymethyl ester with l-arginine. The dimeric form of eNOS was measured by low-temperature sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Serum tetrahydrobiopterin (BH4) concentrations were determined by high-performance liquid chromatography. Protein and mRNA expression of GTP cyclohydrolase 1 (GTPCH1), key enzyme of BH4 synthesis, were examined. Results Losartan attenuated glomerular ROS production in DM. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in DM glomeruli. Losartan reversed the decreased GTPCH1 and decreased dimeric form of eNOS and glomerular NO production by increased BH4 bioavailability. Conclusions. ARB improved the NOS uncoupling in diabetic nephropathy by increasing BH4 bioavailability.

Low-dose paclitaxel ameliorates renal fibrosis in rat UUO model by inhibition of TGF- β /Smad activity

Transforming growth factor-β (TGF-β) has a pivotal function in the progression of renal fibrosis in a wide variety of renal diseases. Smad proteins have been identified to have an important function in regulating the expression of extracellular matrix (ECM) proteins through TGF-β signaling pathway. Aberrant TGF-β/Smad signaling can be modulated by stabilization of microtubules with paclitaxel. In this study, we investigated if paclitaxel can attenuate tubulointerstitial fibrosis in a rat model of unilateral ureteral obstruction (UUO). Rats in groups of six were subjected to UUO and received low-dose intraperitoneal injection of paclitaxel (0.3 mg/kg) twice a week. They were killed at day 7 and 14 after UUO or Sham operation. TGF-β signaling cascade and status of various ECM proteins were evaluated by RT–PCR, western blotting and immunohistochemical or immunofluorescence staining. The paclitaxel treatment markedly suppressed Smad2 and Smad3 phosphorylation. This was associated with attenuated expression of integrin-linked kinase, collagens I and III, fibronectin (FN) and α-smooth muscle actin, and a substantial decrease in renal fibrosis in animals that underwent UUO and received paclitaxel. These data indicate that the low-dose paclitaxel ameliorates renal tubulointerstitial fibrosis by modulating TGF-β signaling, and thus, the paclitaxel may have some therapeutic value in humans.

Olmesartan Ameliorates Progressive Glomerular Injury in Subtotal Nephrectomized Rats through Suppression of Superoxide Production

Angiotensin type 1 receptor blockers are more effective than other antihypertensive agents in slowing the progression of renal disease. Angiotensin II (Ang II) induces production of NAD(P)H oxidase–dependent superoxide in vascular and mesangial cells, but the direct role of Ang II in glomerular superoxide production remains unknown. Here we examined the effect of Ang II on superoxide production both ex vivo and in vivo. Ang II increased superoxide generation in isolated normal glomeruli in a dose-dependent manner, and co-incubation with olmesartan, an angiotensin type 1 receptor blocker, suppressed such increase. Subtotal nephrectomized rats (Nx, n=8) showed impaired renal function, increased glomerular sclerosis, and significantly high superoxide production in glomeruli. These changes were inhibited in olmesartan-treated (n=8), but not hydralazine-treated (n=8) Nx rats. Oxidative stress and nitrosative stress were observed in Nx glomeruli, as evidenced by increased levels of carbonyl protein and nitrotyrosine formation, respectively. These changes were inhibited by 8-week treatment with olmesartan. The apoptosis observed in Nx glomeruli was also suppressed by olmesartan. Superoxide generation in Nx glomeruli was blocked by an NAD(P)H oxidase inhibitor, diphenylene iodinium. The mRNA expression levels of two NAD(P)H oxidase subunits were increased in Nx, and olmesartan significantly reduced the mRNA expression levels. These results indicate that Ang II directly induced superoxide production through activation of NAD(P)H oxidase, and olmesartan would inhibit superoxide production and oxidative stress independent of its blood pressure–lowering effect. These findings support the notion that superoxide plays a primary role in glomerular injury in chronic kidney disease.

Isohumulones Derived from Hops Ameliorate Renal Injury via an Anti-Oxidative Effect in Dahl Salt-Sensitive Rats

Previous studies have reported that isohumulones, the bitter compounds in beer, improve insulin resistance and hyperlipidemia in several animal models. In this study, we examined whether isohumulones ameliorate renal injury. Dahl salt-sensitive hypertensive rats were fed a low-salt diet (LS), a high-salt diet (HS) or a high-salt diet containing 0.3% isohumulones (HS+IH) for 4 weeks. Urinary nitrite/nitrate (NOx) excretion was measured at 4 weeks along with blood pressure and urinary protein excretion. Renal injury was evaluated histologically and reactive oxygen species (ROS) and nitric oxide (NO) production in the renal cortex was visualized. Oxidative stress and NO synthase (NOS) expression were evaluated by immunohistochemical staining and Western blot analysis. Mean blood pressure was significantly decreased in the HS+IH group compared with the HS group at 4 weeks (158.1±8.7 vs. 177.5±3.7 mmHg; p<0.05). Isohumulones prevented the development of proteinuria in the HS+IH group compared with the HS group at 2 weeks (61.7±26.8 vs. 117.2±9.8 mg/day; p<0.05). Glomerulosclerosis and interstitial fibrosis scores were significantly decreased in the HS+IH group compared with the HS group (0.61±0.11 vs. 1.55±0.23, 23.7±6.8 vs. 36.1±3.5%; p<0.05 for both). In the HS group, increased ROS and decreased NO were observed in glomeruli in vivo. Isohumulones reduced the ROS production, leading to the restoration of bioavailable NO. Urinary NOx excretion was significantly increased in the HS+IH group compared with the HS group. Furthermore, renal nitrotyrosine was increased in the HS group compared with the LS group, and this effect was prevented by isohumulones. Renal NOS expression did not differ among the three groups. These results suggest that isohumulones may prevent the progression of renal injury caused by hypertension via an anti-oxidative effect.

Blockade of serotonin 2A receptor improves glomerular endothelial function in rats with streptozotocin-induced diabetic nephropathy

BackgroundSerotonin (5-HT) is involved in vascular inflammation and atherosclerogenesis. Serum 5-HT concentrations are elevated in diabetes, and 5-HT is involved in diabetic vasculopathies. Sarpogrelate hydrochloride, a 5-HT2A receptor antagonist, has renoprotective effects, but its effect in diabetic nephropathy is not elucidated. The aim of this study was to examine the effects of sarpogrelate on endothelial dysfunction in rats with streptozotocin (STZ)-induced diabetes.MethodsRats with STZ-induced diabetes were either untreated or treated with sarpogrelate (30 mg/kg P.O.) for 8 weeks. At the end of the experiment, we measured urinary albumin excretion, serum adiponectin concentration and platelet-derived microparticles. Intraglomerular coagulation was detected by immunostaining for platelets. Production of renal reactive oxygen species (ROS) and nitric oxide (NO) was investigated by confocal laser microscopy and used as an index of glomerular endothelial dysfunction.ResultsDiabetic nephropathy was associated with enhanced production of ROS and diminished bioavailable NO in the glomeruli. Treatment with sarpogrelate improved ROS/NO imbalance in glomeruli, suppressed platelet aggregation in glomeruli, reduced platelet-derived microparticles, increased serum adiponectin level and reduced the level of albuminuria, compared with non-treated diabetic rats.ConclusionsOur results indicate that sarpogrelate improves endothelial function in rats with STZ-induced diabetes through a reduction of glomerular platelet activation and an increase in serum adiponectin concentrations and suggest that sarpogrelate is potentially useful for the treatment of diabetic nephropathy.

C/EBP-β Modulates Transcription of Tubulointerstitial Nephritis Antigen in Obstructive Uropathy

Tubulointerstitial injury leading to fibrosis is a common pathway of many renal diseases. During this type of injury, modeled by unilateral ureteral obstruction (UUO), cells undergo epithelial-to-mesenchymal transition (EMT), a process that is mediated by various cytokines that modulate the biology of extracellular matrix proteins. Here, we studied the tubulointerstitial nephritis antigen (TINag), a tubular basement membrane protein, in the UUO model of tubulointerstitial injury. We observed upregulation of type IV collagen but downregulation of both laminin and TINag in obstructed kidneys. TINag downregulation was a result of oxidative stress; in the proximal tubular epithelial cell line HK-2, TINag expression and its promoter activity decreased after treatment with H2O2. We identified multiple CCAAT/enhancer binding protein beta (C/EBP-beta) motifs in the TINag promoter and observed that oxidant stress perturbed interactions between TINag DNA and C/EBP-beta protein. Oxidant stress reduced nuclear translocation of C/EBP-beta in HK-2 cells, which was restored by antioxidants. In addition, overexpression of C/EBP-beta restored the H2O2-induced reduction of TINag promoter activity and expression. Furthermore, in vivo, renal obstruction reduced nuclear expression of C/EBP-beta. Cells grown on a TINag substratum maintained their normal epithelial phenotype and cytoskeletal organization, similar to those grown on type IV collagen, and demonstrated reduced synthesis of fibronectin. Taken together, these findings suggest that altered interactions between C/EBP-beta and TINag play a critical role in the pathophysiology of renal injury after obstruction.

Pioglitazone Enhances the Antihypertensive and Renoprotective Effects of Candesartan in Zucker Obese Rats Fed a High-Protein Diet

The metabolic syndrome is a risk factor for the development of chronic kidney disease. Angiotensin II type 1 receptor blockers (ARBs) and thiazolidinediones (TZDs) provide renovascular protection, probably in the metabolic syndrome. However, the effect of both agents administered together in patients with metabolic syndrome remains to be determined. The aim of this study was to assess the effects of ARB plus TZD combination therapy in Zucker obese rats fed a high-protein diet, an animal model of metabolic syndrome and renal injury. Zucker obese rats were fed a high-protein diet (OHP; n=6), a high-protein diet containing candesartan, an ARB (OHP+C; n=6), or a high-protein diet containing both candesartan and pioglitazone (OHP+CP; n=6) for 12 weeks. Systolic blood pressure and urinary protein excretion were measured throughout the study, and renal histology and immunohistochemistry were assessed at 12 weeks. OHP rats developed hypertension (157±4 mmHg) and proteinuria (178±44 mg/d), and these conditions were significantly ameliorated by candesartan (to 143±3 mmHg and 84±25 mg/d, respectively). Pioglitazone enhanced the antihypertensive and anti-proteinuric effects of candesartan (121±3 mmHg, 16±8 mg/d, respectively). Histologically, candesartan ameliorated glomerulosclerosis, podocyte injury, interstitial fibrosis and monocyte/macrophage infiltration into the tubulointerstitium in the kidneys of OHP rats. Pioglitazone abrogated residual interstitial fibrosis in the kidneys of OHP+C rats. Our results suggested that pioglitazone augmented the antihypertensive, anti-proteinuric and possibly renal anti-fibrotic actions of candesartan in Zucker obese rats fed a high-protein diet. The combination therapy of ARB and TZD may protect against renal injury in patients with metabolic syndrome.

Role of Extracellular Matrix Renal Tubulo-interstitial Nephritis Antigen (TINag) in Cell Survival Utilizing Integrin αvβ3/Focal Adhesion Kinase (FAK)/Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B-Serine/Threonine Kinase (AKT) Signaling Pathway

Tubulo-interstitial nephritis antigen (TINag) is an extracellular matrix protein expressed in tubular basement membranes. Combined mutations in TINag and nephrocystin-1 genes lead to nephronophthisis with reduced cell survival. Because certain extracellular matrix proteins are known to modulate cell survival, studies were initiated in Lewis rats lacking TINag to assess if they are more susceptible to cisplatin-induced injury. Cisplatin induced a higher degree of tubular cell damage and apoptosis in regions where TINag is expressed in a parental Wistar strain. This was accompanied by an accentuated increase in serum creatinine and Kim-1 RNA and renal expression of Bax, p53, and its nuclear accumulation, mtDNA fragmentation, and a decrease of Bcl-2. Cisplatin induced fulminant apoptosis of HK-2 cells with increased caspase3/7 activity, mtDNA fragmentation, and a reduced cell survival. These effects were partially reversed in cells maintained on TINag substratum. Far Western/solid phase assays established TINag binding with integrin αvβ3 comparable with vitronectin. Transfection of cells with αv-siRNA accentuated cisplatin-induced apoptosis, aberrant translocation of cytochrome c and Bax, and reduced cell survival. The αv-siRNA decreased expression of integrin-recruited focal adhesion kinase (FAK) and p-FAK, while increasing the expression of p53 and p-p53. Similarly, p-AKT was reduced although ILK was unaffected. Inhibition of PI3K had similar adverse cellular effects. These effects were ameliorated in cells on TINag substratum. In vivo, a higher degree of decrease in the expression of p-FAK and pAKT was observed in Lewis rats following cisplatin treatment. These in vivo and in vitro studies demonstrate an essential role of TINag in cellular survival to maintain proper tubular homeostasis utilizing integrin αvβ3 and downstream effectors.