Naoki Kashihara

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.

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoys effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoys inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.

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.

Phenotypic Modulation of the Mesangium Reflected by Contractile Proteins in Diabetes

The phenotypic change of the mesangial cell is considered to play a pivotal role in the accumulation of extracellular matrix in diabetic nephropathy. This investigation was undertaken to evaluate the expression of the various isoforms of contractile proteins in the streptozocin (STZ)-induced diabetic rat kidney and in renal biopsy specimens from patients with diabetic nephropathy. Specific antibodies to myosin heavy chain isoforms (SM1, SM2, SMemb), caldesmon, and α-smooth muscle actin and cDNAs for SMemb were used. Increased expression of SMemb at the mRNA and protein levels was demonstrated at 1 week after STZ administration in the rat. Both levels were increased at 4 weeks. Mesangial staining of caldesmon was observed at 4 weeks and that of α-smooth muscle actin at 24 weeks. Immunohistochemical mesangial staining of the contractile proteins was pronounced in patients with diabetic nephropathy in contrast to the trace mesangial staining in normal control subjects. These results indicate that the phenotypic change in mesangial cells occurs in the early stages of diabetes and that several stages in phenotypic changes may exist. Expression of the contractile protein isoforms, especially SMemb, should serve as a new marker for the subsequent glomerular hypertrophy and sclerosis.

Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling

Augmented Wnt signaling has been implicated in many fibrotic diseases including obstructive nephropathy. Soluble form Klotho has been reported to function as a secreted Wnt antagonist. In this study, we tested whether Klotho protein could reduce renal fibrosis by inhibition of Wnt signaling. Transgenic mice that overexpressed Klotho, wild-type mice, and Klotho hetero mutant mice underwent unilateral ureteral obstruction (UUO). In some Klotho hetero mutant mice, Klotho-encoding plasmid was transferred into the skeletal muscle by electroporation. UUO induced activation of Wnt signaling in wild-type but less in Klotho transgenic mice. Enhanced tubulointerstitial fibrosis in wild-type mice was also attenuated in Klotho transgenic mice. In contrast, Wnt signaling and concomitant tubulointerstitial fibrosis were further augmented in Klotho hetero mutant mice after UUO compared with wild-type mice. In Klotho-encoding plasmid-transfected Klotho hetero mutant mice, however, Wnt signaling was markedly reduced accompanied by a decrease in extracellular matrix deposition after UUO. In vitro studies showed that stimulation of Wnt3a induced prolonged cell cycle arrest at G(2)/M phase, with a resultant increase in production of fibrogenic cytokines. Cotreatment with Klotho bypassed the G(2)/M arrest and reduced fibrogenic cytokine production. In conclusion, Klotho is a critical negative regulator of Wnt signaling and a suppressor of renal fibrosis in the obstructed kidney model.

Role of membrane-type matrix metalloproteinase 1 (MT-1-MMP), MMP-2, and its inhibitor in nephrogenesis

Extracellular matrix (ECM) proteins, their integrin receptors, and matrix metalloproteinases (MMPs), the ECM-degrading enzymes, are believed to be involved in various biological processes, including embryogenesis. In the present study, we investigated the role of membrane type MMP, MT-1-MMP, an activator pro-MMP-2, in metanephric development. Also, its relationship with MMP-2 and its inhibitor, TIMP-2, was studied. Since mRNAs of MT-1-MMP and MMP-2 are respectively expressed in the ureteric bud epithelia and mesenchyme, they are ideally suited for juxtacrine/paracrine interactions during renal development. Northern blot analyses revealed a single ∼4.5-kb mRNA transcript of MT-1-MMP, and its expression was developmentally regulated. Inclusion of MT-1-MMP antisense oligodeoxynucleotide (ODN) in the culture media induced dysmorphogenetic changes in the embryonic metanephros. MMP-2 antisense ODN also induced similar changes, but they were relatively less; on the other hand TIMP-2 antisense ODN induced a mild increase in the size of explants. Concomitant exposure of MT-1-MMP and MMP-2 antisense ODNs induced profound alterations in the metanephroi. Treatment of TIMP-2 antisense ODN to metanephroi exposed to MT-1-MMP/MMP-2 antisense notably restored the morphology of the explants. Specificity of the MT-1-MMP antisense ODN was reflected in the selective decrease in its mRNA and protein expression. The MT-1-MMP antisense ODN also resulted in a failure in the activation of pro-MMP-2 to MMP-2. These findings suggest that the trimacromolecular complex of MT-1-MMP:MMP-2:TIMP-2 modulates the organogenesis of the metanephros, conceivably by mediating paracrine/juxtacrine epithelial:mesenchymal interactions.Extracellular matrix (ECM) proteins, their integrin receptors, and matrix metalloproteinases (MMPs), the ECM-degrading enzymes, are believed to be involved in various biological processes, including embryogenesis. In the present study, we investigated the role of membrane type MMP, MT-1-MMP, an activator pro-MMP-2, in metanephric development. Also, its relationship with MMP-2 and its inhibitor, TIMP-2, was studied. Since mRNAs of MT-1-MMP and MMP-2 are respectively expressed in the ureteric bud epithelia and mesenchyme, they are ideally suited for juxtacrine/paracrine interactions during renal development. Northern blot analyses revealed a single approximately 4.5-kb mRNA transcript of MT-1-MMP, and its expression was developmentally regulated. Inclusion of MT-1-MMP antisense oligodeoxynucleotide (ODN) in the culture media induced dysmorphogenetic changes in the embryonic metanephros. MMP-2 antisense ODN also induced similar changes, but they were relatively less; on the other hand TIMP-2 antisense ODN induced a mild increase in the size of explants. Concomitant exposure of MT-1-MMP and MMP-2 antisense ODNs induced profound alterations in the metanephroi. Treatment of TIMP-2 antisense ODN to metanephroi exposed to MT-1-MMP/MMP-2 antisense notably restored the morphology of the explants. Specificity of the MT-1-MMP antisense ODN was reflected in the selective decrease in its mRNA and protein expression. The MT-1-MMP antisense ODN also resulted in a failure in the activation of pro-MMP-2 to MMP-2. These findings suggest that the trimacromolecular complex of MT-1-MMP:MMP-2:TIMP-2 modulates the organogenesis of the metanephros, conceivably by mediating paracrine/juxtacrine epithelial:mesenchymal interactions.

Rap1b GTPase Ameliorates Glucose-Induced Mitochondrial Dysfunction

The role of tubular injury in diabetic nephropathy is relatively unknown, despite that apoptosis of tubular epithelial cells is commonly observed in human renal biopsies. The GTPase Ras-proximate-1 (Rap1b) is upregulated in the hyperglycemic state and is known to increase B-Raf, an antiapoptotic effector protein. In this study, the effects of high glucose on renal tubular apoptosis and the potential ability for Rap1b to ameliorate these effects were investigated. In the kidneys of diabetic mice, apoptotic tubular cells and dysmorphic mitochondria were observed, Bcl-2 expression was decreased, and Bax expression was increased. Total Rap1b expression was slightly increased, but its associated GTPase activity was significantly decreased. In vitro, high extracellular glucose led to decreased Bcl-2 expression, reduced Rap1b GTPase activity, and increased levels of both Bax and GTPase activating protein in a proximal tubular cell line (HK-2). These changes were accompanied by increased DNA fragmentation, decreased high molecular weight mitochondrial DNA, altered mitochondrial morphology and function, disrupted Bcl-2-Bax and Bcl-2-Rap1b interactions, and reduced cell survival. Overexpression of Rap1b partially prevents these abnormalities. Furthermore, the BH4 domain of Bcl-2 was found to be required for successful protein-protein interaction between Bcl-2 and Rap1b. In summary, these data suggest that Rap1b ameliorates glucose-induced mitochondrial dysfunction in renal tubular cells.

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.

Glomerular cell apoptosis in human lupus nephritis

Disturbed apoptosis is proposed to be involved in the pathogenesis of systemic lupus erythematosus. However, the role of renal cell apoptosis in the pathogenesis and progression of human lupus nephritis is still controversial. We have investigated glomerular cell apoptosis and the clinicopathological relationship between apoptosis and immunoserological or histological findings in 22 patients with lupus nephritis using electron microscopy and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Resident glomerular cells as well as infiltrating neutrophils undergoing apoptosis were observed in 12 of 20 patients with lupus nephritis using electron microscopy. TUNEL-positive cells were recognized in 93% of patients with diffuse proliferative lupus nephritis (class IV) in contrast to the 20% of patients with class V. The number of TUNEL-positive cells in glomeruli significantly correlated with the level of immunoserological activity of lupus, such as anti-double-stranded DNA autoantibody and consumption of plasma complement. There was a positive correlation between glomerular cell apoptosis and the degree of proliferation in lupus nephritis. These data suggest that apoptosis is increased, but not decreased in glomeruli from patients with lupus nephritis. The signals that could induce glomerular cell apoptosis in lupus nephritis will need to be identified.