Keiji Isshiki

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC β inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC β inhibitor ameliorates not only early diabetes‐induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor β1 (TGF‐βΙ) and extracellular matrix (ECM) proteins in streptozoto‐cin‐induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long‐term effects of a PKC β inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC β inhibitor reduced urinary albumin excretion rates and inhibited glo‐merular PKC activation in diabetic db/db mice. Administration of a PKC β inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF‐β and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long‐term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC β inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.—Koya, D., Haneda, M., Nakagawa, H., Isshiki, K., Sato, H., Maeda, S., Sugimoto, T., Yasuda, H., Kashiwagi, A., Ways, D. K., King, G. L., Kikkawa, R. Amelioration of accelerated diabetic mesangial expansion by treatment with A PKC b inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J. 14, 439–447 (2000)

Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney

Mitochondrial oxidative damage is a basic mechanism of aging, and multiple studies demonstrate that this process is attenuated by calorie restriction (CR). However, the molecular mechanism that underlies the beneficial effect of CR on mitochondrial dysfunction is unclear. Here, we investigated in mice the mechanisms underlying CR-mediated protection against hypoxia in aged kidney, with a special focus on the role of the NAD-dependent deacetylase sirtuin 1 (Sirt1), which is linked to CR-related longevity in model organisms, on mitochondrial autophagy. Adult-onset and long-term CR in mice promoted increased Sirt1 expression in aged kidney and attenuated hypoxia-associated mitochondrial and renal damage by enhancing BCL2/adenovirus E1B 19-kDa interacting protein 3-dependent (Bnip3-dependent) autophagy. Culture of primary renal proximal tubular cells (PTCs) in serum from CR mice promoted Sirt1-mediated forkhead box O3 (Foxo3) deacetylation. This activity was essential for expression of Bnip3 and p27Kip1 and for subsequent autophagy and cell survival of PTCs under hypoxia. Furthermore, the kidneys of aged Sirt1+/- mice were resistant to CR-mediated improvement in the accumulation of damaged mitochondria under hypoxia. These data highlight the role of the Sirt1-Foxo3 axis in cellular adaptation to hypoxia, delineate a molecular mechanism of the CR-mediated antiaging effect, and could potentially direct the design of new therapies for age- and hypoxia-related tissue damage.

SIRT1 inhibits transforming growth factor beta-induced apoptosis in glomerular mesangial cells via Smad7 deacetylation.

SIRT1, a class III histone deacetylase, is considered a key regulator of cell survival and apoptosis through its interaction with nuclear proteins. In this study, we have examined the likelihood and role of the interaction between SIRT1 and Smad7, which mediates transforming growth factor β (TGFβ)-induced apoptosis in renal glomerular mesangial cells. Immunoprecipitation analysis revealed that SIRT1 directly interacts with the N terminus of Smad7. Furthermore, SIRT1 reversed acetyl-transferase (p300)-mediated acetylation of two lysine residues (Lys-64 and -70) on Smad7. In mesangial cells, the Smad7 expression level was reduced by SIRT1 overexpression and increased by SIRT1 knockdown. SIRT1-mediated deacetylation of Smad7 enhanced Smad ubiquitination regulatory factor 1 (Smurf1)-mediated ubiquitin proteasome degradation, which contributed to the low expression of Smad7 in SIRT1-overexpressing mesangial cells. Stimulation by TGFβ or overexpression of Smad7 induced mesangial cell apoptosis, as assessed by morphological apoptotic changes (nuclear condensation) and biological apoptotic markers (cleavages of caspase3 and poly(ADP-ribose) polymerase). However, TGFβ failed to induce apoptosis in Smad7 knockdown mesangial cells, indicating that Smad7 mainly mediates TGFβ-induced apoptosis of mesangial cells. Finally, SIRT1 overexpression attenuated both Smad7- and TGFβ-induced mesangial cell apoptosis, whereas SIRT1 knockdown enhanced this apoptosis. We have concluded that Smad7 is a new target molecule for SIRT1 and SIRT1 attenuates TGFβ-induced mesangial cell apoptosis through acceleration of Smad7 degradation. Our results suggest that up-regulation of SIRT1 deacetylase activity is a potentially useful therapeutic strategy for prevention of TGFβ-related kidney disease through its effect on cell survival.

Characterization of protein kinase C β isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization

Retinal neovascularization is a major cause of blindness and requires the activities of several signaling pathways and multiple cytokines. Activation of protein kinase C (PKC) enhances the angiogenic process and is involved in the signaling of vascular endothelial growth factor (VEGF). We have demonstrated a dramatic increase in the angiogenic response to oxygen-induced retinal ischemia in transgenic mice overexpressing PKCβ2 isoform and a significant decrease in retinal neovascularization in PKCβ isoform null mice. The mitogenic action of VEGF, a potent hypoxia-induced angiogenic factor, was increased by 2-fold in retinal endothelial cells by the overexpression of PKCβ1 or β2 isoforms and inhibited significantly by the overexpression of a dominant-negative PKCβ2 isoform but not by the expression of PKC α, δ, and ζ isoforms. Association of PKCβ2 isoform with retinoblastoma protein was discovered in retinal endothelial cells, and PKCβ2 isoform increased retinoblastoma phosphorylation under basal and VEGF-stimulated conditions. The potential functional consequences of PKCβ-induced retinoblastoma phosphorylation could include enhanced E2 promoter binding factor transcriptional activity and increased VEGF-induced endothelial cell proliferation.

Reduction of Diabetes-Induced Oxidative Stress, Fibrotic Cytokine Expression, and Renal Dysfunction in Protein Kinase Cβ–Null Mice

Diabetes induces the activation of several protein kinase C (PKC) isoforms in the renal glomeruli. We used PKC-β−/− mice to examine the action of PKC-β isoforms in diabetes-induced oxidative stress and renal injury at 8 and 24 weeks of disease. Diabetes increased PKC activity in renal cortex of wild-type mice and was significantly reduced (<50% of wild-type) in diabetic PKC-β−/− mice. In wild-type mice, diabetes increased the translocation of PKC-α and -β1 to the membrane, whereas only PKC-α was elevated in PKC-β−/− mice. Increases in urinary isoprostane and 8-hydroxydeoxyguanosine, parameters of oxidative stress, in diabetic PKC-β−/− mice were significantly reduced compared with diabetic wild-type mice. Diabetes increased NADPH oxidase activity and the expressions of p47phox, Nox2, and Nox4 mRNA levels in the renal cortex and were unchanged in diabetic PKC-β−/− mice. Increased expression of endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, connective tissue growth factor (CTGF), and collagens IV and VI found in diabetic wild-type mice was attenuated in diabetic PKC-β−/− mice. Diabetic PKC-β−/− mice were protected from renal hypertrophy, glomerular enlargement, and hyperfiltration observed in diabetic wild-type mice and had less proteinuria. Lack of PKC-β can protect against diabetes-induced renal dysfunction, fibrosis, and increased expressions of Nox2 and -4, ET-1, VEGF, TGF-β, CTGF, and oxidant production.

Role of Altered Renal Lipid Metabolism in the Development of Renal Injury Induced by a High-Fat Diet

Metabolic syndrome is associated with increased risk of chronic kidney disease, and the renal injury in patients with metabolic syndrome may be a result of altered renal lipid metabolism. We fed wild-type or insulin-sensitive heterozygous peroxisome proliferator-activated receptor gamma-deficient (PPARgamma(+/-)) mice a high-fat diet for 16 weeks. In wild-type mice, this diet induced core features of metabolic syndrome, subsequent renal lipid accumulation, and renal injury including glomerulosclerosis, interstitial fibrosis, and albuminuria. Renal lipogenesis accelerated, determined by increased renal mRNA expression of the lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase (ACC) and by increased ACC activity. In addition, renal lipolysis was suppressed, determined by reduced mRNA expression of the lipolytic enzyme carnitine palmitoyl acyl-CoA transferase 1 and by reduced activity of AMP-activated protein kinase. In PPARgamma(+/-) mice, renal injury, systemic metabolic abnormalities, renal accumulation of lipids, and the changes in renal lipid metabolism were attenuated. Thus, a high-fat diet leads to an altered balance between renal lipogenesis and lipolysis, subsequent renal accumulation of lipid, and renal injury. We suggest that renal lipid metabolism could serve as a new therapeutic target to prevent chronic kidney disease in patients with metabolic syndrome.

Exendin-4 has an anti-hypertensive effect in salt-sensitive mice model

The improvement of salt-sensitive hypertension is a therapeutic target for various vascular diseases. Glucagon-like peptide 1 (GLP-1), an incretin peptide, has been reported to have natriuretic effect as well as blood glucose lowering effect, although its exact mechanism and clinical usefulness remain unclear. Here, we examined anti-hypertensive effect of exendin-4, a GLP-1 analog, in salt-sensitive obese db/db mice and angiotensin II (angII)-infused C57BLK6/J mice. The treatment of exendin-4 for 12 weeks inhibited the development of hypertension in db/db mice. In db/db mice, the urinary sodium excretion was delayed and blood pressure was elevated in response to a high-salt load, whereas these were attenuated by exendin-4. In db/db mice, intra-renal angII concentration was increased. Furthermore, exendin-4 prevented angII-induced hypertension in non-diabetic mice and inhibited angII-induced phosphorylation of ERK1/2 in cultured renal cells. Considered together, our results indicate that exendin-4 has anti-hypertensive effects through the attenuation of angII-induced high-salt sensitivity.

Reduction in Microalbuminuria as an Integrated Indicator for Renal and Cardiovascular Risk Reduction in Patients With Type 2 Diabetes

OBJECTIVE—Microalbuminuria in diabetic patients is a predictor for diabetic nephropathy and cardiovascular disease. The aim of this study is to investigate the clinical impact of reducing microalbuminuria in type 2 diabetic patients in an observational follow-up study. RESEARCH DESIGN AND METHODS—We enrolled 216 type 2 diabetic patients with microalbuminuria during an initial 2-year evaluation period and observed them for the next 8 years. Remission and a 50% reduction of microalbuminuria were defined as a shift to normoalbuminuria and a reduction <50% from the initial level of microalbuminuria. The association between reducing microalbuminuria and first occurrence of a renal or cardiovascular event and annual decline rate of estimated glomerular filtration rate (eGFR) was evaluated. RESULTS—Twelve events occurred in 93 patients who attained a 50% reduction of microalbuminuria during the follow-up versus 35 events in 123 patients without a 50% reduction. The cumulative incidence rate of events was significantly lower in patients with a 50% reduction. A pooled logistic regression analysis revealed that the adjusted risk for events in subjects after a 50% reduction was 0.41 (95% CI 0.15–0.96). In addition, the annual decline rate of eGFR in patients with a 50% reduction was significantly slower than in those without such a reduction. The same results were also found in the analysis regarding whether remission occurred. CONCLUSIONS—The present study provides clinical evidence implying that a reduction of microalbuminuria in type 2 diabetic patients is an integrated indicator for renal and cardiovascular risk reduction.

Enhanced sodium sensitivity and disturbed circadian rhythm of blood pressure in essential hypertension.

Objective To assess whether an association between sodium-sensitive hypertension and metabolic syndrome exists; and whether, in patients with metabolic syndrome, the nocturnal fall of blood pressure decreases and salt restriction affects the circadian blood pressure rhythm. Methods Japanese patients with essential hypertension, who were treated without any antihypertensive agent, were maintained on a high-sodium diet and a low-sodium diet for 1 week each. On the last day of each diet, the 24-h blood pressures were measured. A diagnosis of metabolic syndrome was made according to the International Diabetes Foundation definition Results Among the 56 patients with essential hypertension, 15 patients were complicated with metabolic syndrome while 41 patients were not. The nocturnal blood pressure fall was significant in patients without metabolic syndrome, while it was not so in patients with metabolic syndrome. Only in patients with metabolic syndrome was the nocturnal blood pressure fall enhanced by sodium restriction. The prevalence of sodium-sensitive hypertension in patients with metabolic syndrome was significantly higher than in those without metabolic syndrome (70.6 versus 36.0%, respectively; P = 0.017). A multiple logistic regression analysis revealed central obesity to be an independent risk factor for sodium-sensitive hypertension (odds ratio, 1.41; 95% confidence interval, 1.04–1.91). Conclusions In patients with essential hypertension, an inter-relationship exists among metabolic syndrome, enhanced sodium sensitivity of the blood pressure and non-dipping. The elevated risk of cardiovascular diseases in patients with metabolic syndrome may be related to sodium-sensitive hypertension and non-dipping.

Obesity-Mediated Autophagy Insufficiency Exacerbates Proteinuria-induced Tubulointerstitial Lesions

Obesity is an independent risk factor for renal dysfunction in patients with CKDs, including diabetic nephropathy, but the mechanism underlying this connection remains unclear. Autophagy is an intracellular degradation system that maintains intracellular homeostasis by removing damaged proteins and organelles, and autophagy insufficiency is associated with the pathogenesis of obesity-related diseases. We therefore examined the role of autophagy in obesity-mediated exacerbation of proteinuria-induced proximal tubular epithelial cell damage in mice and in human renal biopsy specimens. In nonobese mice, overt proteinuria, induced by intraperitoneal free fatty acid-albumin overload, led to mild tubular damage and apoptosis, and activated autophagy in proximal tubules reabsorbing urinary albumin. In contrast, diet-induced obesity suppressed proteinuria-induced autophagy and exacerbated proteinuria-induced tubular cell damage. Proximal tubule-specific autophagy-deficient mice, resulting from an Atg5 gene deletion, subjected to intraperitoneal free fatty acid-albumin overload developed severe proteinuria-induced tubular damage, suggesting that proteinuria-induced autophagy is renoprotective. Mammalian target of rapamycin (mTOR), a potent suppressor of autophagy, was activated in proximal tubules of obese mice, and treatment with an mTOR inhibitor ameliorated obesity-mediated autophagy insufficiency. Furthermore, both mTOR hyperactivation and autophagy suppression were observed in tubular cells of specimens obtained from obese patients with proteinuria. Thus, in addition to enhancing the understanding of obesity-related cell vulnerability in the kidneys, these results suggest that restoring the renoprotective action of autophagy in proximal tubules may improve renal outcomes in obese patients.