Masakazu Haneda

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.

Mitogen-Activated Protein Kinase Cascade Is Activated in Glomeruli of Diabetic Rats and Glomerular Mesangial Cells Cultured Under High Glucose Conditions

The activation of protein kinase C (PKC) found in diabetic glomeruli and glomerular mesangial cells cultured under high glucose conditions has been proposed to contribute to the development of diabetic nephropathy. However, the abnormalities distal to PKC have not been fully elucidated yet. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) cascade, an important kinase cascade downstream to PKC and an activator of cytosolic phospholipase A2 (cPLA2) by direct phosphorylation, is activated in glomeruli isolated from streptozotocin-induced diabetic rats. MAPK cascade was also activated in glomerular mesangial cells cultured under high glucose (27.8 mmol/1) conditions for 5 days, and the activation of MAPK cascade was inhibited by treating the cells with calphostin C, an inhibitor of PKC. Furthermore, the activities of cPLA2 also increased in cells cultured under the same conditions and this activation was inhibited by both calphostin C and PD 098059, an inhibitor of MEK (MAPK or extracellular signal-regulated kinase [ERK] kinase). These results indicate that MAPK cascade is activated in glomeruli and mesangial cells under the diabetic state possibly through the activation of PKC. Activated MAPK, in turn, may induce various functional changes of mesangial cells at least through the activation of cPLA2 and contribute to the development of diabetic nephropathy.

Diabetic neuropathy and nerve regeneration.

Diabetic neuropathy is the most common peripheral neuropathy in western countries. Although every effort has been made to clarify the pathogenic mechanism of diabetic neuropathy, thereby devising its ideal therapeutic drugs, neither convinced hypotheses nor unequivocally effective drugs have been established. In view of the pathologic basis for the treatment of diabetic neuropathy, it is important to enhance nerve regeneration as well as prevent nerve degeneration. Nerve regeneration or sprouting in diabetes may occur not only in the nerve trunk but also in the dermis and around dorsal root ganglion neurons, thereby being implicated in the generation of pain sensation. Thus, inadequate nerve regeneration unequivocally contributes to the pathophysiologic mechanism of diabetic neuropathy. In this context, the research on nerve regeneration in diabetes should be more accelerated. Indeed, nerve regenerative capacity has been shown to be decreased in diabetic patients as well as in diabetic animals. Disturbed nerve regeneration in diabetes has been ascribed at least in part to all or some of decreased levels of neurotrophic factors, decreased expression of their receptors, altered cellular signal pathways and/or abnormal expression of cell adhesion molecules, although the mechanisms of their changes remain almost unclear. In addition to their steady-state changes in diabetes, nerve injury induces injury-specific changes in individual neurotrophic factors, their receptors and their intracellular signal pathways, which are closely linked with altered neuronal function, varying from neuronal survival and neurite extension/nerve regeneration to apoptosis. Although it is essential to clarify those changes for understanding the mechanism of disturbed nerve regeneration in diabetes, very few data are now available. Rationally accepted replacement therapy with neurotrophic factors has not provided any success in treating diabetic neuropathy. Aside from adverse effects of those factors, more rigorous consideration for their delivery system may be needed for any possible success. Although conventional therapeutic drugs like aldose reductase (AR) inhibitors and vasodilators have been shown to enhance nerve regeneration, their efficacy should be strictly evaluated with respect to nerve regenerative capacity. For this purpose, especially clinically, skin biopsy, by which cutaneous nerve pathology including nerve regeneration can be morphometrically evaluated, might be a safe and useful examination.

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.

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.

Mitogen-activated protein kinase phosphatase : a negative regulator of the mitogen-activated protein kinase cascade

Mitogen-activated protein kinases (MAPKs) are activated by various stimuli, such as growth factors, cytokines, or stress, and are considered to be important mediators in intracellular signal transduction networks. The dual-specificity kinases, MAPK kinases (MKKs), which phosphorylate the TXY motif in the catalytic domain of MAPKs, can cause the activation of MAPKs. Recently, a family of dual-specificity phosphatases has been identified, members of which are able to dephosphorylate and inactivate MAPKs. The studies cited in this review have revealed that these MAPK phosphatases might play an important role in various cellular functions by downregulating the MAPK cascade.

Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells

SummaryType IV collagen production by cultured glomerular mesangial cells and the effect of glucose on it were evaluated in order to explore the possible contribution of mesangial cells to the accumulation of type IV collagen in mesangial matrix typically seen in diabetes. Type IV collagen was measured quantitatively by enzyme-linked immunosorbent assay. The majority of type IV collagen was secreted into culture media and secreted-type IV collagen increased with cell growth in early log phase and decreased in late log phase and after confluency. By exposing the cells to high concentrations of glucose (27.8 mmol/l), both secreted- and cell-associated-type IV collagens increased significantly compared with the cells cultured under normal glucose concentrations (5.6 mmol/l) or under equivalent concentrations of mannitol, resulting in a significant increase in total type IV collagen accumulation from 32.1±6.4 (under 5.6 mmol/l glucose) to 51.0±4.6 μg/dish (mean ± SD, n=4) on day 4, from 113.6±6.6 to 156.8±7.1 on day 6, from 248.5±15.2 to 310.0±12.6 on day 8 and from 372.4±14.8 to 507.9±17.2 on day 12. These results indicate the importance of glucose-induced alteration of mesangial cell function in the development of diabetic mesangial expansion.

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.

Abnormal Angiogenesis in Diabetic Nephropathy

Diabetic retinopathy is the leading cause of blindness in the Western world (1) and is characterized by abnormal angiogenesis driven by several factors, including tissue ischemia and hyperglycemia. This abnormal angiogenesis results in new vessels that are often immature and play a pathological role in retinopathy, contributing to both vitreous hemorrhage and fibrosis (2). In addition, increased vascular permeability leading to plasma leakage accounts for the development of macula edema, disrupting visual function (2). These evidences have led to the development of several therapeutic strategies targeting angiogenesis in diabetic retinopathy (3). Abnormal angiogenesis also occurs in diabetic nephropathy; therefore, the overriding question is whether new vessel formation in the kidney plays a pathological role in diabetic nephropathy similar to that observed in retinopathy. Intriguingly, the progression of both diabetic retinopathy and nephropathy is altered by vascular growth factor signaling through receptor tyrosine kinases, specifically involving the vascular endothelial growth factor (VEGF)-A and angiopoietin families. This review discusses abnormal angiogenesis and the role of both VEGF-A and angiopoietins in diabetic nephropathy. ### Evidence of abnormal angiogenesis in diabetic nephropathy. In 1987, Osterby and Nyberg (4) described abnormal blood vessels in glomeruli of patients with long-term type 1 diabetes, and later these findings were shown to occur in type 2 diabetic patients (5,6) (Fig. 1 A ). The abnormal vessels occupied 1–5% of glomerular capillary area, they were occasionally dilated, and the glomerular basement membrane adjacent to them was found to be focally extremely thin. Abnormal vessels were also present in Bowmans capsule or in the glomerular vascular pole, the latter of which could often be detected as an “extra efferent arteriole” (4,7). Min and Yamanaka (8) then performed detailed analyses of computer-generated three-dimensional images in 94 patients with diabetic nephropathy and found the presence of extravessels. Intriguingly, in this study the abnormal vessels anastomosed to …