Shogo Koshikawa

Stimulatory and Inhibitory regulation of lipolysis by the NPR-A/cGMP/PKG and NPR-C/Gi pathways in rat cultured adipocytes.

OBJECTIVE Recent studies have suggested the abundant expression of natriuretic peptide receptor in adipose tissue. This study was designed to investigate the levels of natriuretic receptor-A (NPR-A) and NPR-C gene expression during the process of preadipocyte differentiation and its role in adipogenesis and lipid metabolism. METHODS We measured mRNA levels of NPR-A and NPR-C during the process of rat preadipocyte differentiation in vitro. We also measured the effects of ANP and C-ANP, a ligand for NPR-C, on preadipocyte differentiation. In addition, we assessed the effects of ANP and C-ANP on lipolysis and the cellular mechanism. RESULTS The mRNA levels of NPR-A and NPR-C on day 3, 6, 10 are (-26%, +226%), (+6%, +568%), and (+207%, +3232%) respectively as compared with day 1. ANP (10(-)(7) M) and 8-bromo-cGMP (10(-)(4) M) significantly increased Oil Red positive area and cell number of matured-adipocytes. ANP and 8-bromo-cGMP also increased the mRNA levels of adipocyte-related genes such as PPARgamma, leptin, and adiponectin on day 3, whereas C-ANP did not change these parameters. ANP (10(-)(9)-10(-)(6) M) increased intracellular cGMP levels and promoted lipolysis in adipocytes and the effects were abolished by HS-142-1, and KT5823. Conversely C-ANP (10(-)(6) M) decreased intracellular cAMP levels and lipolysis and its effect was inhibited by PTX. CONCLUSION Results suggest that ANP may promote adipocyte differentiation and lipolysis via the NPR-A/cGMP/PKG pathway. Direct action of ANP via NPR-C in adipogenesis may be either absent or barely present, but ANP may play a counter regulatory role in lipolysis via NPR-C/Gi pathway.

Fasudil, a Rho-kinase inhibitor, reverses L-NAME exacerbated severe nephrosclerosis in spontaneously hypertensive rats.

Background In this study, we tested the hypothesis that long-term Rho-kinase inhibition would reverse nitro-L-arginine methyl ester-exacerbated nephrosclerosis in spontaneously hypertensive rats and attempted to elucidate the mechanism involved. Methods Five groups (each n = 8) were studied: untreated spontaneously hypertensive rats; nitro-L-arginine methyl ester (50 mg/l in drinking water, for 3 weeks)-treated spontaneously hypertensive rats; nitro-L-arginine methyl ester with fasudil (10 mg/kg/day)-treated spontaneously hypertensive rats; nitro-L-arginine methyl ester for 3 weeks followed by fasudil for 3 weeks-treated spontaneously hypertensive rats (same doses), and nitro-L-arginine methyl ester for 3 weeks followed by untreated for 3 weeks. We examined renal function, blood pressure, histological features, oxidative stress markers, and mRNA expression in the renal cortex. Results Nitro-L-arginine methyl ester-treated spontaneously hypertensive rats had higher blood pressure, proteinuria, and serum creatinine and lower creatinine clearance, urinary NO3/NO2 ratio, and urinary cGMP excretion compared with control spontaneously hypertensive rats (all Ps < 0.05). Nitro-L-arginine methyl ester-treated spontaneously hypertensive rats also had increased free radical metabolites and abnormal morphological findings with increased nicotinamide adenine dinucleotide phosphate oxidase activity, phosphorylation of myosin phosphatase targeting subunit-1, and mRNA expression of RhoA, RhoB, RhoC, collagen I and III, transforming growth factor-β, nicotinamide adenine dinucleotide phosphate subunit, endothelial nitric oxide synthase, plasminogen activator inhibitor, and intercellular adhesion molecule-1 in the renal cortex compared with control spontaneously hypertensive rats. Long-term co-treatment with fasudil slightly improved these indices, but most of them were not statistically significant. Late fasudil treatment significantly improved kidney function, morphological changes, and alterations of mRNA expression in the renal cortex, although late untreated controls did not show any improvement. Conclusion These results suggest that Rho-kinase inhibition partly reverses hypertensive glomerulosclerosis. The renoprotective effect of the Rho-kinase inhibitor may have multiple mechanisms including inhibition of extracellular matrix production, oxidative stress, adhesion molecule production, and antifibrinolysis.

Natriuretic peptide/natriuretic peptide receptor-A (NPR-A) system has inhibitory effects in renal fibrosis in mice

OBJECT This study was designed to examine whether natriuretic peptide/natriuretic peptide receptor-A (NPR-A) system attenuates renal fibrosis in a unilateral ureteral obstruction (UUO) model and also examined the mechanism involved. METHODS Three groups were studied: untreated UUO in wild-type mice; untreated UUO in NPR-A KO mice; and ANP treated (0.05 microg/kg/min) UUO in wild-type mice. We measured histological and immunohistochemical findings (alpha-SMA and F4/80), tissue cGMP levels, various mRNA expression levels by real-time PCR analysis, and transcription factor levels (AP-1 and NF-kappaB) in renal tissue. RESULTS Compared with wild-type UUO mice, NPRA-KO UUO mice had abnormal morphological findings (fibrous area: +26%, alpha-SMA expression: +30%) with lower tissue cGMP levels and increases in the mRNA expression levels of TGF-beta, collagen I, collagen III, PAI-1, renin and angiotensinogen, whereas there were no differences in F4/80 positive cells or the mRNA expression levels of ICAM-1, osteopontin, or MCP-1 between the two groups. In contrast, ANP pre-treatment significantly improved morphological changes with increase of tissue cGMP levels and reduction in the mRNA expression level of TGF-beta, collagen I, collagen III, PAI-1, ICAM-1, osteopontin, MCP-1, renin, and angiotensinogen. NPRA-KO UUO mice had higher AP-1 levels than wild-type UUO mice and ANP pre-treatment reduced AP-1 and NF-kappaB activity. CONCLUSION The endogenous natriuretic peptide/NPR-A system may inhibit renal fibrosis partly via inhibition of the angiotensin/AP-1/TGF-beta/collagen pathway and exogenous ANP pre-treatment may inhibit it partly via both the angiotensin/AP-1/TGF-beta/collagen and NF-kappaB/inflammatory pathways.