Yoshikazu Miyasato

The serine protease prostasin regulates hepatic insulin sensitivity by modulating TLR4 signalling

The effects of high-fat diet (HFD) and postprandial endotoxemia on the development of type 2 diabetes are not fully understood. Here we show that the serine protease prostasin (PRSS8) regulates hepatic insulin sensitivity by modulating Toll-like receptor 4 (TLR4)-mediated signalling. HFD triggers the suppression of PRSS8 expression by inducing endoplasmic reticulum (ER) stress and increases the TLR4 level in the liver. PRSS8 releases the ectodomain of TLR4 by cleaving it, which results in a reduction in the full-length form and reduces the activation of TLR4. Liver-specific PRSS8 knockout (LKO) mice develop insulin resistance associated with the increase in hepatic TLR4. Restoration of PRSS8 expression in livers of HFD, LKO and db/db mice decreases the TLR4 level and ameliorates insulin resistance. These results identify a novel physiological role for PRSS8 in the liver and provide new insight into the development of diabetes resulting from HFD or metabolic endotoxemia.

The Serine Protease Inhibitor Camostat Mesilate Attenuates the Progression of Chronic Kidney Disease through its Antioxidant Effects

Background/Aims: We have so far demonstrated the renoprotective effect of camostat mesilate (CM) in 5/6 nephrectomized rats at least partly through its antioxidant effect. However, precise mechanisms were not fully clarified. Therefore, we now examined the renoprotective and antioxidant mechanisms of CM by using the adenine-induced chronic kidney disease (CKD) rat model. Methods: In protocol 1, we analyzed the effect of CM on CKD. Rats were fed on a 0.75% adenine diet for 3 weeks to induce CKD followed by the experimental period with vehicle, CM, or hydralazine (HYD) treatment for 5 weeks. In protocol 2, we examined the safety of CM and HYD on the normal rats. In addition, we explored free radical scavenging activities of CM and its metabolites in vitro using electron paramagnetic resonance (EPR) spectroscopy. Results: CM, but not HYD, significantly reduced the serum creatinine levels, although both treatments showed similar reduction in the blood pressure. CM decreased mRNA expression and protein levels of fibrotic markers, the severity of renal fibrosis, the accumulation of oxidative stress, and the expression of NADPH oxidase components in the kidney. In the protocol 2, there were no statistically significant differences in general parameters except for the systolic blood pressure in HYD group. EPR study revealed that CM and its metabolites have potent hydroxyl radical scavenging activities in vitro. Conclusion: Our findings indicate that CM significantly ameliorates the progression of CKD partly through its antioxidant effect independently from its blood pressure-lowering effect. Our results suggest the possibility that CM could be a new therapeutic agent that could arrest the progression of CKD.

Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response

Cisplatin-induced acute kidney injury (AKI) has been recognized as one of cisplatin’s serious side effects, limiting its use in cancer therapy. Sirtuin 1 (SIRT1) and SIRT3 play protective roles against cisplatin-induced kidney injury. However, the role of SIRT7 in cisplatin-induced kidney injury is not yet known. In this study, we found that Sirt7 knockout (KO) mice were resistant to cisplatin-induced AKI. Furthermore, our studies identified that loss of SIRT7 decreases the expression of tumor necrosis factor-α (TNF-α) by regulating the nuclear expression of the transcription factor nuclear factor kappa B. It has been reported that cisplatin-induced nephrotoxicity is mediated by TNF-α. Our results indicate that SIRT7 plays an important role in cisplatin-induced AKI and suggest the possibility of SIRT7 as a novel therapeutic target for cisplatin-induced nephrotoxicity.

Doxycycline attenuates cisplatin-induced acute kidney injury through pleiotropic effects

Cisplatin (CDDP) is a widely-used chemotherapeutic drug for solid tumors, but its nephrotoxicity is a major dose-limiting factor. Doxycycline (Dox) is a tetracycline antibiotic that has been commonly used in a variety of infections. Dox has been shown to possess several other properties, including antitumor, anti-inflammatory, antioxidative, and matrix metalloproteinase (MMP)-inhibiting actions. We, therefore, investigated whether Dox exerts renoprotective effects in CDDP-induced acute kidney injury (AKI). Twelve-week-old male C57BL/6J mice were divided into the following groups: 1) control, 2) Dox (2 mg/ml in drinking water), 3) CDDP (25 mg/kg body weight, intraperitoneally), and 4) CDDP+Dox. After seven days of pretreatment with Dox, CDDP was administered and the animals were killed at day 1 or day 3. We evaluated renal function along with renal histological damage, inflammation, oxidative stress, and apoptosis. MMP and serine protease activities in the kidney tissues were assessed using zymography. Administration of CDDP exhibited renal dysfunction and caused histological damage predominantly in the proximal tubules. Dox did not affect either expression of CDDP transporters or the accumulation of CDDP in renal tissues; however, it significantly ameliorated renal dysfunction and histological changes together with reduced detrimental responses, such as oxidative stress and inflammation in the kidneys. Furthermore, Dox inhibited the activity of MMP-2 and MMP-9, as well as serine proteases in the kidney tissues. Finally, Dox markedly mitigated apoptosis in renal tubules. Thus, Dox ameliorated CDDP-induced AKI through its pleiotropic effects. Our results suggest that Dox may become a novel strategy for the prevention of CDDP-induced AKI in humans.

Successful treatment of rapidly progressive immunoglobulin A nephropathy with human immunodeficiency virus infection by steroid pulse therapy and tonsillectomy.

A 43-year-old man was admitted to our hospital with rapid decline in kidney function. He was in good control of HIV infection under antiretroviral therapy. Physical examination findings were unremarkable. Laboratory data showed an abnormal kidney function (Fig. 1). Serologic studies were negative. HIV RNA was undetectable. Renal biopsy revealed endocapillary cellular proliferation and mesangial matrix accumulation. 25% of glomeruli had cellular crescents. Immunofluorescence showed granular pattern of IgA in mesangial regions. A diagnosis of IgA nephropathy (M0,S1,E1,T1 in Oxford criteria) was made. After three courses of methylprednisolone pulse plus oral prednisolone therapy, the patient showed improvement in kidney function; however, proteinuria and haematuria persisted. We added tonsillectomy with another course of methylprednisolone pulse therapy. After 22 months from admission his kidney function remained stable, with significant improvement on proteinuria and haematuria.

A serine protease inhibitor attenuates aldosterone-induced kidney injuries via the suppression of plasmin activity

Emerging evidence has suggested that aldosterone has direct deleterious effects on the kidney independently of its hemodynamic effects. However, the detailed mechanisms of these direct effects remain to be elucidated. We have previously reported that camostat mesilate (CM), a synthetic serine protease inhibitor, attenuated kidney injuries in Dahl salt-sensitive rats, remnant kidney rats, and unilateral ureteral obstruction rats, suggesting that some serine proteases would be involved in the pathogenesis of kidney injuries. The current study was conducted to investigate the roles of serine proteases and the beneficial effects of CM in aldosterone-related kidney injuries. We observed a serine protease that was activated by aldosterone/salt in rat kidney lysate, and identified it as plasmin with liquid chromatography-tandem mass spectrometry. Plasmin increased pro-fibrotic and inflammatory gene expressions in rat renal fibroblast cells. CM inhibited the protease activity of plasmin and suppressed cell injury markers induced by plasmin in the fibroblast cells. Furthermore, CM ameliorated glomerulosclerosis and interstitial fibrosis in the kidney of aldosterone/salt-treated rats. Our findings indicate that plasmin has important roles in kidney injuries that are induced by aldosterone/salt, and that serine protease inhibitor could provide a new strategy for the treatment of aldosterone-associated kidney diseases in humans.

MPS 01-03 SERINE PROTEASE INHIBITION ATTENUATES SALT-SENSITIVE HYPERTENSION AND PODOCYTE INJURY IN A RAT MODEL OF METABOLIC SYNDROME

Objective: Chronic kidney disease caused by metabolic syndrome (MetS) is characterized by proteinuria, Na retention and hypertension. In the setting of proteinuria, plasminogen/plasmin filtered through damaged glomeruli could activate the epithelial Na channel (ENaC) leading to hypertension, independently of aldosterone. In this study, we evaluated effects of a synthetic serine protease (SP) inhibitor, camostat mesilate (CM) which inhibits the plasmin activity, on salt-sensitive hypertension in a rat model of MetS with high-salt (HS) diet. In addition, we studied protective effects of CM against podocyte injury in vitro. Design and Method: Five-week-old SHR and control WKY were divided into WKY, SHR, and SHR + CM (Experiment 1), and 13-week-old SHR/ND mcr-cp (model rats for MetS) were divided into normal chow (NS), HS (8.0% NaCl diet), and HS + CM (Experiment 2). After systolic BP measurement and 24 h urine collection were performed for 4 weeks, rats were sacrificed for histological examination. Urinary plasmin activities were evaluated by zymography. Cultured murine podocytes in high aldosterone and glucose media were treated with CM. Results: In Experiment 1, although SHR displayed hypertension, urinary protein excretion and plasmin activity were not substantially increased. Accordingly, CM did not prevent hypertension in SHR (SBP (mmHg): SHR 162 vs. SHR + CM 158). In Experiment 2, HS diet induced severe hypertension, marked proteinuria and plasmin activation in urine in SHR/ND mcr-cp. These changes were significantly suppressed by the treatment with CM (SBP (mmHg): HS 230 vs. HS + CM 178). CM increased urinary sodium/potassium ratio, indicating that CM inhibited the ENaC activity. Although CM mitigated apoptosis of podocytes in vivo and in vitro, CM did not inhibit the activity of Omi/HtrA2 that is a mitochondrial SP associated with apoptosis. Conclusions: In conclusion, CM could exert significant antihypertensive and renoprotective effects in a rat model of MetS, suggesting that SP inhibition could be a new therapeutic strategy against salt-sensitive hypertension in MetS.