Terumasa Nakagawa

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.

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.

PS 10-06 ALDOSTERONE MODULATES EXPRESSION OF AN ACID-SECRETING AMMONIA TRANSPORTER Rhcg IN INTERCALATED CELLS OF THE COLLECTING DUCTS

Objective: Main adverse effects of renin-angiotensin-aldosterone system (RAAS) blockers are metabolic acidosis and hyperkalemia, which limit the use of RAAS blockers for treatment of patients with chronic kidney disease. The mechanism of urinary acid secretion by the RAAS has not been fully understood. Rhesus blood group C glycoprotein (Rhcg) is an ammonia transporter which cooperates with H+-ATPase to secrete H+ from the intercalated cells of the collecting duct in the kidney. We hypothesize that aldosterone increases acid secretion by regulating Rhcg. Design and Method: In the present study, effects of aldosterone on the regulation of Rhcg were examined in vivo and in vitro. Mice were continuously administered aldosterone (40 &mgr;g/body/day) or vehicle for a week. Urinary pH, ammonia excretion, and titratable acids were measured. Membrane fraction of the whole kidney was extracted. IN-IC cells (a rat intercalated cell line) were treated with aldosterone (10–6 M) for 24 h, and then mRNA, whole cell protein, and membrane fraction were extracted. The Rhcg mRNA and protein expressions were measured by real-time PCR and Western blotting, respectively. The expression of serum and glucocorticoid-regulated kinase 1 (Sgk1) mRNA was also measured in IN-IC cells. Results: Administration of aldosterone increased ammonia in urine and Rhcg protein expression in membrane fraction of the whole kidney by 90%, respectively. In IN-IC cells, treatment with aldosterone increased expressions of Rhcg protein in membrane fraction by 220% and Sgk1 mRNA by 200%, respectively. Similarly to the endogenous Rhcg expression, aldosterone increased the expression of Flag-tagged mice Rhcg in membrane fraction. Conclusions: These results suggest that RAAS blockers cause metabolic acidosis, in part, by inhibiting the effect of aldosterone on membrane distribution of Rhcg in the intercalated cells of the collecting ducts.

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.