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Serine proteases (SPs) play physiological roles in the kidney. We previously reported that a synthetic SP inhibitor, camostat mesilate (CM), suppressed sodium reabsorption in the renal tubule and showed natriuretic effects in aldosterone-infused rats. Here, we aimed to explore novel physiological roles of SPs in the renal tubule and understand the mechanism of actions of SP inhibitors, by administering CM to healthy rats. Sprague–Dawley rats were classified into control and CM (subcutaneous sustained-release pellet) groups and sacrificed on day 7. CM significantly increased urine volumes by approximately two-fold in a urinary sodium- and osmolyte excretion-independent manner, indicating the occurrence of free water excretion. Serum vasopressin, potassium, and calcium levels and the osmolality in the renal medulla, which all affect free water reabsorption in the renal tubule, remained unchanged after CM administration. CM decreased urinary exosomal AQP2 excretion, suggesting suppression of AQP2 activity in the collecting duct. These changes were reversed by desmopressin infusion. Water diuresis caused by CM was independent of its action on prostasin or TMPRSS4. Our results revealed the association of SP inhibition with free water handling and demonstrated that CM administration exerted diuretic effects with AQP2 downregulation, suggesting SP inhibitors as a new class of aquaretic drugs.

Metabolic syndrome (MetS) is associated with chronic kidney disease and proteinuria. Previously, we reported that a synthetic serine protease inhibitor, camostat mesilate (CM), mitigated hypertension and proteinuria in rodent disease models. The present study evaluated the anti-hypertensive and anti-proteinuric effects of CM in MetS model rats (SHR/ND mcr-cp). Rats were divided into normal salt-fed (NS), high salt-fed (HS), HS and CM-treated (CM), and HS and hydralazine-treated (Hyd) groups. Rats were sacrificed after four weeks of treatment. Severe hypertension and proteinuria were observed in the HS group. Although CM and Hyd equally alleviated hypertension, CM suppressed proteinuria and glomerular sclerosis more efficiently than Hyd. The HS group revealed a decrease in podocyte number and podocyte-specific molecules, together with an increase in glomerular apoptotic cells and apoptosis-related proteins in the kidney. These changes were significantly attenuated by CM, but not by Hyd. Furthermore, CM ameliorated the apoptotic signals in murine cultured podocytes stimulated with the high glucose and aldosterone medium. In conclusion, CM could exert renoprotective effects in MetS model rats, together with the inhibition of podocyte apoptosis. Our study suggests that serine protease inhibition may become a new therapeutic strategy against MetS-related hypertension and renal injuries.

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.

ObjectivesOwing to recent changes in our understanding of the underlying cause of chronic kidney disease (CKD), the importance of lifestyle modification for preventing the progression of kidney dysfunction and complications has become obvious. In addition, effective cooperation between general physicians (GPs) and nephrologists is essential to ensure a better care system for CKD treatment. In this cluster-randomized study, we studied the effect of behavior modification on the outcome of early- to moderate-stage CKD.DesignStratified open cluster-randomized trial.SettingA total of 489 GPs belonging to 49 local medical associations (clusters) in Japan.ParticipantsA total of 2,379 patients (1,195 in group A (standard intervention) and 1,184 in group B (advanced intervention)) aged between 40 and 74 years, who had CKD and were under consultation with GPs.InterventionAll patients were managed in accordance with the current CKD guidelines. The group B clusters received three additional interventions: patients received both educational intervention for lifestyle modification and a CKD status letter, attempting to prevent their withdrawal from treatment, and the group B GPs received data sheets to facilitate reducing the gap between target and practice.Main outcome measureThe primary outcome measures were 1) the non-adherence rate of accepting continuous medical follow-up of the patients, 2) the collaboration rate between GPs and nephrologists, and 3) the progression of CKD.ResultsThe rate of discontinuous clinical visits was significantly lower in group B (16.2% in group A vs. 11.5% in group B, p = 0.01). Significantly higher referral and co-treatment rates were observed in group B (pConclusionOur care system achieved behavior modification of CKD patients, namely, significantly lower discontinuous clinical visits, and behavior modification of both GPs and nephrologists, namely significantly higher referral and co-treatment rates, resulting in the retardation of CKD progression, especially in patients with proteinuric Stage 3 CKD.Trial registrationThe University Hospital Medical Information Network clinical trials registry UMIN000001159.

Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker.We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria.The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCA-negative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and CRP. After glucocorticoid treatment, thrombinuria in patients with CresGN rapidly disappeared but proteinuria and hematuria persisted.Thrombinuria was specific for glomerular inflammation, was unaffected by systemic inflammation or coagulation, and demonstrated good diagnostic accuracy for CresGN including ANCA-negative cases. Thrombinuria measurement may provide risk-free diagnosis and screening for CresGN.

Atrial natriuretic peptide (ANP) is known to influence NaCl transport in the medullary thick ascending limbs (MAL), where the largest NaCl reabsorption occurs among distal nephron segments in response to arginine vasopressin (AVP). In the present study, we investigated the effect of ANP on bicarbonate (HCO3 (-)) transport in the MAL using an isolated tubule perfusion technique. The HCO3 (-) concentration was measured using free-flow ultramicro-fluorometer. We first observed basal HCO3 (-) reabsorption in both long- and short-looped MALs (lMALs, and sMALs, respectively). AVP inhibited HCO3 (-) reabsorption in both lMALs and sMALs, whereas ANP did not change HCO3 (-) transport. However, in the presence of AVP, ANP restored the HCO3 (-) reabsorption inhibited by AVP both in lMAL and sMAL. The effects of ANP on HCO3 (-) transport was mimicked by cyclic GMP. The mRNA expression level of the vasopressin V2 receptor in lMALs was significantly higher than in sMALs, whereas expression of the V1a receptor was unchanged. In summary, AVP inhibits HCO3 (-) transport, and ANP counteracts the action of AVP on HCO3 (-) transport both in lMALs and sMALs.