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Background and purposeDiabetic nephropathy is a common complications related to high morbidity and mortality in type 2 diabetes. We investigated the action of the dual modulator, PTUPB, a soluble epoxide hydrolase and cyclooxygenase-2 inhibitor against diabetic nephropathy.Experimental approachSixteen-week-old type 2 diabetic and proteinuric obese ZSF1 rats were treated with vehicle, PTUPB or enalapril for 8 weeks. Measurements were made of epoxyeicosatrienoic acids, thromboxane B2 (TBX2 ) and prostaglandin E2 (PGE2 ) in the kidney of these and lean ZSF1 rats along with their blood pressure.Key resultObese ZSF1 rats were diabetic with fivefold higher fasting blood glucose levels and markedly higher HbA1c levels compared with lean ZSF1 rats. PTUPB nor enalapril reduced fasting blood glucose or HbA1c but alleviated the development of diabetic nephropathy. In PTUPB-treated obese ZSF1 rats, glomerular nephrin expression was preserved. Enalapril also alleviated diabetic nephropathy. Diabetic renal injury in obese ZSF1 rats was accompanied by renal inflammation with six to sevenfold higher urinary MCP-1 (CCR2) level and renal infiltration of CD-68 positive cells. PTUPB and enalapril significantly reduced urinary MCP-1 levels and renal mRNA expression of cytokines. Both PTUPB and enalapril lowered blood pressure. PTUPB but not enalapril decreased hyperlipidaemia and liver injury in obese ZSF1 rats.Conclusion and implicationsOverall, the dual modulator PTUPB does not treat hyperglycaemia but can effectively alleviate hypertension, diabetic nephropathy, hyperlipidaemia and liver injury in type 2 diabetic rats. Our data further demonstrate that the renal actions of PTUPB are comparable with a current standard diabetic nephropathy treatment.

Kidney injury from antiangiogenic chemotherapy is a significant clinical challenge, and we currently lack the ability to effectively treat it with pharmacological agents. Thus, we set out to investigate whether simultaneous soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) inhibition using a dual sEH/COX-2 inhibitor PTUPB could be an effective strategy for treating antiangiogenic therapy-induced kidney damage. We used a multikinase inhibitor, sorafenib, which is known to cause serious renal side effects. The drug was administered to male Sprague-Dawley rats that were on a high-salt diet. Sorafenib was administered over the course of 56 days. The study included three experimental groups; 1) control group (naïve rats), 2) sorafenib group [rats treated with sorafenib only (20 mg/kg/day p.o.)], and 3) sorafenib + PTUPB group (rats treated with sorafenib only for the initial 28 days and subsequently coadministered PTUPB (10 mg/kg/day i.p.) from days 28 through 56). Blood pressure was measured every 2 weeks. After 28 days, sorafenib-treated rats developed hypertension (161 ± 4 mmHg). Over the remainder of the study, sorafenib treatment resulted in a further elevation in blood pressure through day 56 (200 ± 7 mmHg). PTUPB treatment attenuated the sorafenib-induced blood pressure elevation and by day 56, blood pressure was 159 ± 4 mmHg. Urine was collected every 2 weeks for biochemical analysis. After 28 days, sorafenib rats developed pronounced proteinuria (9.7 ± 0.2 P/C), which intensified significantly (35.8 ± 3.5 P/C) by the end of day 56 compared with control (2.6 ± 0.4 P/C). PTUPB mitigated sorafenib-induced proteinuria, and by day 56, it reduced proteinuria by 73%. Plasma and kidney tissues were collected on day 56. Kidney histopathology revealed intratubular cast formation, interstitial fibrosis, glomerular injury, and glomerular nephrin loss at day 56 in sorafenib-treated rats. PTUPB treatment reduced histological features by 30%-70% compared with the sorafenib-treated group and restored glomerular nephrin levels. Furthermore, PTUPB also acted on the glomerular permeability barrier by decreasing angiotensin-II-induced glomerular permeability to albumin. Finally, PTUPB improved in vitro the viability of human mesangial cells. Collectively, our data demonstrate the potential of using PTUPB or dual sEH/COX-2 inhibition as a therapeutic strategy against sorafenib-induced glomerular nephrotoxicity.

Introduction: Renal fibrosis associated with inflammation is a critical pathophysiological event in chronic kidney disease (CKD). We have developed DM509 which acts concurrently as a farnesoid X receptor agonist and a soluble epoxide hydrolase inhibitor and investigated DM509 efficacy as an interventional treatment using the unilateral ureteral obstruction (UUO) mouse model. Methods: Male mice went through either UUO or sham surgery. Interventional DM509 treatment (10mg/kg/d) was started three days after UUO induction and continued for 7 days. Plasma and kidney tissue were collected at the end of the experimental protocol. Results: UUO mice demonstrated marked renal fibrosis with higher kidney hydroxyproline content and collagen positive area. Interventional DM509 treatment reduced hydroxyproline content by 41% and collagen positive area by 65%. Renal inflammation was evident in UUO mice with elevated MCP-1, CD45-positive immune cell positive infiltration, and profibrotic inflammatory gene expression. DM509 treatment reduced renal inflammation in UUO mice. Renal fibrosis in UUO was associated with epithelial-tomesenchymal transition (EMT) and DM509 treatment reduced EMT. UUO mice also had tubular epithelial barrier injury with increased renal KIM-1, NGAL expression. DM509 reduced tubular injury markers by 25-50% and maintained tubular epithelial integrity in UUO mice. Vascular inflammation was evident in UUO mice with 9 to 20-fold higher ICAM and VCAM gene expression which was reduced by 40-50% with DM509 treatment. Peritubular vascular density was reduced by 35% in UUO mice and DM509 prevented vascular loss. Discussion: Interventional treatment with DM509 reduced renal fibrosis and inflammation in UUO mice demonstrating that DM509 is a promising drug that combats renal epithelial and vascular pathological events associated with progression of CKD. [ABSTRACT FROM AUTHOR]

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency. [ABSTRACT FROM AUTHOR]

The renal collecting duct is known to play a critical role in many physiological processes, including systemic water–electrolyte homeostasis, acid–base balance, and the salt sensitivity of blood pressure. ClC-K2 (ClC-Kb in humans) is a Cl−-permeable channel expressed on the basolateral membrane of several segments of the renal tubule, including the collecting duct intercalated cells. ClC-Kb mutations are causative for Bartters' syndrome type 3 manifested as hypotension, urinary salt wasting, and metabolic alkalosis. However, little is known about the significance of the channel in the collecting duct with respect to the normal physiology and pathology of Bartters' syndrome. In this review, we summarize the available experimental evidence about the signaling determinants of ClC-K2 function and the regulation by systemic and local factors as well as critically discuss the recent advances in understanding the collecting-duct-specific roles of ClC-K2 in adaptations to changes in dietary Cl− intake and maintaining systemic acid–base homeostasis. [ABSTRACT FROM AUTHOR]

R2738 --> 855.3 --> Epac is a relatively new direct downstream effector of cAMP. Two known Epac isoforms, Epac1 and Epac2 are abundantly expressed in the renal epithelial cells of the proximal tubule and the collecting duct. We recently demonstrated that Epac1‐/‐ and Epac2‐/‐ mice develop polyuria largely due to decreased NHE‐3 expression in the proximal tubule. In contrast, AQP2‐dependent water reabsorption in the collecting duct was unaffected. In this study, we utilized double Epac knockout mice to determine whether both isoforms contribute complementarily to the urinary production and concentrating ability. We found that Epac1&2‐/‐ produced significantly higher 24h urinary volume and lower osmolarities compared to the values in single Epac knockouts. These differences were further exacerbated in response to water deprivation test suggesting a defect in the collecting duct. Consistently, Epac1&2 blockade with ESI‐09 inhibited AQP2 translocation to the apical plasma membrane in CD cells in response to AVP. At the same time, inhibition of a single Epac isoform fails to do so. Overall, our results demonstrate that concomitant Epac1‐/‐ and Epac2‐/‐deletion compromises reabsorption in both PT and CD, thus leading to a much greater in urine volume and decrease urinary concentrating ability than the single isoform Epac knockout. [ABSTRACT FROM AUTHOR]

R3619 --> 727.15 --> The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid‐secreting A‐ and base secreting B‐type of intercalated cells cannot be easily separated in functional studies despite virtually mirror opposite localization of their transport acid‐base systems. Here, we used BCECF‐sensitive intracellular pH (pHi) measurements in split‐opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell‐specific ClC‐K2‐/‐ mice to demonstrate that ClC‐K2 inhibition enables to distinguish signals from A‐ and B‐ types of intercalated cells. We show that ClC‐K2 Cl‐ channel is expressed on the basolateral side of intercalated cells, where it governs Cl‐‐dependent H+/HCO3‐ transport. ClC‐K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC‐K2 ‐/‐ mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pHi in AE1‐positive A‐type and decreased pHi in pendrin‐positive B‐type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H+ secretion in A‐type and decreased proton transport in B‐type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Concluding, we show that inhibition of ClC‐K2 can be employed to discriminate between A‐ and B‐type of intercalated cells in split‐opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A‐ and B‐types in response to systemic acid/base stimuli. [ABSTRACT FROM AUTHOR]

The Ca 2+ -permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K + transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K + balance. We used balance metabolic cage experiments and systemic measurements with different K + feeding regimens [high (5% K + ), regular (0.9% K + ), and low (<0.01% K + )] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4 fl/fl -Pax8Cre) and their littermate controls (TRPV4 fl/fl ). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca 2+ influx. There were no differences in plasma electrolytes, urinary volume, and K + levels at baseline. In contrast, plasma K + levels were significantly elevated in TRPV4 fl/fl -Pax8Cre mice on high K + intake. K + -loaded knockout mice exhibited lower urinary K + levels than TRPV4 fl/fl mice, which was accompanied by higher aldosterone levels by day 7 . Moreover, TRPV4 fl/fl -Pax8Cre mice had more efficient renal K + conservation and higher plasma K + levels in the state of dietary K + deficiency. H + -K + -ATPase levels were significantly increased in TRPV4 fl/fl -Pax8Cre mice on a regular diet and especially on a low-K + diet, pointing to augmented K + reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H + -K + -ATPase activity, in split-opened collecting ducts from TRPV4 fl/fl -Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K + balance and urinary K + excretion during variations in dietary K + intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K + transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K + intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K + levels in both states of K + load and deficiency.

The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid-secreting A- and base secreting B-type of intercalated cells cannot be easily separated in functional studies. We used BCECF-sensitive intracellular pH (pH i ) measurements in split-opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell-specific ClC-K2 -/- mice to demonstrate that ClC-K2 inhibition enables to distinguish signals from A- and B-intercalated cells. We show that ClC-K2 Cl - channel is expressed on the basolateral side of intercalated cells, where it governs Cl - -dependent H + /HCO 3 - transport. ClC-K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC-K2 -/- mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pH i in AE1-positive A-type and decreased pH i in pendrin-positive B-type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H + secretion in A-type and decreased proton transport in B-type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Overall, we show that inhibition of ClC-K2 can be employed to discriminate between A- and B-type of intercalated cells in split-opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A- and B-type in response to systemic acid/base stimuli., (© 2022 Federation of American Societies for Experimental Biology.)