Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct.

The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.

TRPV4 expression in the renal tubule is necessary for maintaining whole body K+ homeostasis.

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.

TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium.

Mechanosensitive TRPV4 channel plays a dominant role in maintaining [Ca2+ ]i homeostasis and flow-sensitive [Ca2+ ]i signaling in the renal tubule. Polycystic kidney disease (PKD) manifests as progressive cyst growth due to cAMP-dependent fluid secretion along with deficient mechanosensitivity and impaired TRPV4 activity. Here, we tested how regulation of renal TRPV4 function by dietary K+ intake modulates the rate of cystogenesis and mechanosensitive [Ca2+ ]i signaling in cystic cells of PCK453 rats, a homologous model of human autosomal recessive PKD (ARPKD). One month treatment with both high KCl (5% K+ ) and KB/C (5% K+ with bicarbonate/citrate) diets significantly increased TRPV4 levels when compared to control (0.9% K+ ). High KCl diet caused an increased TRPV4-dependent Ca2+ influx, and partial restoration of mechanosensitivity in freshly isolated monolayers of cystic cells. Unexpectedly, high KB/C diet induced an opposite effect by reducing TRPV4 activity and worsening [Ca2+ ]i homeostasis. Importantly, high KCl diet decreased cAMP, whereas high KB/C diet further increased cAMP levels in cystic cells (assessed as AQP2 distribution). At the systemic level, high KCl diet fed PCK453 rats had significantly lower kidney-to-bodyweight ratio and reduced cystic area. These beneficial effects were negated by a concomitant administration of an orally active TRPV4 antagonist, GSK2193874, resulting in greater kidney weight, accelerated cystogenesis, and augmented renal injury. High KB/C diet also exacerbated renal manifestations of ARPKD, consistent with deficient TRPV4 activity in cystic cells. Overall, we demonstrate that TRPV4 channel activity negatively regulates cAMP levels in cystic cells thus attenuating (high activity) or accelerating (low activity) ARPKD progression.

Modus operandi of ClC-K2 Cl- Channel in the Collecting Duct Intercalated Cells.

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.

Salt-sensitive hypertension after reversal of unilateral ureteral obstruction.

The incidence of ureter obstruction is increasing and patients recovering from this kidney injury often progress to chronic kidney injury. There is evidence that a long-term consequence of recovery from ureter obstruction is an increased risk for salt-sensitive hypertension. A reversal unilateral ureteral obstruction (RUUO) model was used to study long-term kidney injury and salt-sensitive hypertension. In this model, we removed the ureteral obstruction at day 10 in mice. Mice were divided into four groups: (1) normal salt diet, (2) high salt diet, (3) RUUO normal salt diet, and (4) RUUO high salt diet. At day 10, the mice were fed a normal or high salt diet for 4 weeks. Blood pressure was measured, and urine and kidney tissue collected. There was a progressive increase in blood pressure in the RUUO high salt diet group. RUUO high salt group had decreased sodium excretion and glomerular injury. Renal epithelial cell injury was evident in RUUO normal and high salt mice as assessed by neutrophil gelatinase-associated lipocalin (NGAL). Kidney inflammation in the RUUO high salt group involved an increase in F4/80 positive macrophages; however, CD3+ positive T cells were not changed. Importantly, RUUO normal and high salt mice had decreased vascular density. RUUO was also associated with renal fibrosis that was further elevated in RUUO mice fed a high salt diet. Overall, these findings demonstrate long-term renal tubular injury, inflammation, decreased vascular density, and renal fibrosis following reversal of unilateral ureter obstruction that could contribute to impaired sodium excretion and salt-sensitive hypertension.

Dual soluble epoxide hydrolase inhibitor - farnesoid X receptor agonist interventional treatment attenuates renal inflammation and fibrosis.

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-to-mesenchymal 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.

Evolving concepts of TRPV4 in controlling flow-sensitivity of the renal nephron.

Kidneys are central for whole body water and electrolyte balance by first filtering plasma at the glomeruli and then processing the filtrate along the renal nephron until the final urine is produced. Renal nephron epithelial cells mediate transport of water and solutes which is under the control of systemic hormones as well as local mechanical stimuli arising from alterations in fluid flow. TRPV4 is a mechanosensitive Ca2+ channel abundantly expressed in different segments of the renal nephron. The accumulated evidence suggests a critical role for TRPV4 in sensing variations in flow rates. In turn, TRPV4 activation triggers numerous downstream cellular responses stimulated by elevated intracellular Ca2+ concentrations [Ca2+]i. In this review, we discuss the recent concepts in flow-mediated regulation of solute homeostasis by TRPV4 in different segments of renal nephron. Specifically, we summarize the evidence for TRPV4 involvement in endocytosis-mediated albumin uptake in the proximal tubule, reactive oxygen species (ROS) generation in the ascending loop of Henle, and maintaining K+ homeostasis in the connecting tubule/collecting duct. Finally, we outline the function and significance of TRPV4 in the setting of polycystic kidney disease.

ClC-K2 Cl- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

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 (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-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. 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.

Epac1-/- and Epac2-/- mice exhibit deficient epithelial Na+ channel regulation and impaired urinary Na+ conservation.

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1-/- and Epac2-/- mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.

Dual sEH/COX-2 Inhibition Using PTUPB-A Promising Approach to Antiangiogenesis-Induced Nephrotoxicity.

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.

Multitarget molecule, PTUPB, to treat diabetic nephropathy in rats.

BACKGROUND AND PURPOSE: Diabetic 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 APPROACH: Sixteen-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 RESULT: Obese 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 IMPLICATIONS: Overall, 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.

Dual soluble epoxide hydrolase inhibitor/PPAR-γ agonist attenuates renal fibrosis.

Renal fibrosis is a contributor to chronic kidney disease and an important predictor of long-term prognosis. We developed a dual soluble epoxide hydrolase inhibitor-PPAR-γ agonist (sEHi/PPAR-γ), RB394, and investigated its ability to attenuate renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. RB394 efficacy was compared to an sEH inhibitor (sEHi), a PPAR-γ agonist rosiglitazone (Rosi), or their combination (sEHi + Rosi). All interventional treatments were administrated in drinking water 3 days after UUO induction surgery and continued for 7 days. UUO mice developed renal fibrosis with higher collagen formation and RB394 significantly attenuated fibrosis (P < 0.05). Renal expression of α-smooth muscle actin (α-SMA) was elevated in UUO mice and all treatments except sEHi significantly attenuated renal α-SMA expression. Renal mRNA expression fibrotic and fibrosis regulators were higher in UUO mice and RB394 and sEHi + Rosi treatments attenuated their expression. Renal inflammation was evident in UUO mice with increased infiltration of CD45 and F4/80 positive cells. RB394 and sEHi + Rosi treatments attenuated renal inflammation in UUO mice. UUO mice had renal tubular and vascular injury. Renal tubular and vascular injuries were attenuated to a greater extent by RB394 and sEHi + Rosi than sEHi or Rosi treatment alone. Renal mRNA expression of oxidative stress markers were significantly higher in UUO mice (P < 0.05). RB394 and sEHi + Rosi attenuated expression of oxidative stress markers to a greater extent than other interventional treatments (P < 0.05). These findings demonstrate that RB394 can attenuate renal fibrosis by reducing renal inflammation, oxidative stress, tubular injury, and vascular injury. In conclusion, RB394 demonstrates exciting potential as a therapeutic for renal fibrosis and chronic kidney disease.

Epoxyeicosatrienoic Acid Analog EET-A Blunts Development of Lupus Nephritis in Mice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disorder that causes life threatening renal disease and current therapies are limited with serious side-effects. CYP epoxygenase metabolites of arachidonic acid epoxyeicosatrienoic acids (EETs) demonstrate strong anti-inflammatory and kidney protective actions. We investigated the ability of an orally active EET analog, EET-A to prevent kidney injury in a mouse SLE model. Twenty-weeks old female NZBWF1 (SLE) and age-matched NZW/LacJ (Non SLE) were treated with vehicle or EET-A (10 mg/kg/d, p.o.) for 14 weeks and urine and kidney tissues were collected at the end of the protocol. SLE mice demonstrated marked renal chemotaxis with 30-60% higher renal mRNA expression of CXC chemokine receptors (CXCR) and CXC chemokines (CXCL) compared to Non SLE mice. In SLE mice, the elevated chemotaxis is associated with 5-15-fold increase in cytokine mRNA expression and elevated inflammatory cell infiltration in the kidney. SLE mice also had elevated BUN, serum creatinine, proteinuria, and renal fibrosis. Interestingly, EET-A treatment markedly diminished renal CXCR and CXCL renal mRNA expression in SLE mice. EET-A treatment also reduced renal TNF-α, IL-6, IL-1ß, and IFN-γ mRNA expression by 70-80% in SLE mice. Along with reductions in renal chemokine and cytokine mRNA expression, EET-A reduced renal immune cell infiltration, BUN, serum creatinine, proteinuria and renal fibrosis in SLE mice. Overall, we demonstrate that an orally active EET analog, EET-A prevents renal injury in a mouse model of SLE by reducing inflammation.

A dual farnesoid X receptor/soluble epoxide hydrolase modulator treats non-alcoholic steatohepatitis in mice.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are the most prevalent metabolic liver disorders and a serious global health burden. NAFLD/NASH pathogenesis and progression are highly multi-factorial and likely demand a combination of multiple mechanisms to provide a more effective treatment. We have developed a dual farnesoid X receptor agonist (FXRA)/soluble epoxide hydrolase inhibitor (sEHi) to simultaneously address two validated and complementary modes of action in NASH treatment. Here we report the in vivo profiling for this FXRA/sEHi in toxin- and diet-induced rodent NASH models. In streptozotocin-induced NASH as a proof-of-concept study, the experimental FXRA/sEHi drug robustly prevented hepatic steatosis and fibrosis, and improved lipid homeostasis as well as biochemical markers of liver health. In methionine-choline-deficient high-fat diet-induced NASH, FXRA/sEHi treatment reduced hepatic steatosis and fibrosis to levels similar to healthy animals and demonstrated anti-inflammatory activity confirming that dual FXRA/sEHi modulation produces a triad of complementary anti-NASH effects. Our results validate dual FXRA/sEHi modulation as an effective therapeutic strategy to treat NASH and advocates for a combinational drug therapeutic approach for multifactorial liver diseases.