Kir7.1 knockdown and inhibition alter renal electrolyte handling but not the development of hypertension in Dahl salt-sensitive rats.

High K+ supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K+ (Kir) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. Kir7.1 is a member of the ATP-regulated subfamily of Kir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of Kir7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of Kir7.1, we generated a knockout of Kir7.1 (Kcnj13) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific Kir7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 (Kcnj13-/-) resulted in embryonic lethality. Heterozygous Kcnj13+/- rats revealed an increase in K+ excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal Kir7.1 expression when dietary K+ was increased. K+ supplementation also demonstrated that Kcnj13+/- rats excreted more K+ on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13+/- rats excrete less Na+. Interestingly, chronic infusion of ML418 significantly increased Na+ and Cl- excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of Kir7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension.NEW & NOTEWORTHY To investigate the role of the Kir7.1 channel in salt-sensitive hypertension, its function was examined using complementary genetic and pharmacological approaches. The results revealed that although reducing Kir7.1 expression had some impact on maintaining K+ and Na+ balance, it did not lead to a significant change in the development or magnitude of salt-induced hypertension. Hence, it is probable that Kir7.1 works in conjunction with other basolateral K+ channels to fine-tune membrane potential.

ß-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the ß-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated ß-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine ß-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated ß-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of ß-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the ß-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the ß-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated ß-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.

SGLT2 inhibition effect on salt-induced hypertension, RAAS, and Na+ transport in Dahl SS rats.

Na+-glucose cotransporter-2 (SGLT2) inhibitors are the new mainstay of treatment for diabetes mellitus and cardiovascular diseases. Despite the remarkable benefits, the molecular mechanisms mediating the effects of SGLT2 inhibitors on water and electrolyte balance are incompletely understood. The goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function via affecting the renin-angiotensin-aldosterone system (RAAS) and Na+ channels/transporters along the nephron in Dahl salt-sensitive rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 wk blunted the development of salt-induced hypertension as evidenced by lower blood pressure and a left shift of the pressure natriuresis curve. Urinary flow rate, glucose excretion, and Na+- and Cl--to-creatinine ratios increased in Dapa-treated compared with vehicle-treated rats. To define the contribution of the RAAS, we measured various hormones. Despite apparent effects on Na+- and Cl--to-creatinine ratios, Dapa treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of Dapa on renal Na+ channels and transporters using RT-PCR, Western blot analysis, and patch clamp. Neither mRNA nor protein expression levels of renal transporters (SGLT2, Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter 2, Na+-Cl- cotransporter, and α-, ß-, and γ-epithelial Na+ channel subunits) changed significantly between groups. Furthermore, electrophysiological experiments did not reveal any difference in Dapa treatment on the conductance and activity of epithelial Na+ channels. Our data suggest that SGLT2 inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the RAAS or the expression or activity of the main Na+ channels and transporters.NEW & NOTEWORTHY The present study indicates that Na+-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na+ excretion without secondary effects on the expression and function of other Na+ transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

Astrocytic responses to high glucose impair barrier formation in cerebral microvessel endothelial cells.

Hyperglycemic conditions are prodromal to blood-brain barrier (BBB) impairment. The BBB comprises cerebral microvessel endothelial cells (CMECs) that are surrounded by astrocytic foot processes. Astrocytes express high levels of gap junction connexin 43 (Cx43), which play an important role in autocrine and paracrine signaling interactions that mediate gliovascular cross talk through secreted products. One of the key factors of the astrocytic "secretome" is vascular endothelial growth factor (VEGF), a potent angiogenic factor that can disrupt BBB integrity. We hypothesize that high-glucose conditions change the astrocytic expression of Cx43 and increase VEGF secretion leading to impairment of CMEC barrier properties in vitro and in vivo. Using coculture of neonatal rat astrocytes and CMEC, we mimic hyperglycemic conditions using high-glucose (HG) feeding media and show a significant decrease in Cx43 expression and the corresponding increase in secreted VEGF. This result was confirmed by the analyses of Cx43 and VEGF protein levels in the brain cortex samples from the type 2 diabetic rat (T2DN). To further characterize inducible changes in BBB, we measured transendothelial cell electrical resistance (TEER) and tight junction protein levels in cocultured conditioned astrocytes with isolated rat CMEC. The coculture monolayer's integrity and permeability were significantly compromised by HG media exposure, which was indicated by decreased TEER without a change in tight junction protein levels in CMEC. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross talk between astrocytes and CMEC, which could be one explanation for cerebral BBB disruption in diabetic conditions.

Sexual dimorphism in the progression of type 2 diabetic kidney disease in T2DN rats.

Diabetic kidney disease (DKD) is a common complication of diabetes, which frequently leads to end-stage renal failure and increases cardiovascular disease risk. Hyperglycemia promotes renal pathologies such as glomerulosclerosis, tubular hypertrophy, microalbuminuria, and a decline in glomerular filtration rate. Importantly, recent clinical data have demonstrated distinct sexual dimorphism in the pathogenesis of DKD in people with diabetes, which impacts both severity- and age-related risk factors. This study aimed to define sexual dimorphism and renal function in a nonobese type 2 diabetes model with the spontaneous development of advanced diabetic nephropathy (T2DN rats). T2DN rats at 12- and over 48-wk old were used to define disease progression and kidney injury development. We found impaired glucose tolerance and glomerular hyperfiltration in T2DN rats to compare with nondiabetic Wistar control. The T2DN rat displays a significant sexual dimorphism in insulin resistance, plasma cholesterol, renal and glomerular injury, urinary nephrin shedding, and albumin handling. Our results indicate that both male and female T2DN rats developed nonobese type 2 DKD phenotype, where the females had significant protection from the development of severe forms of DKD. Our findings provide further evidence for the T2DN rat strain's effectiveness for studying the multiple facets of DKD.

NOX4-dependent regulation of ENaC in hypertension and diabetic kidney disease.

NADPH oxidase 4 (NOX4) is the most abundant NOX isoform in the kidney; however, its importance for renal function has only recently emerged. The NOX4-dependent pathway regulates many factors essential for proper sodium handling in the distal nephron. However, the functional significance of this pathway in the control of sodium reabsorption during the initiation of chronic kidney disease is not established. The goal of this study was to test Nox4-dependent ENaC regulation in two models: SS hypertension and STZ-induced type 1 diabetes. First, we showed that genetic ablation of Nox4 in Dahl salt-sensitive (SS) rat attenuated a high-salt (HS)-induced increase in epithelial Na+ channel (ENaC) activity in the cortical collecting duct. We also found that H2 O2 upregulated ENaC activity, and H2 O2 production was reduced in both the renal cortex and medulla in SSNox4-/- rats fed an HS diet. Second, in the streptozotocin model of hyperglycemia-induced renal injury ENaC activity in hyperglycemic animals was elevated in SS but not SSNox4-/- rats. NaCl cotransporter (NCC) expression was increased compared to healthy controls, while expression values between SS and SSNox4-/- groups were similar. These data emphasize a critical contribution of the NOX4-mediated pathway in maladaptive upregulation of ENaC-mediated sodium reabsorption in the distal nephron in the conditions of HS- and hyperglycemia-induced kidney injury.

Behavioral, metabolic, and renal outcomes of 1-month isolation in adolescent male Dahl salt-sensitive rats.

Social contact deficit is considered a stressful circumstance associated with various neural, hormonal, genetic, immune, and behavioral effects. A growing body of clinical and basic science evidence suggests that social isolation is linked to a higher risk of various neurological, cardiovascular, and metabolic diseases, including hypertension, diabetes mellitus, and obesity. However, the impact of the deficit of social interaction on kidney function is not well established. The Dahl salt-sensitive (SS) rat is a classical model of salt-induced hypertension and associated kidney injury. In this study, we investigated the effect of 30 days of social isolation (SI) on blood and urine electrolytes and metabolic, physiological, and behavioral parameters in adolescent male Dahl SS rats fed a normal 0.4% NaCl diet. SI rats demonstrated increased behavioral excitability compared with rats kept in groups. We also observed increased food consumption and a decrease in plasma leptin levels in the SI group without differences in water intake and weight gain compared with grouped animals. No changes in the level of blood and urine electrolytes, 24-h urine output, creatinine clearance, and albumin/creatinine ratio were identified between the SI and grouped rats. These findings indicate that 30 days of social isolation of adolescent Dahl SS rats affects metabolic parameters but has no apparent influence on kidney function.

Genetic mutation of Kcnj16 identifies Kir5.1-containing channels as key regulators of acute and chronic pH homeostasis.

Acute and chronic homeostatic pH regulation is critical for the maintenance of optimal cellular function. Renal mechanisms dominate global pH regulation over longer time frames, and rapid adjustments in ventilation compensate for acute pH and CO2 changes. Ventilatory CO2 and pH chemoreflexes are primarily determined by brain chemoreceptors with intrinsic pH sensitivity likely driven by K+ channels. Here, we studied acute and chronic pH regulation in Kcnj16 mutant Dahl salt-sensitive (SS Kcnj16-/-) rats; Kcnj16 encodes the pH-sensitive inwardly rectifying K+ 5.1 (Kir5.1) channel. SS Kcnj16-/- rats hyperventilated at rest, likely compensating for a chronic metabolic acidosis. Despite their resting hyperventilation, SS Kcnj16-/- rats showed up to 45% reduction in the ventilatory response to graded hypercapnic acidosis vs. controls. SS Kcnj16-/- rats chronically treated with bicarbonate or the carbonic anhydrase inhibitor hydrochlorothiazide had partial restoration of arterial pH, but there was a further reduction in the ventilatory response to hypercapnic acidosis. SS Kcnj16-/- rats also had a nearly absent hypoxic ventilatory response, suggesting major contributions of Kir5.1 to O2- and CO2-dependent chemoreflexes. Although previous studies demonstrated beneficial effects of a high-K+ diet (HKD) on cardiorenal phenotypes in SS Kcnj16-/- rats, HKD failed to restore the observed ventilatory phenotypes. We conclude that Kir5.1 is a key regulator of renal H+ handling and essential for acute and chronic regulation of arterial pH as determinants of the ventilatory CO2 chemoreflex.-Puissant, M. M., Muere, C., Levchenko, V., Manis, A. D., Martino, P., Forster, H. V., Palygin, O., Staruschenko, A., Hodges, M. R. Genetic mutation of Kcnj16 identifies Kir5.1-containing channels as key regulators of acute and chronic pH homeostasis.

Progression of diabetic kidney disease in T2DN rats.

Diabetic kidney disease (DKD) is one of the leading pathological causes of decreased renal function and progression to end-stage kidney failure. To explore and characterize age-related changes in DKD and associated glomerular damage, we used a rat model of type 2 diabetic nephropathy (T2DN) at 12 wk and older than 48 wk. We compared their disease progression with control nondiabetic Wistar and diabetic Goto-Kakizaki (GK) rats. During the early stages of DKD, T2DN and GK animals revealed significant increases in blood glucose and kidney-to-body weight ratio. Both diabetic groups had significantly altered renin-angiotensin-aldosterone system function. Thereafter, during the later stages of disease progression, T2DN rats demonstrated a remarkable increase in renal damage compared with GK and Wistar rats, as indicated by renal hypertrophy, polyuria accompanied by a decrease in urine osmolarity, high cholesterol, a significant prevalence of medullary protein casts, and severe forms of glomerular injury. Urinary nephrin shedding indicated loss of the glomerular slit diaphragm, which also correlates with the dramatic elevation in albuminuria and loss of podocin staining in aged T2DN rats. Furthermore, we used scanning ion microscopy topographical analyses to detect and quantify the pathological remodeling in podocyte foot projections of isolated glomeruli from T2DN animals. In summary, T2DN rats developed renal and physiological abnormalities similar to clinical observations in human patients with DKD, including progressive glomerular damage and a significant decrease in renin-angiotensin-aldosterone system plasma levels, indicating these rats are an excellent model for studying the progression of renal damage in type 2 DKD.

Vibrodissociation method for isolation of defined nephron segments from human and rodent kidneys.

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease.

Relationship between the renin-angiotensin-aldosterone system and renal Kir5.1 channels.

Kir5.1 (encoded by the Kcnj16 gene) is an inwardly rectifying K+ (Kir) channel highly expressed in the aldosterone-sensitive distal nephron of the kidney, where it forms a functional channel with Kir4.1. Kir4.1/Kir5.1 channels are responsible for setting the transepithelial voltage in the distal nephron and collecting ducts and are thereby major determinants of fluid and electrolyte distribution. These channels contribute to renal blood pressure control and have been implicated in salt-sensitive hypertension. However, mechanisms pertaining to the impact of K ir4.1/Kir5.1-mediated K+ transport on the renin-angiotensin-aldosterone system (RAAS) remain unclear. Herein, we utilized a knockout of Kcnj16 in the Dahl salt-sensitive rat (SSKcnj16-/-) to investigate the relationship between Kir5.1 and RAAS balance and function in the sensitivity of blood pressure to the dietary Na+/K+ ratio. The knockout of Kcnj16 caused substantial elevations in plasma RAAS hormones (aldosterone and angiotensin peptides) and altered the RAAS response to changing the dietary Na+/K+ ratio. Blocking aldosterone with spironolactone caused rapid mortality in SSKcnj16-/- rats. Supplementation of the diet with high K+ was protective against mortality resulting from aldosterone-mediated mechanisms. Captopril and losartan treatment had no effect on the survival of SSKcnj16-/- rats. However, neither of these drugs prevented mortality of SSKcnj16-/- rats when switched to high Na+ diet. These studies revealed that the knockout of Kcnj16 markedly altered RAAS regulation and function, suggesting Kir5.1 as a key regulator of the RAAS, particularly when exposed to changes in dietary sodium and potassium content.

Increased ENaC activity during kidney preservation in Wisconsin solution.

BACKGROUND: The invention of an effective kidney preservation solution capable of prolonging harvested kidney viability is the core of kidney transplantation procedure. Researchers have been working on upgrading the preservation solution quality aiming at prolonging storage time while maintaining utmost organ viability and functionality. For many years, the University of Wisconsin (UW) solution has been considered the gold standard solution for kidney preservation. However, the lifespan of kidney preservation in the UW solution is still limited. Its impact on the epithelial Na+ channel (ENaC) activity and its mediated processes is unknown and the primary goal of this study. METHODS: Kidneys harvested from 8 weeks old Sprague Dawley rats were divided into 4 groups depending upon the period of preservation in UW solution. Additional analysis was performed using dogs' kidneys. ENaC activity was measured using patch clamp technique; protein expression and mRNA transcription were tested through Western blot and RT-qPCR, respectively. A colorimetric LDH level estimation was performed at different time points during UW solution preservation. RESULTS: Kidney preservation in Wisconsin solution caused reduction of the kidney size and weight and elevation of LDH level. ENaC activity increased in both rat and dog kidneys preserved in the UW solution as assessed by patch clamp analysis. On the contrary, ENaC channel mRNA levels remained unchanged. CONCLUSIONS: ENaC activity is significantly elevated in the kidneys during preservation in UW solution, which might affect the immediate post-implantation allograft function and trajectory post-transplant.

A NOX4/TRPC6 Pathway in Podocyte Calcium Regulation and Renal Damage in Diabetic Kidney Disease.

Background Loss of glomerular podocytes is an indicator of diabetic kidney disease (DKD). The damage to these cells has been attributed in part to elevated intrarenal oxidative stress. The primary source of the renal reactive oxygen species, particularly H2O2, is NADPH oxidase 4 (NOX4). We hypothesized that NOX4-derived H2O2 contributes to podocyte damage in DKD via elevation of podocyte calcium.Methods We used Dahl salt-sensitive (SS) rats with a null mutation for the Nox4 gene (SSNox4-/-) and mice with knockout of the nonselective calcium channel TRPC6 or double knockout of TRPC5 and TRPC6. We performed whole animal studies and used biosensor measurements, electron microscopy, electrophysiology, and live calcium imaging experiments to evaluate the contribution of this pathway to the physiology of the podocytes in freshly isolated glomeruli.Results Upon induction of type 1 diabetes with streptozotocin, SSNox4-/- rats exhibited significantly lower basal intracellular Ca2+ levels in podocytes and less DKD-associated damage than SS rats did. Furthermore, the angiotensin II-elicited calcium flux was blunted in glomeruli isolated from diabetic SSNox4-/- rats compared with that in glomeruli from diabetic SS rats. H2O2 stimulated TRPC-dependent calcium influx in podocytes from wild-type mice, but this influx was blunted in podocytes from Trpc6-knockout mice and, in a similar manner, in podocytes from Trpc5/6 double-knockout mice. Finally, electron microscopy revealed that podocytes of glomeruli isolated from Trpc6-knockout or Trpc5/6 double-knockout mice were protected from damage induced by H2O2 to the same extent.Conclusions These data reveal a novel signaling mechanism involving NOX4 and TRPC6 in podocytes that could be pharmacologically targeted to abate the development of DKD.

Protective role of Trpc6 knockout in the progression of diabetic kidney disease.

Diabetic kidney disease (DKD) is a chronic kidney pathology that leads to end-stage renal disease. Previous studies from our laboratory indicate that there is an association between the development of DKD and the transient receptor potential canonical 6 (TRPC6) channel. Trpc6 expression and activity were increased in the streptozotocin (STZ)-treated Dahl Salt-sensitive (Dahl SS) rat, an established model of type 1 diabetes. Here, using a Trpc6 knockout created on the Dahl SS rat background (SSTrpc6-/-), we test the hypothesis that the absence of Trpc6 will protect podocytes and kidney function during the development of DKD. Four groups of animals (control SSWT, SSTrpc6-/-, STZ-treated SSWT, and STZ-SSTrpc6-/-) were utilized in this study. Diabetes development was monitored for 11 wk after STZ injection with periodic weight, glucose, and urinary output measurements. There was an increase in albuminuria and glomerular injury following STZ treatment, which was not different between Dahl SS and SSTrpc6-/- groups. Western blot analysis revealed elevated levels of nephrin in urine samples of STZ-SSWT rats, which was higher compared with STZ-SSTrpc6-/- rats. Furthermore, pathological increases in basal [Ca2+]i levels and foot process damage of podocytes during the development of DKD was attenuated in the STZ-SSTrpc6-/- compared with STZ-SSWT rats. Overall, our data indicate that TRPC6 channel inhibition may have at least partial renoprotective effects, which could lead to the development of new pharmacological tools to treat or prevent the progression of DKD.

Nitric oxide production by glomerular podocytes.

Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration. However, whether changes in NO occur in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. We showed here that podocytes produce NO, and further hypothesized that hypertensive animals would exhibit reduced NO production in these cells in response to various paracrine factors, which might contribute to the damage of glomeruli filtration barrier and development of proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed a low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks) diets and loaded podocytes with either a combination of NO and Ca2+ fluorophores (DAF-FM and Fura Red, respectively) or DAF-FM alone. Changes in fluorescence were observed with confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and hydrogen peroxide (H2O2). Application of Ang II resulted in activation of both NO and intracellular calcium ([Ca2+]i) transients. In contrast, ATP promoted [Ca2+]i transients, but did not have any effects on NO production. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production: the response to Ang II or H2O2 in podocytes of glomeruli isolated from SS rats fed a HS diet was significantly reduced compared to rats fed a LS diet. Therefore, glomerular podocytes from hypertensive rats showed a diminished NO release in response to Ang II or oxidative stress, suggesting that podocytic NO signaling is dysfunctional in this condition and likely contributes to the development of kidney injury.

Characterization of purinergic receptor expression in ARPKD cystic epithelia.

Polycystic kidney diseases (PKDs) are a group of inherited nephropathies marked by formation of fluid-filled cysts along the nephron. Growing evidence suggests that in the kidney formation of cysts and alteration of cystic electrolyte transport are associated with purinergic signaling. PCK/CrljCrl-Pkhd1pck/CRL (PCK) rat, an established model of autosomal recessive polycystic kidney disease (ARPKD), was used here to test this hypothesis. Cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley rat kidney cortical interstitium as assessed by highly sensitive ATP enzymatic biosensors. Confocal calcium imaging of the freshly isolated cystic monolayers revealed a stronger response to ATP in a higher range of concentrations (above 100 µM). The removal of extracellular calcium results in the profound reduction of the ATP evoked transient, which suggests calcium entry into the cyst-lining cells is occurring via the extracellular (ionotropic) P2X channels. Further use of pharmacological agents (α,ß-methylene-ATP, 5-BDBD, NF449, isoPPADS, AZ10606120) and immunofluorescent labeling of isolated cystic epithelia allowed us to narrow down potential candidate receptors. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is shifted in ARPKD cystic epithelia in an age-related manner towards prevalence of P2X4 and/or P2X7 receptors, which opens new avenues for the treatment of this disease.

Role of Rho GDP dissociation inhibitor α in control of epithelial sodium channel (ENaC)-mediated sodium reabsorption.

The epithelial sodium channel (ENaC) is expressed in the aldosterone-sensitive distal nephron where it performs sodium reabsorption from the lumen. We have recently shown that ENaC activity contributes to the development of salt-induced hypertension as a result of deficiency of EGF level. Previous studies revealed that Rho GDP-dissociation inhibitor α (RhoGDIα) is involved in the control of salt-sensitive hypertension and renal injury via Rac1, which is one of the small GTPases activating ENaC. Here we investigated the intracellular mechanism mediating the involvement of the RhoGDIα/Rac1 axis in the control of ENaC and the effect of EGF on ENaC in this pathway. We demonstrated that RhoGDIα is highly expressed in the cortical collecting ducts of mice and rats, and its expression is down-regulated in Dahl salt-sensitive rats fed a high salt diet. Knockdown of RhoGDIα in cultured cortical collecting duct principal cells increased ENaC subunits expression and ENaC-mediated sodium reabsorption. Furthermore, RhoGDIα deficiency causes enhanced response to EGF treatment. Patch clamp analysis reveals that RhoGDIα significantly decreases ENaC current density and prevents its up-regulation by RhoA and Rac1. Inhibition of Rho kinase with Y27632 had no effects on ENaC response to EGF either in control or RhoGDIα knocked down cells. However, EGF treatment increased levels of active Rac1, which was further enhanced in RhoGDIα-deficient cells. We conclude that changes in the RhoGDIα-dependent pathway have a permissive role in the Rac1-mediated enhancement of ENaC activity observed in salt-induced hypertension.

Cross-talk between insulin and IGF-1 receptors in the cortical collecting duct principal cells: implication for ENaC-mediated Na+ reabsorption.

Insulin and IGF-1 are recognized as powerful regulators of the epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron. As previously described, these hormones both acutely increase ENaC activity in freshly isolated split open tubules and cultured principal cortical collecting duct cells. The present study was aimed at differentiating the effects of insulin and IGF-1 on Na+ transport in immortalized mpkCCDcl4 cells and defining their interrelations. We have shown that both insulin and IGF-1 applied basolaterally, but not apically, enhanced transepithelial Na+ transport in the mpkCCDcl4 cell line with EC50 values of 8.8 and 14.5 nM, respectively. Insulin treatment evoked phosphorylation of both insulin and IGF-1 receptors, whereas the effects of IGF-1 were more profound on its own receptor rather than the insulin receptor. AG-1024 and PPP, inhibitors of IGF-1 and insulin receptor tyrosine kinase activity, diminished insulin- and IGF-1-stimulated Na+ transport in mpkCCDcl4 cells. The effects of insulin and IGF-1 on ENaC-mediated currents were found to be additive, with insulin likely stimulating both IGF-1 and insulin receptors. We hypothesize that insulin activates IGF-1 receptors in addition to its own receptors, making the effects of these hormones interconnected.

Impaired epithelial Na+ channel activity contributes to cystogenesis and development of autosomal recessive polycystic kidney disease in PCK rats.

BACKGROUND: Autosomal recessive polycystic kidney disease is a genetic disorder characterized by the development of renal cysts of tubular epithelial cell origin. Epithelial Na(+) channel (ENaC) is responsible for sodium reabsorption in the aldosterone-sensitive distal nephron. Here, we investigated the ENaC expression and activity in cystic tissue taken from rats with autosomal recessive polycystic kidney disease. METHODS: Polycystic kidney (PCK) rats were treated with the selective ENaC inhibitor benzamil given in the drinking water, and after 4 or 12 wk, the severity of morphological malformations in the kidneys was assessed. ENaC and aquaporin-2 expression and ENaC activity were tested with immunohistochemistry and patch-clamp electrophysiology, respectively. RESULTS: Treatment with benzamil exacerbated development of cysts compared with the vehicle-treated animals. In contrast, the 12 wk of treatment with the loop diuretic furosemide had no effect on cystogenesis. Single-channel patch-clamp analysis revealed that ENaC activity in the freshly isolated cystic epithelium was significantly lower than that in the noncystic collecting ducts isolated from PCK or normal Sprague-Dawley rats. Immunohistochemical analysis confirmed that ß-ENaC and aquaporin-2 expressions in cysts are decreased compared with nondilated tubules from PCK rat kidneys. CONCLUSION: We demonstrated that cystic epithelium exhibits low ENaC activity and this phenomenon can contribute to cyst progression.

Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct.

The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 µM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.