Distal Sodium Reabsorption Research Articles

Glomerular and tubular effects of dapagliflozin, eplerenone and their combination in patients with chronic kidney disease: A post-hoc analysis of the ROTATE-3 study.

Sodium-glucose co-transporter 2 inhibitors and mineralocorticoid receptor antagonists reduce albuminuria and the risk of kidney failure. The aim of this study was to investigate the effects of both agents alone and in combination on markers of the glomerular endothelial glycocalyx and tubular function. This post-hoc analysis utilized data of the ROTATE-3 study, a randomized cross-over study in 46 adults with chronic kidney disease and urinary albumin excretion ≥100 mg/24 h, who were treated for 4 weeks with dapagliflozin, eplerenone or its combination. The effects of dapagliflozin, eplerenone and the combination on outcome measures such as heparan sulphate, neuro-hormonal markers and tubular sodium handling were assessed with mixed repeated measures models. The mean percentage change from baseline in heparan sulphate after 4 weeks treatment with dapagliflozin, eplerenone or dapagliflozin-eplerenone was -34.8% (95% CI -52.2, -10.9), -5.9% (95% CI -32.5, 31.3) and -28.1% (95% CI -48.4, 0.1) respectively. The mean percentage change from baseline in plasma aldosterone was larger with eplerenone [38.9% (95% CI 2.8, 87.7)] and dapagliflozin-eplerenone [32.2% (95% CI -1.5, 77.4)], compared with dapagliflozin [-12.5% (95% CI -35.0, 17.8)], respectively. Mean percentage change from baseline in copeptin with dapagliflozin, eplerenone or dapagliflozin-eplerenone was 28.4% (95% CI 10.7, 49.0), 4.2% (95% CI -10.6, 21.4) and 23.8% (95% CI 6.6, 43.9) respectively. Dapagliflozin decreased proximal absolute sodium reabsorption rate by 455.9 mmol/min (95% CI -879.2, -32.6), while eplerenone decreased distal absolute sodium reabsorption rate by 523.1 mmol/min (95% CI -926.1, -120.0). Dapagliflozin-eplerenone decreased proximal absolute sodium reabsorption [-971.0 mmol/min (95% CI -1411.0, -531.0)], but did not affect distal absolute sodium reabsorption [-9.2 mmol/min (95% CI -402.0, 383.6)]. Dapagliflozin and eplerenone exert different effects on markers of glomerular and tubular function supporting the hypothesis that different mechanistic pathways may account for their kidney protective effects.

Mechanisms of enhanced renal and hepatic erythropoietin synthesis by sodium-glucose cotransporter 2 inhibitors.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of major heart failure events, an action that is statistically linked to enhanced erythropoiesis, suggesting that stimulation of erythropoietin and cardioprotection are related to a shared mechanism. Four hypotheses have been proposed to explain how these drugs increase erythropoietin production: (i) renal cortical reoxygenation with rejuvenation of erythropoietin-producing cells; (ii) counterregulatory distal sodium reabsorption leading to increased tubular workload and oxygen consumption, and thus, to localized hypoxia; (iii) increased iron mobilization as a stimulus of hypoxia-inducible factor-2α (HIF-2α)-mediated erythropoietin synthesis; and (iv) direct HIF-2α activation and enhanced erythropoietin gene transcription due to increased sirtuin-1 (SIRT1) signaling. The first two hypotheses assume that the source of increased erythropoietin is the interstitial fibroblast-like cells in the deep renal cortex. However, SGLT2 inhibitors do not alter regional tissue oxygen tension in the non-diabetic kidney, and renal erythropoietin synthesis is markedly impaired in patients with anemia due to chronic kidney disease, and yet, SGLT2 inhibitors produce an unattenuated erythrocytic response in these patients. This observation raises the possibility that the liver contributes to the production of erythropoietin during SGLT2 inhibition. Hypoxia-inducible factor-2α and erythropoietin are coexpressed not only in the kidney but also in hepatocytes; the liver is a major site of production when erythropoietin stimulation is maintained for prolonged periods. The ability of SGLT2 inhibitors to improve iron mobilization by derepressing hepcidin and ferritin would be expected to increase cytosolic ferrous iron, which might stimulate HIF-2α expression in both the kidney and liver through the action of iron regulatory protein 1. Alternatively, the established ability of SGLT2 inhibitors to enhance SIRT1 might be the mechanism of enhanced erythropoietin production with these drugs. In hepatic cell lines, SIRT1 can directly activate HIF-2α by deacetylation, and additionally, through an effect of SIRT in the liver, peroxisome proliferator-activated receptor-γ coactivator-1α binds to hepatic nuclear factor 4 to promote transcription of the erythropoietin gene and synthesis of erythropoietin. Since SIRT1 up-regulation exerts direct cytoprotective effects on the heart and stimulates erythropoietin, it is well-positioned to represent the shared mechanism that links erythropoiesis to cardioprotection during SGLT2 inhibition.

Pattern Recognition versus Pathogenesis: Electrolytes in a Patient with Adrenal Insufficiency.

Pattern Recognition versus Pathogenesis: Electrolytes in a Patient with Adrenal Insufficiency.

FGF-23 (Fibroblast Growth Factor-23) and Cardiorenal Interactions

Animal models implicate FGF-23 (fibroblast growth factor-23) as a direct contributor to adverse cardiorenal interactions such as sodium avidity, diuretic resistance, and neurohormonal activation, but this has not been conclusively demonstrated in humans. Therefore, we aimed to evaluate whether FGF-23 is associated with parameters of cardiorenal dysfunction in humans with heart failure, independent of confounding factors. One hundred ninety-nine outpatients with heart failure undergoing diuretic treatment at the Yale Transitional Care Center were enrolled and underwent blood collection, and urine sampling before and after diuretics. FGF-23 was associated with several metrics of disease severity such as higher home loop diuretic dose and NT-proBNP (N-terminal pro-B-type natriuretic peptide), and lower estimated glomerular filtration rate, serum chloride, and serum albumin. Multivariable analysis demonstrated no statistically significant association between FGF-23 and sodium avidity measured by fractional excretion of sodium, or proximal or distal tubular sodium reabsorption, either before diuretic administration or at peak diuresis (P≥0.11 for all). Likewise, FGF-23 was not independently associated with parameters of diuretic resistance (diuretic excretion, cumulative urine and sodium output, and loop diuretic efficiency [P≥0.33 for all]) or neurohormonal activation (plasma or urine renin [P≥0.36 for all]). Moreover, the upper boundary of the 95% CI of all the partial correlations were ≤0.30, supporting the lack of meaningful correlations. FGF-23 was not associated with mortality in multivariable analysis (P=0.44). FGF-23 was not meaningfully associated with any cardiorenal parameter in patients with heart failure. While our methods cannot rule out a small effect, FGF-23 is unlikely to be a primary driver of cardiorenal interactions.

The Effect of Epidermal Growth Factor Inhibition on Distal Nephron Sodium Reabsorption in Mice

The Effect of Epidermal Growth Factor Inhibition on Distal Nephron Sodium Reabsorption in Mice

Pathogenetical aspects of tubulointerstitial syndrome development in alloxan-induced experimental diabetes mellitus

Annotation. The aim of our study was to explore the pathogenetical aspects of tubulointerstitial syndrome development in alloxan-induced experimental diabetes mellitus. The experiments were carried out on 20 white non-linear mature male rats, 10 with experimental diabetes mellitus (EDМ) induced by intraperitoneal administration of alloxan at a dose of 160 mg/kg of body weight, 10 intact rats served as the control group. 25 days after administration of the diabetogenic substance, the animals were withdrawn from the experiment. The concentration of sodium and potassium ions in urine and blood plasma samples was determined, followed by calculation (considering water-induced 2-hour diuresis and endogenous creatinine clearance) of glomerular filtration rate, electrolyte excretion, their filtration rate, absolute and relative reabsorption, clearance, their proximal and distal renal transport. Removed after decapitation rats’ kidneys were dissected to 3 parts – renal cortex, medulla and papilla, sodium and potassium content was determined in water-extract of the corresponding part of the renal parenchyma, and papillary-cortical, papillary-medullar and medullary-cortical concentration ion gradients were calculated. Significant suppression of papillary-medullar and papillary-cortical concentration sodium gradients, as well as a slight limitation of its medullary-cortical gradient were established. The concentration potassium gradients were significantly reduced. Statistical processing of the obtained data was carried out with the determination of the average value, standard deviations. To assess the probability of the difference between the study groups used non-parametric Mann-Whitney ranking criterion according to the algorithms implemented in the computer program “Statistica for Windows”, “Version 8.0”. There was a decrease of the sodium-potassium ratio in urine, enhanced urinary excretion of potassium and an increase of its content in urine, as well as intensification of absolute transtubular sodium transport due to equivalent augmentation of the filtration charge of this cation, increase of proximal sodium reabsorption and, to a lesser degree, – of distal one. The distal and proximal sodium reabsorption, reduced to a unit of active nephrons, was found to be decreased, and the relative reabsorption of the cation significantly exceeded the control values, contributing to the limitation of natriuresis. The results of the study suggest that in 26-day alloxan-induced experimental diabetes hemodynamic-hyperperfusion overload on the tubular apparatus of the kidney causes the development of relative insufficiency of the proximal and distal tubules, disorders of hormone-dependent reabsorption of cations, limitation of regulatory influence of aldosterone and ADH with further tubulointerstitial disturbances that unable adequate osmotic concentration of urine.

PECULIARITIES OF IONOREGULATORY RENAL FUNCTION OF RATS IN THE DYNAMICS OF EXPERIMENTAL DIABETES MELLITUS DEVELOPMENT WITH UNDERLYING PHARMACOLOGICAL BLOCADE OF RENIN-ANGIOTENSINALDOSTERONE SYSTEM

The aim of the study – to explore the role of the renin-angiotensin-aldosteronesystem (RAAS) in the disturbance of ionoregulatory renal function in alloxan-inducedexperimental diabetes mellitus (EDM).Material and methods. The experiments were carried out on 78 white non-linearmature male rats with 11-, 26- and 46-day long alloxan-induced EDM with underlyingpharmacological blockade of RAAS by administration of kaptopril. The study ofionoregulating function of the kidneys was provided by the clearance method under thecondition of water 2-hour diuresis.Results. Pharmacological blockade of RAAS in rats with alloxan-induced EDM causedan intensification of natriuresis at all stages of the experiment: increased urinaryconcentration of sodium ions, its excretion and clearance. On the 11th day of EDM, thesodium filtration charge increased with the development of hyponatremia, proximal anddistal sodium reabsorption standardized in volume of glomerular filtrate (GF) decreased,kaliuresis was suppressed, and sodium-free water clearance elevated. In case of 26-daylong EDM, the sodium filtration charge decreased, its absolute and relative reabsorption,the distal sodium reabsorption standardized by GF increased. Kaliuresis increased. In46-day long EDM, the sodium filtration charge decreased, and hyponatremia enhanced.Absolute and relative sodium reabsorption reduced due to both – proximal and distal.Kaliuresis augmented, the clearance of sodium-free water declined.Conclusions. The increase in urinary sodium loss during the 11-day EDM is stipulatedby glomerular hyperfiltration, causing a functional weakening of the tubulotubularbalance and relative dysfunction of the distal segment of the nephron, emphasizing therenoprotective effect of RAAS on ionoregulatory function of the kidneys. The decrease inthe total reabsorption potential of the tubular segment of the nephron in the dynamics ofEDM development reflects on the proximal tubules, and preserved tubulotubular balancecertifies functional intactness of the distal tubules in 26-day long EDM. RAAS pathologicalactivation and attenuation of the renal blood flow autoregulation by tubuloglomerularfeedback may serve as an initiating factor in the development of tubular disorders in 26-day long alloxan diabetes with following progression in 46-day long EDM.

Myristoylated alanine-rich C kinase substrate-like protein-1 regulates epithelial sodium channel activity in renal distal convoluted tubule cells.

The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP2, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH2-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.

Pathways of hepatic and renal damage through non-classical activation of the renin-angiotensin system in chronic liver disease.

In liver cirrhosis, renin-angiotensin system (RAS) activation sustains renal sodium retention and hepatic fibrogenesis. New information has recently enlivened the traditional concept of RAS. For instance, renin and prorenin bind their ubiquitous receptors, resulting in the local production of angiotensin (Ang) II; increased serum calcium and calcimimetic agents, through stimulation of extracellular calcium-sensing receptors (CaSR), blunt renin production and lead to natriuretic effects in human and experimental cirrhosis. Alongside systemic production, there is Ang II tissue production within various organs through RAS enzymes different from angiotensin-converting enzyme (ACE), that is chymase, tissue plasminogen activator and several cathepsins. In experimental cirrhosis, inhibition of chymase leads to natriuretic and hepatic antifibrotic effects, without changes in systemic haemodynamics. In the kidney, local RAS coordinates proximal and distal tubular sodium reabsorption. However, renalase, whose plasma and tissue levels are severely altered in experimental cirrhosis, degrades systemic and renal tubule catecholamines, antagonizing the effects of renal RAS. Angiotensinogen-derived natriuretic and vasodilating peptides (Ang1-9, Ang1-7, Ang3-8) and their receptors have been described. Receptor agonists or antagonists are available to affect portal hypertension and sodium retention in cirrhosis. ACE2-dependent generation of Ang1-7 may inhibit experimental liver fibrosis. inhibition of Ang1-7 clearance by means of neprilysin blockade has portal hypotensive and natriuretic effects. Ang1-12, whose production renin does not regulate, is converted to several different angiotensin peptides via chymase. Finally, Ang II behaves as either an antinatriuretic or a natriuretic agent, based on the tissue content of AT1 R and AT2 R receptors, their ratio being prone to pharmacological modulation.

Abstract 081: Ghrelin is Produced by the Kidney and Mediates Distal Tubular Sodium Reabsorption in Rats

Ghrelin is a 28-amino acid peptide hormone that exerts powerful appetite-stimulating effects. Ghrelin receptor expression has been reported in the collecting duct, but the role of ghrelin in the kidney is unknown. The present studies confirmed ghrelin mRNA expression in inner medullary collecting duct cells (N=6) using RT PCR and sequencing of the 320-bp PCR product. Ghrelin peptide expression was also confirmed in these cells using immunohistochemistry and flow cytometry. To test intrarenal ghrelin action, 12-week-old uninephrectomized female Sprague-Dawley rats (N=26) received 3 bolus injections of ghrelin siRNA (N=7) or scrambled siRNA (N=7) into the renal interstitial (RI) space of the remaining kidney, each injection 48h apart. Following the last injection, rats received three, 1h RI infusions of 5% dextrose in water. Mean arterial pressure (MAP), urine sodium (Na + ) excretion (U Na V), glomerular filtration rate (GFR), fractional excretion of Na + (FE Na ), and fractional excretion of lithium (FE Li ) were calculated. In a separate set of rats, renal blood flow (RBF) was measured for 1h (N=6 for both ghrelin siRNA and scrambled siRNA). RI ghrelin siRNA-infused rats demonstrated significantly higher U Na V compared to scrambled siRNA-infused rats for all 3 periods (0.29±0.06 vs. 0.09±0.01, 0.35±0.09 vs. 0.11±0.03, and 0.27±0.07 vs. 0.09±0.02 μmol/min; P<0.01, P<0.05, and P<0.05 respectively) and significantly increased FE Na compared to scrambled siRNA infused rats (0.26±0.05 vs. 0.11±0.02, 0.35±0.06 vs. 0.11±0.03, and 0.24±0.04 vs. 0.10±0.03%; P<0.05, P<0.01, and P<0.05 respectively). There were no differences in in GFR, FE Li , MAP, or RBF. Since the increase in U Na V was not accompanied by similar increases in GFR, the effect of renal ghrelin inhibition suggested a tubular rather than hemodynamic, mechanism of action. To localize the specific tubular site, events distal to the renal proximal tubule were identified by changes in FE Na that occurred independently of changes in FE Li . RI ghrelin siRNA significantly increased FE Na without altering FE Li , suggesting distal nephron-dependent Na + reabsorption. Together, these data introduce ghrelin as a novel intrarenal peptide responsible for tonic Na + reabsorption at the level of the distal nephron.

The Influence of Peptides on the Morphofunctional State of Kidneys in Old Rats

More than a quarter of the elderly and senile age population suffers from kidney pathology. For this reason, a prophylaxis of kidney diseases with safe and effective nephroprotectors is a priority of gerontology. This work studies the influence of the polypeptide kidney complex (PKC) and peptides AED, EDL, and AEDG, on the functional state of kidneys in old rats. The administration of the PKC and peptides AED and EDL increased diuresis by 1.2–1.4 times. The PKC and peptide AED reduced the urine protein level and protein excretion by 1.5–2.8 times. The PKC and peptides AED and EDL increased distal sodium transport by 1.2–1.3 times. The peptides AED and EDL increased sodium excretion by 1.3 and 1.6 times respectively. The renal effects of the peptide AEDG resulted in a 21% reduction of glomerular filtration rate, a 3.1-fold decrease in the urine protein level, and a 2.5-fold decrease in protein excretion. The peptide AEDG reduced absolute sodium reabsorption by 1.3 times and increased distal sodium transport by 1.4 times. The realization of glomerular–tubular and tubular–tubular balances is verified by the correlation between the glomerular filtration rate (GFR) and absolute, proximal, and distal sodium reabsorption. In kidney tissue, stimulation of the antioxidant enzyme activity on the background of inhibition of the intensity peroxidation processes was observed, which, together with morphological data, reflects the absence of nephrotoxic effects. PKC and peptides AED, EDL, and AEDG may be considered nephroprotective agents in kidney aging.

Impaired pressure natriuresis and non-dipping blood pressure in rats with early type 1 diabetes mellitus.

Key points Type 1 diabetes mellitus increases cardiovascular risk; hypertension amplifies this risk, while pressure natriuresis regulates long‐term blood pressure.We induced type 1 diabetes in rats by streptozotocin injection and demonstrated a substantial impairment of pressure natriuresis: acute increases in blood pressure did not increase renal medullary blood flow, tubular sodium reabsorption was not downregulated, and proximal tubule sodium reabsorption, measured by lithium clearance, was unaffected.Insulin reduced blood glucose in diabetic rats, and rescued the pressure natriuresis response without influencing lithium clearance, but did not restore medullary blood flow.Radiotelemetry showed that diastolic blood pressure was increased in diabetic rats, and its diurnal variation was reduced.Increases in medullary blood flow and decreases in distal tubule sodium reabsorption that offset acute rises in BP are impaired in early type 1 diabetes, and this impairment could be a target for preventing hypertension in type 1 diabetes. Type 1 diabetes mellitus (T1DM) substantially increases cardiovascular risk, and hypertension amplifies this risk. Blood pressure (BP) and body sodium homeostasis are linked. T1DM patients have increased total exchangeable sodium, correlating directly with BP. Pressure natriuresis is an important physiological regulator of BP. We hypothesised that pressure natriuresis would be impaired, and BP increased, in the early phase of T1DM. Male Sprague‐Dawley rats were injected with streptozotocin (30–45 mg/kg) or citrate vehicle. After 3 weeks, pressure natriuresis was induced by serial arterial ligation. In non‐diabetic controls, this increased fractional excretion of sodium from ∼1% to ∼25% of the filtered load (P < 0.01); in T1DM rats, the response was significantly blunted, peaking at only ∼3% (P < 0.01). Mechanistically, normal lithium clearance suggested that distal tubule sodium reabsorption was not downregulated with increased BP in T1DM rats. The pressure dependence of renal medullary perfusion, considered a key factor in the integrated response, was abolished. Insulin therapy rescued the natriuretic response in diabetic rats, restoring normal downregulation of tubular sodium reabsorption when BP was increased. However, the pressure dependence of medullary perfusion was not restored, suggesting persistent vascular dysfunction despite glycaemic control. Radiotelemetry showed that T1DM did not affect systolic BP, but mean diastolic BP was ∼5 mmHg higher than in non‐diabetic controls (P < 0.01), and normal diurnal variation was reduced. In conclusion, functional impairment of renal sodium and BP homeostasis is an early manifestation of T1DM, preceding hypertension and nephropathy. Early intervention to restore pressure natriuresis in T1DM may complement reductions in cardiovascular risk achieved with glycaemic control.

A13202 Renal sodium handling in relation to environmental and genetic factors in untreated Chinese

Objectives: To evaluate the contributions of environmental and genetic cues to renal sodium handling in a population-based study. Methods: Our study subjects were suspected hypertensive patients being off antihypertensive medication for at least 2 weeks and referred to our hypertension clinic for 24-hour ambulatory blood pressure monitoring. We collected serum and 24-hour urine for measurement of sodium, creatinine and lithium concentration. Sodium and creatinine concentrations were measured by coupled plasma mass spectrometry. Fractional excretion of lithium (FELi) and fractional distal reabsorption rate of sodium (FDRNa) were calculated as markers of proximal and distal sodium handling, respectively. We used the ABI SNapShot method and genotyped 12 SNPs from candidate genes (ADD1, ADD2, ADD3, LSS, ABCB1, HSD3B1, NEDD4L, AGTR1 and WNK1) and 6 SNPs (rs10502933, rs2345088, rs12513375, rs16817782, rs Results: Our study included 1409 untreated patients and 664 men (mean age, 51.0 years). In adjusted analysis, FELi was positively associated with age but negatively with the ratio of the total cholesterol and high density lipid (P ≤ 0.003). FDRNa was negatively with current smoking and waist circumference. After adjusting the host and environmental factors and after Bonferroni correction, among the 18 genetic variations, only AGTR1 rs2131127 was significantly associated with FELi (P = 0.032). In AGTR1 rs2131127 CC homozygotes (n = 162) had lower daytime systolic blood pressure than T allele carriers (131.4 vs. 133.6 mmHg, P = 0.04). Season and humidity together explained ∼1.3% and ∼3.5% of the variation of the fractional excretion of lithium and distal reabsorption of sodium, respectively, while genetic cues just 1.10% or less. Conclusion: Environmental compared with genetic cues are critical drivers of renal sodium handling.

Discovery, characterization, and preclinical development of a Kir4.1 (KCNJ10) inhibitor for the treatment of hypertension

Kir4.1 (encoded by KCNJ10) inward rectifier potassium channels are emerging as key regulators of sodium reabsorption in the distal nephron of the kidney. Kir4.1 is expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use. These human validation data and recent animal studies suggest that Kir4.1 might represent a novel diuretic target for the management of hypertension through inhibition of distal nephron sodium reabsorption. The aim of this study was to develop a selective small‐molecule inhibitor for evaluating the therapeutic potential of renal Kir4.1 channels. From a high‐throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of ~0.9 μM and heterotetrameric Kir4.1/5.1 with an IC50 of ~ 9 μM. Lead optimization failed to identify analogs with improved potency toward Kir4.1. The selectivity screening against other Kir channels revealed that VU992 does not block Kir1.1, Kir2.x, Kir6.1/SUR1 and Kir7.1 channels. The voltage dependent unblock suggested that VU992 probably binds in the pore of the Kir4.1 channel. Computational docking analysis using a Kir4.1 homology model revealed several possible interacting residues in the pore. Experimental binding site analysis using mutagenesis and patch clamp electrophysiology suggested that the pore lining glutamic acid residue at 158 (E158) is crucial for the binding of VU992 to the Kir4.1. Mutating E158 to corresponding asparagine residue in Kir1.1 (E158N) abolished the VU992 block completely. Pharmacokinetic studies in rats showed that VU992 does not cross the blood brain barrier and therefore should not inhibit Kir4.1 channels expressed in the CNS. Finally, oral administration of VU992 dose‐dependently increased urine output and Na+ and K+ loss in volume‐loaded, freely behaving rats. This study shows proof‐of‐concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. Its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron.Support or Funding InformationNIH 5R21 NS073097‐01S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simulating a virtual population's sensitivity to salt and uninephrectomy.

Salt sensitivity, with or without concomitant hypertension, is associated with increased mortality. Reduced functional renal mass plays an important role in causing salt-sensitive hypertension for many individuals. Factors that are important in the condition of decreased renal mass and how they affect blood pressure (BP) or salt sensitivity are unclear. We used HumMod, an integrative mathematical model of human physiology, to create a heterogeneous population of 1000 virtual patients by randomly varying physiological parameters. We examined potential physiological mechanisms responsible for the change in BP in response to high-salt diet (8× change in salt intake for three weeks) with full kidney mass and again after the removal of one kidney in the same group of virtual patients. We used topological data analysis (TDA), a clustering algorithm tool, to analyse the large dataset and separate patient subpopulations. TDA distinguished five unique clusters of salt-sensitive individuals (more than 15 mmHg change in BP with increased salt). While these clusters had similar BP responses to salt, different collections of variables were responsible for their salt sensitivity, e.g. greater reductions in glomerular filtration rate (GFR) or impairments in the renin-angiotensin system. After simulating uninephrectomy in these virtual patients, the three most salt-sensitive clusters were associated with a blunted increase in renal blood flow (RBF) and higher increase in loop and distal sodium reabsorption when compared with the salt-resistant population. These data suggest that the suppression of sodium reabsorption and renin-angiotensin system is key for salt resistance, and RBF in addition to GFR may be an important factor when considering criteria for kidney donors. Here, we show that in our model of human physiology, different derangements result in the same phenotype. While these concepts are known in the experimental community, they were derived here by considering only the data obtained from our virtual experiments. These methodologies could potentially be used to discover patterns in patient sensitivity to dietary change or interventions and could be a revolutionary tool in personalizing medicine.

NEDD4-family E3 ligase dysfunction due to PKHD1/Pkhd1 defects suggests a mechanistic model for ARPKD pathobiology

Autosomal recessive polycystic kidney disease (ARPKD) is an important childhood nephropathy, occurring 1 in 20,000 live births. The major clinical phenotypes are expressed in the kidney with dilatation of the collecting ducts, systemic hypertension, and progressive renal insufficiency, and in the liver with biliary dysgenesis, portal tract fibrosis, and portal hypertension. The systemic hypertension has been attributed to enhanced distal sodium reabsorption in the kidney, the structural defects have been ascribed to altered cellular morphology, and fibrosis to increased TGF-β signaling in the kidney and biliary tract, respectively. The pathogenic mechanisms underlying these abnormalities have not been determined. In the current report, we find that disrupting PKHD1 results in altered sub-cellular localization and function of the C2-WWW-HECT domain E3 family of ligases regulating these processes. We also demonstrate altered activity of RhoA and increased TGF-β signaling and ENaC activity. Linking these phenomena, we found that vesicles containing the PKHD1/Pkhd1 gene product, FPC, also contain the NEDD4 ubiquitin ligase interacting protein, NDFIP2, which interacts with multiple members of the C2-WWW-HECT domain E3 family of ligases. Our results provide a mechanistic explanation for both the cellular effects and in vivo phenotypic abnormalities in mice and humans that result from Pkhd1/PKHD1 mutation.

Compensatory Distal Reabsorption Drives Diuretic Resistance in Human Heart Failure.

Understanding the tubular location of diuretic resistance (DR) in heart failure (HF) is critical to developing targeted treatment strategies. Rodents chronically administered loop diuretics develop DR due to compensatory distal tubular sodium reabsorption, but whether this translates to human DR is unknown. We studied consecutive patients with HF (n=128) receiving treatment with loop diuretics at the Yale Transitional Care Center. We measured the fractional excretion of lithium (FELi), the gold standard for in vivo assessment of proximal tubular and loop of Henle sodium handling, to assess sodium exit after loop diuretic administration and FENa to assess the net sodium excreted into the urine. The mean±SD prediuretic FELi was 16.2%±9.5%, similar to that in a control cohort without HF not receiving diuretics (n=52; 16.6%±9.2%; P=0.82). Administration of a median of 160 (interquartile range, 40-270) mg intravenous furosemide equivalents increased FELi by 12.6%±10.8% (P<0.001) but increased FENa by only 4.8%±3.3%. Thus, only 34% (interquartile range, 15.6%-75.7%) of the estimated diuretic-induced sodium release did not undergo distal reabsorption. After controlling for urine diuretic levels, the increase in FELi explained only 6.4% of the increase in FENa (P=0.002). These data suggest that administration of high-dose loop diuretics to patients with HF yields meaningful increases in sodium exit from the proximal tubule/loop of Henle. However, little of this sodium seems to reach the urine, consistent with findings from animal models that indicate that distal tubular compensatory sodium reabsorption is a primary driver of DR.

Aldosterone Modulates the Association between NCC and ENaC

Distal sodium transport is a final step in the regulation of blood pressure. As such, understanding how the two main sodium transport proteins, the thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC), are regulated is paramount. Both are expressed in the late distal nephron; however, no evidence has suggested that these two sodium transport proteins interact. Recently, we established that these two sodium transport proteins functionally interact in the second part of the distal nephron (DCT2). Given their co-localization within the DCT2, we hypothesized that NCC and ENaC interactions might be modulated by aldosterone (Aldo). Aldo treatment increased NCC and αENaC colocalization (electron microscopy) and interaction (coimmunoprecipitation). Finally, with co-expression of the Aldo-induced protein serum- and glucocorticoid-inducible kinase 1 (SGK1), NCC and αENaC interactions were increased. These data demonstrate that Aldo promotes increased interaction of NCC and ENaC, within the DCT2 revealing a novel method of regulation for distal sodium reabsorption.

Physiological Sensitivity to Salt and Uninephrectomy

Salt sensitivity, with or without concomitant hypertension, is associated with increased mortality. Reduced functional renal mass plays an important role in causing salt sensitive hypertension for many individuals but the mechanisms are unclear. Moreover, different derangements in human physiology can result in the same phenotype. Methodologies that could discover patterns in sensitivity to dietary change or interventions would be a revolutionary tool in personalizing medicine. We used HumMod, an integrative mathematical model of human physiology, to simulate population responses to changes in salt intake with or without reduced kidney mass (uninephrectomy). A heterogeneous population of 1000 virtual patients was created by randomly varying important physiological parameters. We examined potential physiological mechanisms responsible for the chronic blood pressure (BP) response to salt (8‐fold change in salt intake for 3 weeks) with full kidney mass and again with a single kidney in the same group of virtual patients. We also used topological data analysis (TDA), a new clustering algorithm tool, to separate patient subpopulations. In the entire population, BP increased with increased salt intake (14 ± 3 mmHg). This was associated with small changes in glomerular filtration rate (GFR) but significant increases in renal blood flow, total peripheral resistance, and distal tubular sodium reabsorption. Interestingly, TDA distinguished 5 unique clusters of salt sensitive individuals (&gt; 15 mmHg change in BP with increased salt). While these clusters had similar BP responses to salt, they were significantly different in the mechanisms of salt sensitivity (e.g. greater reductions in GFR or impairments in the renin angiotensin system.) After kidney removal in these same individuals, the three most salt sensitive clusters were associated with a blunted increase in renal blood flow (RBF), exaggerated increases in loop and distal sodium reabsorption, and greater sodium and volume retention as compared to the salt resistant population. These data suggest that the suppression of sodium reabsorption and renin angiotensin system is key for salt resistance, while RBF in addition to GFR may be an important factor when considering criteria for potential kidney donors.Support or Funding InformationSupported by NSF EPS‐0903787, P20 GM104357, and NIH PO1 HL51971.

OS 08-05 NOVEL MOLECULAR INSIGHTS IN URINARY EXOSOME PROTEIN PROFILING IN THIAZIDE INDUCED HYPONATREMIA

Objective: Thiazide diuretics are amongst most widely prescribed and effective anti-hypertensive medicines worldwide. Thiazides however cause Thiazide-Induced Hyponatremia (TIH), a novel and potentially important paradigm of dysregulated distal nephron sodium and water reabsorption. A priori TIH must result from excessive saliuresis and/or water reabsorption. The water and electrolyte transporter composition of Urinary Exosomes (UE) reflects their cellular origin and are a promising way to study renal dysfunction. This study assessed the expression of aquaporin 2 (AQP2), thiazide - sensitive sodium chloride co-transporter (NCC) and Phospho NCC (PNCC) in the UE of TIH patients. Advancements in understanding of thiazide-sensitive pathways may further uncover the molecular mechanisms underlying sodium and water trafficking. Design and Method: 100 patients with severe TIH donated urine samples during acute TIH and at two months post thiazide cessation. Matched normonatraemic controls were recruited both on and off thiazides (groups 1 & 2 respectively). UE from each patient group were isolated and AQP2, NCC and PNCC were evaluated by immunoblotting. Protein expression was normalized by urinary creatinine (UCr) and results expressed as units of optical density/UCr. Results: UE expression of AQP2 was higher in acute TIH compared to convalescent (32.93 vs 28.71, P Conclusions: This study highlights the utility of UE analysis to probe the molecular pathophysiology of TIH. Increased AQP2 and reduced NCC expression suggests that TIH results from increased water reabsorption and sodium wasting in the distal nephron. TIH studies may further inform the design of new thiazide medicines less prone to cause hyponatremia.