Distal Convoluted Tubule Cells Research Articles

Identification of adenylyl cyclase isoforms mediating parathyroid hormone- and calcitonin-stimulated cyclic AMP accumulation in distal tubule cells

BackgroundThe distal convoluted tubule (DCT) is an important nephron site for parathyroid hormone (PTH) and calcitonin regulation of urinary divalent cation excretion. These hormones exert their effects on the DCT in substantial part through activation of adenylyl cyclase (AC); however, it is unknown which AC isoforms are involved.MethodsTo examine this, two mouse DCT cell lines were studied: 209 and D1 cells. AC isoform mRNA expression was analyzed by real-time PCR. Cyclic AMP was measured using enzyme immunoassay.ResultsCalcitonin, but not PTH, stimulated cAMP accumulation in 209 cells, while PTH, but not calcitonin, increased cAMP content in D1 cells. Both cell types expressed AC3, AC4, AC6, AC7, and AC9 mRNA; in both cell types, AC6 mRNA was most abundant, followed by AC9, then AC3 and AC7, with relatively very small amounts of AC4 mRNA. Microdissected mouse DCT had a similar pattern of AC isoform mRNA expression although AC5 mRNA was detected. Individual siRNA knockdown of AC6 and AC9 reduced calcitonin-stimulated cAMP accumulation in 209 cells and PTH-induced cAMP levels in D1 cells. Knockdown of AC3 had no effect on hormonal augmentation of cAMP in either cell line. Surprisingly, knockdown of AC7 increased calcitonin-induced cAMP accumulation in 209 cells as well as PTH-stimulated cAMP content in D1 cells.ConclusionsTaken together, these findings indicate that AC6 and AC9 mediate calcitonin- and PTH-stimulated cAMP accumulation in DCT cells, while activation of AC7 may paradoxically reduce the stimulatory effects of PTH and calcitonin on cultured DCT cAMP levels.

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor and NaCl transport mechanisms in the renal distal nephron.

A key role of aldosterone and mineralocorticoid receptor is to regulate fluid volume and K+ homeostasis in the body by acting on the renal distal nephron. Global responses of the kidney to elevated aldosterone levels are determined by the coordinate action of different constituent tubule cells, including principal cells, intercalated cells and distal convoluted tubule cells. Recent studies on genetic mutations causing aldosterone overproduction have identified the molecules involved in aldosterone biosynthesis in the adrenal gland, and there is also increasing evidence for mechanisms and signaling pathways regulating the balance between renal NaCl reabsorption and K+ secretion, the two major effects of aldosterone. In particular, recent studies have demonstrated that mineralocorticoid receptor in intercalated cells is selectively regulated by phosphorylation, which prevents ligand binding and activation. Moreover, the ubiquitin ligase complex composed of Kelch-like 3 and Cullin 3 acts downstream of angiotensin II and plasma K+ alterations, regulating Na-Cl cotransporter independently of aldosterone in distal convoluted tubule cells. These and other effects are integrated to produce appropriate kidney responses in a high-aldosterone state, and are implicated in fluid and electrolyte disorders in humans. This review summarizes the current knowledge on mechanisms modulating mineralocorticoid receptor and its downstream effectors in the distal nephron.

Renal function of cyclin M2 Mg2+ transporter maintains blood pressure.

Epidemiological studies have shown that magnesium intake/excretion is inversely correlated with blood pressure (BP), and artificial supplementation of magnesium was able to prevent hypertension. However, there has been no molecular genetic study showing the importance of magnesium homeostasis in BP regulation. We analyzed magnesium content and BP of mice lacking genes encoding cyclin M (CNNM) Mg transporter family proteins. Systemic heterozygotes and the kidney-specific homozygotes for Cnnm2-deficient alleles are both viable and show hypomagnesemia, indicating the important function of CNNM2 in maintaining magnesium homeostasis in the kidney. Endogenous CNNM2 localizes at the basolateral membrane of kidney distal convoluted tubule cells, which play important roles not only in magnesium reabsorption but also in BP control. The BP of these viable strains is significantly reduced; the SBP values by telemetric measurements are 121.7 ± 2.8 mmHg in wild-type, and 110.2 ± 2.7 and 109.7 ± 3.6 mmHg in systemic heterozygotes and kidney-specific homozygotes, respectively. Analyses of mice lacking CNNM Mg transporters clearly demonstrated abnormalities in BP values, confirming the importance of magnesium homeostasis in maintaining BP.

Transcriptomic analysis and plasma metabolomics in Aldh16a1-null mice reveals a potential role of ALDH16A1 in renal function

ALDH16A1 is a novel member of the ALDH superfamily that is enzymatically-inactive and highly expressed in the kidney. Recent studies identified an association between a rare missense single nucleotide variant (SNV) in the ALDH16A1 gene and elevated serum uric acid levels and gout. The present study explores the mechanisms by which ALDH16A1 influences uric acid homeostasis in the kidney. We generated and validated a mouse line with global disruption of the Aldh16a1 gene through gene targeting and performed RNA-seq analyses in the kidney of wild-type (WT) and Aldh16a1 knockout (KO) mice, along with plasma metabolomics. We found that ALDH16A1 is expressed in proximal and distal convoluted tubule cells in the cortex of the kidney and in zone 3 hepatocytes. RNA-seq and gene ontology enrichment analyses showed that cellular lipid and lipid metabolic processes are up-regulated. Three transporters localized in the apical membrane of the proximal convoluted tubule of the kidney known to influence urate/uric acid homeostasis were found to be up-regulated (Abcc4, Slc16a9) or down-regulated (Slc17a3). An initial metabolomics analysis in plasma revealed an altered lipid profile in KO mice that is in agreement with our RNA-seq analysis. This is the first study demonstrating a functional role of ALDH16A1 in the kidney.

CD8+ T cells stimulate Na-Cl co-transporter NCC in distal convoluted tubules leading to salt-sensitive hypertension

Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8+ T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8+ T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8+ T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K+ channel Kir4.1, and stimulation of the Cl− channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension.

Potassium Sensing by Renal Distal Tubules Requires Kir4.1.

The mammalian distal convoluted tubule (DCT) makes an important contribution to potassium homeostasis by modulating NaCl transport. The thiazide-sensitive Na+/Cl- cotransporter (NCC) is activated by low potassium intake and by hypokalemia. Coupled with suppression of aldosterone secretion, activation of NCC helps to retain potassium by increasing electroneutral NaCl reabsorption, therefore reducing Na+/K+ exchange. Yet the mechanisms by which DCT cells sense plasma potassium concentration and transmit the information to the apical membrane are not clear. Here, we tested the hypothesis that the potassium channel Kir4.1 is the potassium sensor of DCT cells. We generated mice in which Kir4.1 could be deleted in the kidney after the mice are fully developed. Deletion of Kir4.1 in these mice led to moderate salt wasting, low BP, and profound potassium wasting. Basolateral membranes of DCT cells were depolarized, nearly devoid of conductive potassium transport, and unresponsive to plasma potassium concentration. Although renal WNK4 abundance increased after Kir4.1 deletion, NCC abundance and function decreased, suggesting that membrane depolarization uncouples WNK kinases from NCC. Together, these results indicate that Kir4.1 mediates potassium sensing by DCT cells and couples this signal to apical transport processes.

Sodium-chloride cotransporter activity regulated by extracellular potassium

The thiazide-sensitive sodium-chloride cotransporter (NCC) is exclusively expressed in the apical membrane of the renal distal convoluted tubule (DCT), and it is important for maintaining fluid and salt balance. NCC is responsible for electroneutral NaCl reabsorption, and its activity is determined by phosphorylation, which was reported to be regulated by WNK-stimulated Ste20-type kinases, Ste20-related proline alanine-rich kinase, and oxidative stress response 1 kinase. WNK kinases have chloride-binding sites, and WNK4 has the highest sensitivity to intracellular chloride concentration ([Cl−]i) compared with WNK1 and WNK3. NCC dysfunction often comes together with abnormal urinary potassium excretion, which is not only highlighted by Mendelian disorders such as familial hyperkalemic hypertension (FHHt) and Gitelman syndrome but also presented more frequently by long-term usage of thiazides, specific inhibitors of NCC. Recent studies have shown that extracellular potassium (K+) can modulate DCT cell membrane voltage and in turn intracellular Cl−, which regulates phosphorylation of WNK kinases. Additional Cl− independent mechanisms were also reported by several groups. This paper is a brief review of the recent discoveries on mechanisms of NCC regulation by extracellular potassium.

Extracellular K+ rapidly controls NaCl cotransporter phosphorylation in the native distal convoluted tubule by Cl- -dependent and independent mechanisms.

High dietary potassium (K+ ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K+ , similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl- fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl- -dependent. The Cl- -dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl- independent pathway may include additional signalling cascades. A high dietary potassium (K+ ) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration ([K+ ]ex ) modulate NCC phosphorylation via a Cl- -dependent modulation of the with no lysine (K) kinases (WNK)-STE20/SPS-1-44 related proline-alanine-rich protein kinase (SPAK)/oxidative stress-related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K+ ]ex , with the most prominent effects occurring around physiological plasma [K+ ]. Cellular Cl- conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K+ ]ex . However, NCC dephosphorylation triggered by high [K+ ]ex is neither blocked by removing extracellular Cl- , nor by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid. The response to [K+ ]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K+ ]ex directly and rapidly controls NCC phosphorylation by Cl- -dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.

Abstract 092: Novel Mechanism of Salt-sensitive Hypertension: CD8 + T Cells Stimulate Sodium Chloride Co-transporter NCC in Kidney

Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention is an important question, which if answered, could reveal a critical relationship between adaptive immunity and pathogenesis of salt-sensitive hypertension. In the present study, we propose a novel mechanism of salt-sensitive hypertension via the distal nephron: that in salt-sensitive hypertension, renal infiltrating T cells interact with distal convoluted tubule cells (mDCTs), which leads to up-regulation of the sodium-chloride-co-transporter (NCC), and thus enhancement of sodium retention. In vivo tests used DOCA-salt mice (SBP~190mmHg on a high salt diet) and T cell-adoptive transferred mice (SBP~170mmHg on a high salt diet). Results confirmed enhanced NCC & p-NCC expression (>2.5X) in the kidneys of both mouse models. We found significantly more T cells infiltrating the kidneys of DOCA-salt & T cell-transferred mice compared to their controls. It is noteworthy that in both models a great number of CD8 + T cells (CD8Ts) in the renal tubulointerstitium surround DCT segments which have higher levels of NCC expression. Direct contact between these two cell types was confirmed by super-resolution structured illumination microscopy. In vitro, we co-cultured mDCTs with T cells, and found that CD8Ts, but not CD4Ts, significantly increased NCC expression (>3X) in mDCTs via “direct cell-cell contact”. An intracellular sodium-indicator detected higher NCC-mediated sodium uptake (>1.5X) in mDCTs treated with CD8Ts compared to untreated controls. Moreover, these effects were mediated by increased (>2.2X) functional chloride channel Clc-k in the cell membrane. Clc-k knockdown by siRNA blunted chloride efflux and abolished CD8T-induced NCC activation and sodium retention in mDCTs. Taken together, our findings suggest a novel role for CD8 + T cells in enhancement of salt retention contributing to salt-sensitive hypertension: that CD8 + T cells directly contact mDCTs, stimulate NCC by upregulating the chloride channel Clc-k on the mDCT plasma membrane, thereby increasing chloride efflux, which leads to compensatory chloride influx via NCC activation at the cost of increasing sodium influx.

Interleukin-17A Regulates Renal Sodium Transporters and Renal Injury in Angiotensin II-Induced Hypertension.

Angiotensin II-induced hypertension is associated with an increase in T-cell production of interleukin-17A (IL-17A). Recently, we reported that IL-17A(-/-) mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 expression after 2 weeks of angiotensin II infusion compared with wild-type mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, the time when the blood pressure reduction in IL-17A(-/-) mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium-chloride cotransporter and the epithelial sodium channel and protected mice from glomerular and tubular injury. In human proximal tubule (HK-2) cells, IL-17A increased sodium hydrogen exchanger 3 expression through a serum and glucocorticoid-regulated kinase 1-dependent pathway. In mouse distal convoluted tubule cells, IL-17A increased sodium-chloride cotransporter activity in a serum and glucocorticoid-regulated kinase 1/Nedd4-2-dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of serum and glucocorticoid-regulated kinase 1 at serine 78, and treatment with a serum and glucocorticoid-regulated kinase 1 inhibitor blocked the effects of IL-17A on sodium hydrogen exchanger 3 and sodium-chloride cotransporter. Interestingly, both HK-2 and mouse distal convoluted tubule 15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.

Signaling pathways activated during acute aldosterone stimulation in kidney distal convoluted tubule cells

The kidney distal convoluted tubule (DCT) plays an important role in modulating body sodium balance and blood pressure. Long‐term effects of aldosterone to increase sodium reabsorption in the DCT by modulating the function of the NaCl cotransporter (NCC) and the epithelial sodium channel (ENaC) are well described. However, potential effects of aldosterone to acutely modulate NCC or ENaC function in the DCT and the signaling cascades responsible remain largely uncharacterized. Here, we used stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics to map global changes in protein phosphorylation in mouse kidney convoluted tubule cells (mpkDCT) following acute (30 min) treatment with aldosterone (1μM). A total of 8136 unique proteins, containing 14838 different phosphopeptides were identified. Of these, 148 phosphopeptides were significantly up regulated, and 96 phosphopeptides were significantly downregulated in abundance following acute aldosterone treatment. Motif analysis indicated that basophilic motifs were preferential targets for upregulated sites upon aldosterone stimulation, whereas proline‐directed motifs were prominent for downregulated sites. Analysis of motif prediction indicated that the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) and CAMKK dependent pathways may be involved in aldosterone‐induced signaling response by the DCT. Acute application of aldosterone did not significantly alter cAMP levels in mpkDCT cells. Ongoing analysis and pharmacological inhibition studies will help clarify and verify these systems biology findings and determine novel new acute aldosterone‐mediated signaling networks important for blood pressure regulation.

Acid-Induced Chloride Current in Distal Convoluted Tubule

The distal convoluted tubule (DCT) of the kidney is responsible for the fine-tuning of urinary filtrate before excretion. The DCT is known to facilitate the movement of Na, K, Ca, Mg, and Cl via transporters and ion channels. Recent research has focussed on the functional activity of TRP channels in the DCT, with heavy emphasis on TRPM6/7 (Mg at neutral pH 7.4; Na at acidic pH) and TRPV5/6 (Ca). Several studies have suggested that these channels should give rise to large macroscopic currents in DCT cells. Therefore we undertook this study to determine the ionic species representing the dominant current in mouse DCT (MDCT) cells via whole-cell voltage-clamp electrophysiology. Using ionic permeation studies and analysis of reversal potential changes, our results unequivocally demonstrate that MDCT cells exhibit a significant Cl current under both neutral and acidic extracellular solutions. We further identified that this current is rapidly activating, differentiating it from the endogenous acid-induced anion current discovered in HEK293 cells. We noted that currents elicited under acidic pH were substantially larger than those at neutral pH, and thus hypothesized that extracellular acid induced a different, larger Cl current in MDCT cells. This was supported pharmacologically, where only the acid-induced Cl current was blocked by furosemide, a clinically prescribed loop-diuretic. To further understand the significance of a large acid-induced Cl current in the late nephron, we investigated inner medullary collecting duct (IMCD) cells as they represent the most acidic portion of the nephron. IMCD current was not only acid-induced, but shared an identical I-V relationship, pH sensitivity, and time-course of activation with the acid-induced induced Cl current discovered in MDCT cells. We thus conclude that the late nephron contains a large acid-induced Cl current that may play a significant role in anion reabsorption.

A Systems Level Analysis of Vasopressin-mediated Signaling Networks in Kidney Distal Convoluted Tubule Cells.

The kidney distal convoluted tubule (DCT) plays an essential role in maintaining body sodium balance and blood pressure. The major sodium reabsorption pathway in the DCT is the thiazide-sensitive NaCl cotransporter (NCC), whose functions can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. Here we use a systems biology approach centered on stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells to map global changes in protein phosphorylation upon acute treatment with a VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). 6330 unique proteins, containing 12333 different phosphorylation sites were identified. 185 sites were altered in abundance following dDAVP. Basophilic motifs were preferential targets for upregulated sites upon dDAVP stimulation, whereas proline-directed motifs were prominent for downregulated sites. Kinase prediction indicated that dDAVP increased AGC and CAMK kinase families’ activities and decreased activity of CDK and MAPK families. Network analysis implicated phosphatidylinositol-4,5-bisphosphate 3-kinase or CAMKK dependent pathways in VP-mediated signaling; pharmacological inhibition of which significantly reduced dDAVP induced increases in phosphorylated NCC at an activating site. In conclusion, this study identifies unique VP signaling cascades in DCT cells that may be important for regulating blood pressure.

The art of magnesium transport.

Patients with hypomagnesemia suffer from a wide range of symptoms including muscle cramps, cardiac arrhythmias and epilepsy. Disturbances in body Mg(2+) homeostasis can often be attributed to increased Mg(2+) excretion by the kidney. Within the kidney, the distal convoluted tubule (DCT) segment determines the final Mg(2+) excretion, since no reabsorption takes place beyond this segment of the nephron. On 21(st) of January 2015, Jeroen de Baaij defended his thesis "The Distal Convoluted Tubule: the Art of Magnesium Transport", in which he aimed to identify new genes involved in Mg(2+) reabsorption in the DCT. This review summarizes the main findings of his graduate research. TRPM6 mediates apical Mg(2+) entry into the DCT cell and is highly regulated by EGF, insulin and pH. ATP and flavagline compounds have been characterized as new regulators of TRPM6 activity, providing novel pathways to target Mg(2+) disturbances. Using isolated primary DCT cells from mice, PCBD1 was identified as a new transcriptional regulator of Mg(2+) transport in the DCT. Indeed, patients with PCBD1 mutations were shown to suffer hypomagnesemia and MODY5-like diabetes. Subsequently, the work presented in the thesis focused on the elucidation of the basolateral Mg(2+) extrusion of the DCT cell. In vivo studies using SLC41A3-knockout mice suggest that SLC41A3 may act as Mg(2+) extrusion mechanism. CNNM2 has long been hypothesized to transport Mg(2+) at the basolateral membrane of the DCT. However, by determining the protein topology and homology modeling of the CBS domains, it was argued that CNNM2 is rather an Mg(2+)-sensing mechanism. Follow-up studies using (25)Mg(2+) isotopes showed that CNNM2 increases Mg(2+) uptake when overexpressed in HEK293 cells. Additionally, by knocking down cnnm2 in zebrafish, CNNM2 was demonstrated to be essential for brain development and Mg(2+) homeostasis. Mutations in CNNM2 were shown to cause hypomagnesemia, seizures and intellectual disability. Altogether, this thesis established the importance of Mg(2+) reabsorption in the DCT to health and disease. Combined, continued efforts of clinicians, geneticists, and researchers are necessary to improve the care of hypomagnesemic patients and increase our understanding of Mg(2+) reabsorption in the DCT.

The Effect of WNK4 on the Na+:Cl‐ Cotransporter is modulated by Intracellular Chloride

IntroductionThe effect of WNK4 on the renal Na+:Cl‐ cotransporter, NCC is controversial since several studies in vitro and in vivo have shown negative or positive effects. We hypothesize that both effects coexist and are modulated by the intracellular chloride concentration ([Cl‐]i). Piala et al. (2014) showed that L‐WNK1 is a Cl‐‐sensitive kinase and identified two leucine residues forming the Cl‐‐binding site. These leucines are conserved in WNK isoforms.Materials and MethodsWe used the functional expression system of Xenopus oocytes and HEK293 cells. NCC activity was measured by 22Na+ uptake and/or amino acid threonine phosphorylation in the absence or presence of wild type or mutant WNK4, in control conditions, or after [Cl‐]i depleting maneuvers. WNK4 autophosphorylation was also assessed in these conditions.ResultsAt baseline [Cl‐]i ∼ 45 mM, WNK4 autophosphorylation was undetectable and NCC activity was either inhibited or unaffected. [Cl‐]i reduction (25‐30 mM) promoted autophosphorylation transforming WNK4 to an activator of NCC in a kinase‐dependent manner. The L322F substitution transformed WNK4 into a constitutively autophosphorylated kinase that activated NCC without chloride depletion.ConclusionWNK4 is Cl‐ sensitive kinase exerting differential effects on NCC depending on the [Cl‐]i. It is possible that small changes in [Cl‐]i in distal convoluted tubule cells may have a crucial role determining WNK4 effect on NCC, suggesting that diverse physiological processes could converge in the Cl‐modulation of WNK's.

Potassium Modulates Electrolyte Balance and Blood Pressure through Effects on Distal Cell Voltage and Chloride

Dietary potassium deficiency, common in modern diets, raises blood pressure and enhances salt sensitivity. Potassium homeostasis requires a molecular switch in the distal convoluted tubule (DCT), which fails in familial hyperkalemic hypertension (pseudohypoaldosteronism type 2), activating the thiazide-sensitive NaCl cotransporter, NCC. Here, we show that dietary potassium deficiency activates NCC, even in the setting of high salt intake, thereby causing sodium retention and a rise in blood pressure. The effect is dependent on plasma potassium, which modulates DCT cell membrane voltage and, in turn, intracellular chloride. Low intracellular chloride stimulates WNK kinases to activate NCC, limiting potassium losses, even at the expense of increased blood pressure. These data show that DCT cells, like adrenal cells, sense potassium via membrane voltage. In the DCT, hyperpolarization activates NCC via WNK kinases, whereas in the adrenal gland, it inhibits aldosterone secretion. These effects work in concert to maintain potassium homeostasis.

Distal convoluted tubule.

The distal convoluted tubule (DCT) is a short nephron segment, interposed between the macula densa and collecting duct. Even though it is short, it plays a key role in regulating extracellular fluid volume and electrolyte homeostasis. DCT cells are rich in mitochondria, and possess the highest density of Na+/K+-ATPase along the nephron, where it is expressed on the highly amplified basolateral membranes. DCT cells are largely water impermeable, and reabsorb sodium and chloride across the apical membrane via electroneurtral pathways. Prominent among this is the thiazide-sensitive sodium chloride cotransporter, target of widely used diuretic drugs. These cells also play a key role in magnesium reabsorption, which occurs predominantly, via a transient receptor potential channel (TRPM6). Human genetic diseases in which DCT function is perturbed have provided critical insights into the physiological role of the DCT, and how transport is regulated. These include Familial Hyperkalemic Hypertension, the salt-wasting diseases Gitelman syndrome and EAST syndrome, and hereditary hypomagnesemias. The DCT is also established as an important target for the hormones angiotensin II and aldosterone; it also appears to respond to sympathetic-nerve stimulation and changes in plasma potassium. Here, we discuss what is currently known about DCT physiology. Early studies that determined transport rates of ions by the DCT are described, as are the channels and transporters expressed along the DCT with the advent of molecular cloning. Regulation of expression and activity of these channels and transporters is also described; particular emphasis is placed on the contribution of genetic forms of DCT dysregulation to our understanding.

Urine uromodulin estimation in partial bladder outlet obstruction and cyclophosphamide-induced haemorrhagic cystitis models in rats.

Uromodulin (UMOD) is a glycoprotein excreted by the thick ascending limb of the Henle's loop and distal convoluted tubule cells, playing various, yet still unclear roles. An abnormal urinary UMOD excretion is observed in many pathophysiological conditions. The aim of our study was to assess urine UMOD excretion in experimental partial bladder outlet obstruction (PBOO), reflecting BPH in humans, and in cyclophosphamide-induced haemorrhagic cystitis (CP-HC). PBOO and CP-HC rats and two appropriate control groups were studied. The PBOO model was surgically induced by partial proximal urethral obstruction and CP-HC by four i.p. cyclophosphamide administrations (every two days). 24-hour urine collections were performed in both PBOO (on 3rd, 7th, 12th and 15th day after surgery) and CP-HC rats (on 1st, 3rd, 5th and 7th day). UMOD was determined with the ELISA method. Both 24-hour urinary UMOD excretion and urinary UMOD concentrations were determined. In the overall assessment, PBOO rats were characterized by decreased mean urinary UMOD concentration. However, as the urine volume, except for transient drop on 3rd day following PBOO operation, was steadily increasing, the daily urinary uromodulin excretion did not differ from the control one. Contrary to PBOO, CP-HC rats demonstrated mean urinary concentration similar to that of the control rats, while their 24hr UMOD excretion in urine was almost doubled due to urine volume increase (from 1.6 up to almost 3 fold). The highest UMOD urinary output was observed after the 3rd and 4th doses of cyclophosphamide. A reduced urinary UMOD excretion in early PBOO phase may be considered as a marker of distal tubular cells damage due to incomplete bladder emptying and increased pressure retrograding to distal tubules. This effect disappears with structural, adaptive histological changes of the bladder wall leading to an improved voiding. In CP-HC animals, the elevated urinary UMOD level may be associated with complex inflammatory response due to the cytotoxic CP action. UMOD assessment in this model may reflect renal and urological toxicity as UMOD excretion rises with the cumulative cyclophosphamide dose.

Sympathetic stimulation of thiazide-sensitive sodium chloride cotransport in the generation of salt-sensitive hypertension.

Excessive renal efferent sympathetic nerve activity contributes to hypertension in many circumstances. Although both hemodynamic and tubular effects likely participate, most evidence supports a major role for α-adrenergic receptors in mediating the direct epithelial stimulation of sodium retention. Recently, it was reported, however, that norepinephrine activates the thiazide-sensitive NaCl cotransporter (NCC) by stimulating β-adrenergic receptors. Here, we confirmed this effect and developed an acute adrenergic stimulation model to study the signaling cascade. The results show that norepinephrine increases the abundance of phosphorylated NCC rapidly (161% increase), an effect largely dependent on β-adrenergic receptors. This effect is not mediated by the activation of angiotensin II receptors. We used immunodissected mouse distal convoluted tubule to show that distal convoluted tubule cells are especially enriched for β₁-adrenergic receptors, and that the effects of adrenergic stimulation can occur ex vivo (79% increase), suggesting they are direct. Because the 2 protein kinases, STE20p-related proline- and alanine-rich kinase (encoded by STK39) and oxidative stress-response kinase 1, phosphorylate and activate NCC, we examined their roles in norepinephrine effects. Surprisingly, norepinephrine did not affect STE20p-related proline- and alanine-rich kinase abundance or its localization in the distal convoluted tubule; instead, we observed a striking activation of oxidative stress-response kinase 1. We confirmed that STE20p-related proline- and alanine-rich kinase is not required for NCC activation, using STK39 knockout mice. Together, the data provide strong support for a signaling system involving β₁-receptors in the distal convoluted tubule that activates NCC, at least in part via oxidative stress-response kinase 1. The results have implications about device- and drug-based treatment of hypertension.

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

The thiazide-sensitive sodium chloride cotransporter, NCC, is the major NaCl transport protein in the distal convoluted tubule (DCT). The transport activity of NCC can be regulated by phosphorylation, but knowledge of modulation of NCC trafficking by phosphorylation is limited. In this study, we generated novel tetracycline-inducible Madin-Darby canine kidney type I (MDCKI) cell lines expressing NCC to examine the role of NCC phosphorylation and ubiquitylation on NCC endocytosis. In MDCKI-NCC cells, NCC was highly glycosylated at molecular weights consistent with NCC monomers and dimers. NCC constitutively cycles to the apical plasma membrane of MDCKI-NCC cells, with 20-30% of the membrane pool of NCC internalized within 30 min. The use of dynasore, PitStop2, methyl-β-cyclodextrin, nystatin, and filipin (specific inhibitors of either clathrin-dependent or -independent endocytosis) demonstrated that NCC is internalized via a clathrin-mediated pathway. Reduction of endocytosis resulted in greater levels of NCC in the plasma membrane. Immunogold electron microscopy confirmed the association of NCC with the clathrin-mediated internalization pathway in rat DCT cells. Compared with controls, inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr-53, Thr-58, and Ser-71 residues with Asp resulted in increased membrane abundance and reduced rates of NCC internalization. NCC ubiquitylation was lowest in the conditions with greatest NCC phosphorylation, thus providing a mechanism for the reduced endocytosis. In conclusion, our data support a model where NCC is constitutively cycled to the plasma membrane, and upon stimulation, it can be phosphorylated to both increase NCC activity and decrease NCC endocytosis, together increasing NaCl transport in the DCT.