Mouse Distal Convoluted Tubule Cells Research Articles

The Epithelial Na+ Channel Is a Zn2+ Sensitive Renal Na+ Reabsorption Pathway that Mediates Zn2+ Deficiency-induced Hypertension

Background: Zinc (Zn2+) deficiency (ZnD) is comorbid with many chronic diseases including kidney disease and diabetes. Individuals in these vulnerable populations have a higher prevalence of hypertension. Recently, we reported that ZnD induces hypertension by promoting renal Na+ reabsorption. However, the renal Na+ reabsorption mechanisms involved continue to be identified. The renal epithelial Na+ channel (ENaC) plays an essential role in regulating whole-body Na+ and water balance and is involved in hypertension. We hypothesize that ENaC is a Zn2+-sensitive channel that mediates ZnD-induced hypertension. Experimental Design: To establish the role of ENaC in ZnD-induced hypertension, WT C57Bl/6 mice were switched from a Zn2+ adequate to a Zn2+ deficient diet for 10 weeks. During weeks 9 and 10, mice were treated with benzamil, an ENaC inhibitor. Systolic blood pressure (BP) was monitored by tail-cuff plethysmography; Renal ENaC expression was examined by immunohistochemistry and Western blot. To authenticate the Zn2+ sensitivity of ENaC, mouse distal convoluted tubule cells (mDCT) were treated with a Zn2+ chelator (TPEN) or vehicle (ethanol) for 24 hours. A subset of TPEN-treated cellular monolayers were supplemented with Zn2+ for 24 hours to replete intracellular Zn2+. Cellular ENaC expression was examined by Western blot analysis. Results: 1) Blood pressure: Compared to Zn2+ adequate mice (101.4 mm Hg ± 4.225), Zn2+ deficient mice had increased BP (154.6 mm Hg ± 2.422). However, benzamil treatment lowered BP (115.0 mm Hg ± 4.626) of Zn2+ deficient mice. 2)Immunohistochemistry: Next, Zn2+ effects on ENaC expression were examined. In Zn2+ deficient mice, αENaC protein abundance significantly increased compared with Zn2+ adequate mice. 3) Western blot analysis: To directly investigate Zn2+effects on ENaC expression, in vitro studies were performed using mDCT cells. Western blot analysis showed that TPEN-induced ZnD stimulated both αENaC and βENaC protein expression. Conclusion: Collectively, our findings reveal that (1) ENaC is a Zn2+ sensitive renal Na+ reabsorption pathway and (2) ENaC mediates ZnD-induced hypertension. Significance: Understanding the renal mechanisms by which Zn2+ regulates blood pressure may have an important therapeutic impact on hypertension. Funding: R21 DK119879, R01 DK-133698 (National Institute of Diabetes and Digestive and Kidney Diseases); R25DK078381 (NIH NIDDK STEP-UP program);1742339 (National Science Foundation ASK Program). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MiR-125a-5p/miR-125b-5p contributes to pathological activation of angiotensin II-AT1R in mouse distal convoluted tubule cells by the suppression of Atrap

The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.

Abstract 15468: Cd81 Interacts With Sodium Chloride Cotransporter in Lps-Induced Preeclampsia Rats and in Cultured Mouse Renal Distal Convoluted Tubule Cells

Kidney regulates blood pressure mainly through salt/water reabsorption affected by sodium transport proteins along nephron. We have reported that placental CD81 plays an important role in the pathogenesis of preelampsia. In order to explore the connection between CD81 and renal sodium transport proteins in preelampsia, we studied their interactions in vivo and in vitro. On the gestation day 18 of lipopolysaccharide-induced preeclamsia model rats with hypertension and albuminuria, the renal protein abundances of NKCC2 (150±14,% of control, n=5-6/group, mean±SE, t-test, same as below), p-NKCC2(567±70%), NCC (254±41) and WNK4 (196±12) were increased together with an increase of CD81 in kidney (225±46) and urinary exosomes(396±100). Renal NCC (215±30) was elevated while NKCC2 tended to increase at 17000g pellet in LPS group. There was no change of both NKCC2 and NCC at 200,000g pellet. In kidney homogenates, CD81 was coimmunoprecipitated with NKCC2, NCC, not NHE3 while NKCC2, NCC were coimmunoprecipitated with CD81 not CD63. In renal plasma-membrane-enriched fraction NCC, not NKCC2, was also increased in preeclampsia rats. In the apical membrane of renal tubules CD81 was colocalized with NCC while minimum colocalization was found between CD81 and NKCC2. In cultured mouse distal convoluted tubule cells, CD81 was colocalized with NCC while CD81 deletion with siRNA decreased NCC and partially reversed lipopolysaccharide-induced increase of NCC. Furthermore, CD81 was detected only in the urinary exosomes from preeclampsia patients along with increases in NKCC2 (6063±811) and NCC(1085±191)relative to normal pregnant women (n=6/group, t-test). Those findings suggest that CD81 interacts with renal NCC in vivo and in vitro and the increase of renal CD81 may up-regulate NCC, possibly NKCC2 as well, and contribute to the increased blood pressure in preeclampsia.

Abstract 12779: Up-Regulation of the Renal Sodium Transporters is Associated With Increased CD81 in Preeclampsia Rats and Patients

Introduction: the abnormalities of placental CD81 play an important role in preeclampsia (PE), a disorder that links to the impaired placentation in the early stages of pregnancy and the subsequent systemic endothelial cell activation. Hypothesis: up-regulation of the renal sodium transporters is associated with increased CD81 in preeclampsia rats and patients Methods: we examined the renal protein abundance of CD81 and sodium transporters, channels and pump along nephron in rat model with lipopolysaccharide (LPS) -induced PE. A single ultra-low dose of LPS (0.5ug/kg) (PE) or vehicle(control group,CTL) were applied to female SD rats( age: 8-12 wks, n=4/group) on gestational day 5 (GD5) by intraperitoneal injection. Results: The SBP (101±1.6 vs 116±0.8 mmHg, p<0.001, tail-cuff, BP-98A, Softron, Japan) and urine albumin (1885±35 vs 3550±157.8 ug, p<0.001) were higher in PE than CTL while there were no differences in BW, fetus number/weight and serum creatinine and cholesterol concentrations (GD18) between the 2 groups. The renal protein abundances of NKCC2 (168±14 vs 100±13, % of control,P=0.02,immunoblotting) and NCC (161±16 vs 100±13, P=0.04) , among the 8 sodium transport proteins checked, were greater in PE group than in CTL group along with the increase in renal protein abundances of CD81 in PE group relative to CTL group. The protein expressions of MCP-1, IL-6, TNF-a were similar between the 2 groups. CD81 were located in thick ascending limbs of Henle’s loop and distal convoluted tubules and co-immunoprecipitated with NKCC2 and NCC in rat homogenates. CD81 was not altered in placenta, serum, liver in the LPS-PE model indicated that the increase of CD81 is kidney-specific. In PE patients , urinary exosome CD81 was also increased along with increases of NKCC2 and NCC. Furthermore, the increases of CD81 and NCC were confirmed in cultured mouse distal convoluted tubule cells (DCT) stimulated by LPS for 24 hrs at different concentrations. The knockdown of CD81 by siRNA for 48 hrs had no effects on NCC but blocked the increase of NCC induced by LPS for 24 hr. Conclusions: inhibition and decrease of renal CD81 may reverse the increases of renal sodium transporters, therefore improve the hypertension in preeclampsia.

Hypomagnesemia and Hypokalemia: The Possible Role of Vasopressinase in This Rare Presentation of Gestational Diabetes Insipidus

Background: Gestational diabetes insipidus (GDI) is a rare but recognized complication of pregnancy. We are reporting the case of a 33-year-old patient who presented in her 3rd trimester with GDI, severe asymptomatic hypokalemia & hypomagnesemia. This presentation of GDI has not been reported yet to our knowledge. Clinical Case: A 33-year-old G2P1 patient who presented to the hospital due to the discovery of new onset severe asymptomatic hypokalemia on routine labs. Further evaluation revealed K of 2.8 mEq/L, Mg of 1.4 mg/dL & Na of 142mEq/L. She endorsed polyuria & polydipsia and a 24-hour urine collection of 9.35L confirmed diabetes insipidus. Labs also revealed a serum osmolality of 286mOsm/kg & urine K of 15mEq/L. Her urine osmolality was inappropriately low at 138mOsm/kg. She received K & Mg supplementation. In pregnant patients suspected of having GDI who have normal serum Na, water restriction test can be done for further evaluation. It must performed with close monitoring because dehydration can lead to uteroplacental insufficiency. Given her history of previous fetal demise & report of headaches with attempts of water restriction, we opted not to do the test. She was given subcutaneous 1 mcg of desmopressin overnight with her thirst & polyuria improving only briefly. Due to her suboptimal response, we began her on 10 mcg of intranasal spray of desmopressin once in the evening. Her symptoms resolved on this regimen after a few days of observation. Her Mg & eventually K levels reached and remained at normal levels after repletion. The rest of her pregnancy went on without complications and she delivered a healthy male infant via scheduled cesarean section at 36 weeks gestation. She discontinued intranasal desmopressin after 2 weeks and has remained asymptomatic. Labs at follow up 4 weeks later remained normal. It is known that vasopressinase which is made by placental trophoblasts during pregnancy degrade endogenous ADH1. We are hypothesizing that since ADH has been shown to be effective in conserving magnesium in rats by stimulating its uptake in the distal convoluted tubule cells2, a similar mechanism could be present in humans. We believe that the loss of endogenous ADH, leads to the depletion of Mg and consequently hypokalemia in susceptible patients. Conclusion: To our knowledge this is the first case presenting the possible role of vasopressinase in the development of hypomagnesemia & hypokalemia in a patient with GDI. Reference: 1. Gordge MP, Williams DJ, Huggett NJ, Payne NN, and Neild GH. Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum. Clinical Endocrinology, vol. 42, no. 1, pp. 51- 58, 1995. 2. Dai LJ, Bapty B, Ritchie G, Quamme GA. Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells. Am J Physiol. 1998 Feb;274(2):F328-35. doi: 10.1152/ajprenal.1998.274.2.F328. PMID: 9486227.

Activation of the kidney sodium chloride cotransporter by the β2-adrenergic receptor agonist salbutamol increases blood pressure

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In exvivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

Modeling Distal Convoluted Tubule (Patho)Physiology: An Overview of Past Developments and an Outlook Toward the Future.

The kidneys are essential for maintaining electrolyte homeostasis. Blood electrolyte composition is controlled by active reabsorption and secretion processes in dedicated segments of the kidney tubule. Specifically, the distal convoluted tubule (DCT) and connecting tubule are important for regulating the final excretion of sodium, magnesium, and calcium. Studies unravelling the specific function of these segments have greatly improved our understanding of DCT (patho)physiology. Over the years, experimental models used to study the DCT have changed and the field has advanced from early dissection studies with rats and rabbits to the use of various transgenic mouse models. Developments in dissection techniques and cell culture methods have resulted in immortalized mouse DCT cell lines and made it possible to specifically obtain DCT fragments for ex vivo studies. However, we still do not fully understand the complex (patho)physiology of this segment and there is need for advanced human DCT models. Recently, kidney organoids and tubuloids have emerged as new complex cell models that provide excellent opportunities for physiological studies, disease modeling, drug discovery, and even personalized medicine in the future. This review presents an overview of cell models used to study the DCT and provides an outlook on kidney organoids and tubuloids as model for DCT (patho)physiology. Impact statement This study provides a detailed overview of past and future developments on cell models used to study kidney (patho)physiology and specifically the distal convoluted tubule (DCT) segment. Hereby, we highlight the need for an advanced human cell model of this segment and summarize recent advances in the field of kidney organoids and tubuloids with a focus on DCT properties. The findings reported in this review are significant for future developments toward an advanced human model of the DCT that will help to increase our understanding of DCT (patho)physiology.

Abstract 16216: Increases in Renal Nkcc2 and Ncc Preceded the Elevation of Blood Pressure in Mice Treated With Clozapine

One of major side effects of clozapine, a common antipsychotic drug, is hypertension. In order to explore the pathogenesis of the drug-induced hypertension, we examined the renal sodium transport proteins and BP in the C57Bl6/J male mice treated with clozapine at different doses for 1 week. Clozapine at 20mg/kg/day via osmotic mini-pump increased SBP ( 110±0.6,mmHg, femoral artery, under anesthesia) and DBP (76.7±1) relative to vehicle (97.6±1.2/71.2±2.7) while the renal protein expression of NKCC2 (180±21, % of vehicle) and NCC (171±22), and ENaC-α (140±7), β(166±21) and γ(130±7) was increased without changes in NHE3 and α1-NKA (n=5/group). Clozapine at lower doses (5&10mg/kg/day,IP) did not change the conscious SBP and DBP monitored daily by tail-cuff, even on day 7 in 5 mg/kg group (115.61±1.6/90.1±1.3) and in 10mg/kg group (116.8±2/89.0±1.6) relative to vehicle (111.9±1.5/86.9±1.4) (n=4/group). Urinary excretion of Na + & K + and serum concentrations of Na + , K + , Cl - , creatinine were similar in all groups. However, renal protein expression of NKCC2 was increased in 5mg/kg (360±80, % of vehicle) and 10mg/kg (247±50) groups; NCC was increased at 5 mg/kg (175±9); α1-NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups. No increases were found in the protein expression of NHE3 and ENaCs. Those increases in renal NKCC2 &NCC at 5 & 10 mg/kg were consistent with those at 20 mg/kg. AT1R was increased at 5 (221±20) &10mg/kg (255±21) while renin (186±15) and ACE1 (186±5), not ACE2, were increased at 5 mg/kg suggesting an activation of RAAS in kidney. NOX4, not NOX2, was increased at 5 mg/kg (453±69) while NOS3, not NOS1&2, was decreased at 5 (60±21) and 10mg/kg (64±5) groups. TNFα, not IL-6 was increased at 5 (222±18) &10 mg/kg (321±26). In cultured mouse distal convoluted tubule cells, clozapine also increased NCC and α1-NKA at 1nM (169±4;156±7) and 10nm(175±23;183±15) respectively ( 24h,n=4/group). Those changes in kidney, including increases in sodium transport proteins, RAAS components, ROS generation enzymes, inflammation factors and decrease in NO synthase, preceded the elevation of BP suggesting that direct or indirect regulations of clozapine on those proteins may play an important role in the pathogenesis of the drug-induced hypertension.

FP037Functional significance of angiotensin II type 1 receptor signaling in mouse distal convoluted tubule cells

FP037Functional significance of angiotensin II type 1 receptor signaling in mouse distal convoluted tubule cells

Zinc deficiency induces hypertension by promoting renal Na+ reabsorption.

Zn2+ deficiency (ZnD) is a common comorbidity of many chronic diseases. In these settings, ZnD exacerbates hypertension. Whether ZnD alone is sufficient to alter blood pressure (BP) is unknown. To explore the role of Zn2+ in BP regulation, adult mice were fed a Zn2+-adequate (ZnA) or a Zn2+-deficient (ZnD) diet. A subset of ZnD mice were either returned to the ZnA diet or treated with hydrochlorothiazide (HCTZ), a Na+-Cl- cotransporter (NCC) inhibitor. To reduce intracellular Zn2+ in vitro, mouse distal convoluted tubule cells were cultured in N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, a Zn2+ chelator)- or vehicle (DMSO)-containing medium. To replete intracellular Zn2+, TPEN-exposed cells were then cultured in Zn2+-supplemented medium. ZnD promoted a biphasic BP response, characterized by episodes of high BP. BP increases were accompanied by reduced renal Na+ excretion and NCC upregulation. These effects were reversed in Zn2+-replete mice. Likewise, HCTZ stimulated natriuresis and reversed BP increases. In vitro, Zn2+ depletion increased NCC expression. Furthermore, TPEN promoted NCC surface localization and Na+ uptake activity. Zn2+ repletion reversed TPEN effects on NCC. These data indicate that 1) Zn2+ contributes to BP regulation via modulation of renal Na+ transport, 2) renal NCC mediates ZnD-induced hypertension, and 3) NCC is a Zn2+-regulated transporter that is upregulated with ZnD. This study links dysregulated renal Na+ handling to ZnD-induced hypertension. Furthermore, NCC is identified as a novel mechanism by which Zn2+ regulates BP. Understanding the mechanisms of ZnD-induced BP dysregulation may have an important therapeutic impact on hypertension.

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.

Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells

Thiazides block Na+ reabsorption while enhancing Ca2+ reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca2+ uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl− channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl− channels, however the nature of these Cl− channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl− current at extracellular pH7.4. This constitutive Cl− current was more permeable to larger anions (Eisenman sequence I; I−>Br−≥Cl−) and was substantially inhibited by >100mM [Ca2+]o, which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl− current was blocked by NPPB, along with other Cl− channel inhibitors (4,4′-diisothiocyanatostilbene-2,2′-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH<5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl− current. This acid-induced Cl− current was also anion permeable (I−>Br−>Cl−), but was distinguished from the constitutive Cl− current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl− current would be particularly relevant in the acidic IMCD (pH<5.5). To our knowledge, the properties of these Cl− currents are unique and provide the mechanisms to account for the Cl− efflux previously speculated to be present in MDCT cells.

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.

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.

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.

A Role for the Circadian Clock Protein Per1 in the Regulation of the NaCl Co-transporter (NCC) and the with-no-lysine Kinase (WNK) Cascade in Mouse Distal Convoluted Tubule Cells

It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein

The angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP/Agtrap) promotes constitutive internalization of the AT1R so as to specifically inhibit the pathological activation of its downstream signaling yet preserve the base-line physiological signaling activity of the AT1R. Thus, tissue-specific regulation of Agtrap expression is relevant to the pathophysiology of cardiovascular and renal disease. However, the regulatory mechanism of Agtrap gene expression has not yet been fully elucidated. In this study, we show that the proximal promoter region from -150 to +72 of the mouse Agtrap promoter, which contains the X-box, E-box, and GC-box consensus motifs, is able to elicit substantial transcription of the Agtrap gene. Among these binding motifs, we showed that the E-box specifically binds upstream stimulatory factor (Usf) 1 and Usf2, which are known E-box-binding transcription factors. It is indicated that the E-box-Usf1/Usf2 binding regulates Agtrap expression because of the following: 1) mutation of the E-box to prevent Usf1/Usf2 binding reduces Agtrap promoter activity; 2) knockdown of Usf1 or Usf2 affects both endogenous Agtrap mRNA and Agtrap protein expression, and 3) the decrease in Agtrap mRNA expression in the afflicted kidney by unilateral ureteral obstruction is accompanied by changes in Usf1 and Usf2 mRNA. Furthermore, the results of siRNA transfection in mouse distal convoluted tubule cells and those of unilateral ureteral obstruction in the afflicted mouse kidney suggest that Usf1 decreases but Usf2 increases the Agtrap gene expression by binding to the E-box. The results also demonstrate a functional E-box-USF1/USF2 interaction in the human AGTRAP promoter, thereby suggesting that a strategy of modulating the E-box-USF1/USF2 binding has novel therapeutic potential.

Enhanced Angiotensin Receptor-Associated Protein in Renal Tubule Suppresses Angiotensin-Dependent Hypertension

We have previously shown that angiotensin II type 1 receptor-associated protein (ATRAP/Agtrap) interacts with the angiotensin II type 1 receptor and promotes constitutive internalization of the receptor so as to inhibit the pathological activation of its downstream signaling but preserve baseline physiological signaling activity. The present study was designed to investigate the role of renal ATRAP in angiotensin II-dependent hypertension. We generated transgenic mice dominantly expressing ATRAP in the renal tubules, including renal distal tubules. The renal ATRAP transgenic mice exhibited no significant change in blood pressure at baseline on normal salt diet. However, in the renal ATRAP transgenic mice compared with wild-type mice, the following took place: (1) the development of high blood pressure in response to angiotensin II infusion was significantly suppressed based on radiotelemetry, (2) the extent of daily positive sodium balance was significantly reduced during angiotensin II infusion in metabolic cage analysis, and (3) the renal Na+ -Cl- cotransporter activation and α-subunit of the epithelial sodium channel induction by angiotensin II infusion were inhibited. Furthermore, adenoviral overexpression of ATRAP suppressed the angiotensin II-mediated increase in the expression of α-subunit of the epithelial sodium channel in mouse distal convoluted tubule cells. These results indicate that renal tubule-dominant ATRAP activation provokes no evident effects on blood pressure at baseline but exerts an inhibitory effect on the pathological elevation of blood pressure in response to angiotensin II stimulation, thereby suggesting that ATRAP is a potential target of interest in blood pressure modulation under pathological conditions.