Project description:Transcriptional profiling of new born mouse kidney collecting duct (CD) cells comparing the infuence of gestational high salt stress on gene expression remolding of BdkrB2 receptor knockout CD cells with that of BdkrB2 receptor wild type CD cells. The BdkrB2 receptor has been shown to be playing a role in renal vascular tone, kidney secretion and reabsorption function, normal kidney development, while impaired BdkrB2 receptor in kidney shown being associated with renal agenesis and renal dysplasia. Goal was to determine the effects of BdkrB2 receptor knockout together with gestational high salt stress on collecting duct gene expression pattern. Single color microarray experiment, BdkrB2 knockout new born mouse CD cells vs. BdkrB2 WT mosue CD cells with both on gestational high salt stress. Biological replicates: 3 BdkrB2 null replicates, 3 BdkrB2 WT replicates. Expression level of each sample was normalized to WT1 replicate.
Project description:Nitric oxide is a pronatriuretic and prodiuretic factor. The highest renal NO synthase (NOS) activity is found in the inner medullary collecting duct. The collecting duct (CD) is the site of daily fine-tune regulation of sodium balance, and led us to hypothesize that a CD-specific deletion of NOS1 would result in an impaired ability to excrete a sodium load leading to a salt-sensitive blood pressure phenotype. We bred AQP2-CRE mice with NOS1 floxed mice to produce flox control and CD-specific NOS1 knockout (CDNOS1KO) littermates. CDs from CDNOS1KO mice produced 75% less nitrite, and urinary nitrite+nitrate (NOx) excretion was significantly blunted in the knockout genotype. When challenged with high dietary sodium, CDNOS1KO mice showed significantly reduced urine output, sodium, chloride, and NOx excretion, and increased mean arterial pressure relative to flox control mice. In humans, urinary NOx is a newly identified biomarker for the progression of hypertension. These findings reveal that NOS1 in the CD is critical in the regulation of fluid-electrolyte balance, and this new genetic model of CD NOS1 gene deletion will be a valuable tool to study salt-dependent blood pressure mechanisms.
Project description:Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells.For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines.Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells.Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.
Project description:(Pro)renin receptor (PRR), a component of the renin-angiotensin system, has emerged as a new regulator of collecting duct function. The present study was designed to investigate the role of PRR in high salt-induced apoptosis in cultured mouse inner medullary collecting duct cells, mIMCD-K2 cells. Exposure to high NaCl at 550 mosM/kgH2O increased PRR protein abundance, as did exposure to mannitol, sodium gluconate, or choline chloride. This was accompanied by upregulation of the abundance of phosphorylated NF-κB p65 protein. NF-κB inhibition with QNZ, caffeic acid phenethyl ester, or small interfering RNA (siRNA)-mediated silencing of NF-κB p65 attenuated high-NaCl-induced PRR upregulation. Exposure to high salt for 24 h induced apoptosis, as assessed by immunoblotting and immunocytochemistry analysis of cleaved caspase-3 and flow cytometry analysis of the number of apoptotic cells. High-NaCl-induced apoptosis was attenuated by a PRR decoy inhibitor, PRO20, or siRNA-mediated silencing of NF-κB p65. These results show that induction of PRR expression by exposure to high NaCl occurs through activation of NF-κB, thus contributing to cell apoptosis.
Project description:We elucidated the role of collecting duct kinin B2 receptor (B2R) in the development of salt-sensitivity and angiotensin II (ANG II)-induced hypertension. To this end, we used a Cre-Lox recombination strategy to generate mice lacking Bdkrb2 gene for B2R in the collecting duct (Hoxb7-Cre(tg/+):Bdkrb2(flox/flox)). In 3 groups of control (Bdkrb2(flox/flox)) and 3 groups of UB(Bdkrb2-/-) mice, systolic blood pressure (SBP) responses to high salt intake (4 or 8% NaCl; HS) were monitored by radiotelemetry in comparison with standard salt diet (0.4% NaCl) prior to and during subcutaneous ANG II infusion (1000?ng/min/kg) via osmotic minipumps. High salt intakes alone for 2 weeks did not alter SBP in either strain. ANG II significantly increased SBP equally in control (121?±?2 to 156?±?3?mmHg) and UB(Bdkrb2-/-) mice (120?±?2 to 153?±?2?mmHg). The development of ANG II-induced hypertension was exacerbated by 4%HS in both control (125?±?3 to 164?±?5?mmHg) and UB(Bdkrb2-/-) mice (124?±?2 to 162?±?3?mmHg) during 2 weeks. Interestingly, 8%HS caused a more profound and earlier ANG II-induced hypertension in UB(Bdkrb2-/-) (129?±?2 to 166?±?3?mmHg) as compared to control (128?±?2 to 158?±?2?mmHg) and it was accompanied by body weight loss and increased mortality. In conclusion, targeted inactivation of B2R in the renal collecting duct does not cause salt-sensitivity; however, collecting duct B2R attenuates the hypertensive actions of ANG II under conditions of very high salt intake.
Project description:Pathophysiological anomalies in autosomal dominant and recessive forms of polycystic kidney disease (PKD) may derive from impaired function/formation of the apical central monocilium of ductal epithelia such as that seen in the Oak Ridge polycystic kidney or orpk (Ift88(Tg737Rpw)) mouse and its immortalized cell models for the renal collecting duct. According to a previous study, Na/H exchanger (NHE) activity may contribute to hyperabsorptive Na(+) movement in cilium-deficient ("mutant") cortical collecting duct principal cell monolayers derived from the orpk mice compared with cilium-competent ("rescued") monolayers. To examine NHE activity, we measured intracellular pH (pH(i)) by fluorescence imaging with the pH-sensitive dye BCECF, and used a custom-designed perfusion chamber to control the apical and basolateral solutions independently. Both mutant and rescued monolayers exhibited basolateral Na(+)-dependent acid-base transporter activity in the nominal absence of CO(2)/HCO(3)(-). However, only the mutant cells displayed appreciable apical Na(+)-induced pH(i) recoveries from NH(4)(+) prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pH(i) dependence of basolateral cariporide/HOE-694-sensitive NHE activity under our experimental conditions was similar in both mutant and rescued cells, and 3.5- to 4.5-fold greater than apical HOE-sensitive NHE activity in the mutant cells (pH(i) 6.23-6.68). Increased apical NHE activity correlated with increased apical NHE1 expression in the mutant cells, and increased apical localization in collecting ducts of kidney sections from orpk vs. control mice. A kidney-specific conditional cilium-knockout mouse produced a more acidic urine compared with wild-type littermates and became alkalotic by 28 days of age. This study provides the first description of altered NHE activity, and an associated acid-base anomaly in any form of PKD.
Project description:Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid water/glycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-d-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to approximately 30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3/AQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.
Project description:Mice with a collecting duct-specific deletion of endothelin-1 are hypertensive and have impaired Na excretion. Because endothelin-1 activates NO synthase (NOS) in the collecting duct, we hypothesized that impaired renal NO production in knockout mice exacerbates the hypertensive state. Control and knockout mice were treated chronically with N(G)-nitro-l-arginine methyl ester, and blood pressure (BP) and urinary nitrate/nitrite excretion were assessed. On a normal Na diet, knockout systolic BP was 18 mm Hg greater than in controls. N(G)-nitro-l-arginine methyl ester increased BP in control mice by 30 mm Hg and 10 mm Hg in collecting duct-specific deletion of endothelin-1 knockout mice, thereby abolishing the difference in systolic BP between the groups. A high-Na diet increased BP similarly in both groups. Urinary nitrate/nitrite excretion was lower in knockout mice than in controls on normal or high Na intake. In separate experiments, renal perfusion pressure was adjusted in anesthetized mice, and urinary nitrate/nitrite and Na excretion were determined. Similar elevations of BP increased urinary Na and nitrate/nitrite excretion in control mice but to a significantly lesser extent in knockout mice. Isoform-specific NOS activity and expression were determined in renal inner medulla homogenates from control and knockout mice. NOS1 and NOS3 activities were lower in knockout than in control mice given normal or high-Na diets. However, NOS1 or NOS3 protein expressions were similar in both groups on normal or high-Na intake. These data demonstrate that collecting duct-derived endothelin-1 is important in the following: (1) chronic N(G)-nitro-l-arginine methyl ester-induced hypertension; (2) full expression of pressure-dependent changes in sodium excretion; and (3) control of inner medullary NOS1 and NOS3 activity.
Project description:To maintain potassium homeostasis, kidneys exert flow-dependent potassium secretion to facilitate kaliuresis in response to elevated dietary potassium intake. This process involves stimulation of calcium-activated large conductance maxi-K (BK) channels in the distal nephron, namely the connecting tubule and the collecting duct. Recent evidence suggests that the TRPV4 channel is a critical determinant of flow-dependent intracellular calcium elevations in these segments of the renal tubule. Here, we demonstrate that elevated dietary potassium intake (five percent potassium) increases renal TRPV4 mRNA and protein levels in an aldosterone-dependent manner and causes redistribution of the channel to the apical plasma membrane in native collecting duct cells. This, in turn, leads to augmented TRPV4-mediated flow-dependent calcium ion responses in freshly isolated split-opened collecting ducts from mice fed the high potassium diet. Genetic TRPV4 ablation greatly diminished BK channel activity in collecting duct cells pointing to a reduced capacity to excrete potassium. Consistently, elevated potassium intake induced hyperkalemia in TRPV4 knockout mice due to deficient renal potassium excretion. Thus, regulation of TRPV4 activity in the distal nephron by dietary potassium is an indispensable component of whole body potassium balance.
Project description:The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl(-)/HCO(3)(-) exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO(3)(-) concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl(-)/HCO(3)(-) exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH(4)(+) (ammonium) excretion increased significantly, consistent with increased trapping of NH(3) in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl(-)/HCO(3)(-) exchanger(s) is (are) present in the CCD.