scholarly journals Increased Na+/H+exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease

2012 ◽  
Vol 302 (10) ◽  
pp. C1436-C1451 ◽  
Author(s):  
Dragos Olteanu ◽  
Xiaofen Liu ◽  
Wen Liu ◽  
Venus C. Roper ◽  
Neeraj Sharma ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Collecting Duct ◽  
Principal Cell ◽  
Cortical Collecting Duct ◽  
Wild Type ◽  
Polycystic Kidney ◽  
Acid Base ◽  
Collecting Ducts ◽  
Mutant Cells

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 ( Ift88Tg737Rpw) 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 (pHi) 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 CO2/HCO3−. However, only the mutant cells displayed appreciable apical Na+-induced pHirecoveries from NH4+prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pHidependence 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 (pHi6.23–6.68). 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.

Mechanoregulation of intracellular Ca2+ concentration is attenuated in collecting duct of monocilium-impaired orpk mice

2005 ◽  
Vol 289 (5) ◽  
pp. F978-F988 ◽  
Author(s):  
Wen Liu ◽  
Noel S. Murcia ◽  
Yi Duan ◽  
Sheldon Weinbaum ◽  
Bradley K. Yoder ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Primary Cilia ◽  
Collecting Duct ◽  
Polycystic Kidney ◽  
New Findings ◽  
Collecting Ducts ◽  
Before And After ◽  
Luminal Flow

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the progressive dilatation of collecting ducts, the nephron segments responsible for the final renal regulation of sodium, potassium, acid-base, and water balance. Murine models of ARPKD possess mutations in genes encoding cilia-associated proteins, including Tg737 in orpk mice. New findings implicate defects in structure/function of primary cilia as central to the development of polycystic kidney disease. Our group (Liu W, Xu S, Woda C, Kim P, Weinbaum S, and Satlin LM, Am J Physiol Renal Physiol 285: F998–F1012, 2003) recently reported that increases in luminal flow rate in rabbit collecting ducts increase intracellular Ca2+ concentration ([Ca2+]i) in cells therein. We thus hypothesized that fluid shear acting on the apical membrane or hydrodynamic bending moments acting on the cilium increase renal epithelial [Ca2+]i. To further explore this, we tested whether flow-induced [Ca2+]i transients in collecting ducts from mutant orpk mice, which possess structurally abnormal cilia, differ from those in controls. Isolated segments from 1- and 2-wk-old mice were microperfused in vitro and loaded with fura 2; [Ca2+]i was measured by digital ratio fluorometry before and after the rate of luminal flow was increased. All collecting ducts responded to an increase in flow with an increase in [Ca2+]i, a response that appeared to be dependent on luminal Ca2+ entry. However, the magnitude of the increase in [Ca2+]i in 2- but not 1-wk-old mutant orpk animals was blunted. We speculate that this defect in mechano-induced Ca2+ signaling in orpk mice leads to aberrant structure and function of the collecting duct in ARPKD.

Heightened epithelial Na+ channel-mediated Na+ absorption in a murine polycystic kidney disease model epithelium lacking apical monocilia

2006 ◽  
Vol 290 (4) ◽  
pp. C952-C963 ◽  
Author(s):  
Dragos Olteanu ◽  
Bradley K. Yoder ◽  
Wen Liu ◽  
Mandy J. Croyle ◽  
Elisabeth A. Welty ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Collecting Duct ◽  
Short Circuit ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Polycystic Kidney ◽  
Luminal Membrane ◽  
And Function ◽  
Amiloride Analogs

The Tg737° rpk autosomal recessive polycystic kidney disease (ARPKD) mouse carries a hypomorphic mutation in the Tg737 gene. Because of the absence of its protein product Polaris, the nonmotile primary monocilium central to the luminal membrane of ductal epithelia, such as the cortical collecting duct (CCD) principal cell (PC), is malformed. Although the functions of the renal monocilium remain elusive, primary monocilia or flagella on neurons act as sensory organelles. Thus we hypothesized that the PC monocilium functions as a cellular sensor. To test this hypothesis, we assessed the contribution of Polaris and cilium structure and function to renal epithelial ion transport electrophysiology. Properties of Tg737° rpk mutant CCD PC clones were compared with clones genetically rescued with wild-type Tg737 cDNA. All cells were grown as polarized cell monolayers with similarly high transepithelial resistance on permeable filter supports. Three- to fourfold elevated transepithelial voltage ( Vte) and short-circuit current ( Isc) were measured in mutant orpk monolayers vs. rescued controls. Pharmacological and cell biological examination of this enhanced electrical end point in mutant monolayers revealed that epithelial Na+ channels (ENaCs) were upregulated. Amiloride, ENaC-selective amiloride analogs (benzamil and phenamil), and protease inhibitors (aprotinin and leupeptin) attenuated heightened Vte and Isc. Higher concentrations of additional amiloride analogs (ethylisopropylamiloride and dimethylamiloride) also revealed inhibition of Vte. Cell culture requirements and manipulations were also consistent with heightened ENaC expression and function. Together, these data suggest that ENaC expression and/or function are upregulated in the luminal membrane of mutant, cilium-deficient orpk CCD PC monolayers vs. cilium-competent controls. When the genetic lesion causes loss or malformation of the monocilium, ENaC-driven Na+ hyperabsorption may explain the rapid emergence of severe hypertension in a majority of patients with ARPKD.

scholarly journals The isolated polycystin-1 cytoplasmic COOH terminus prolongs ATP-stimulated Cl– conductance through increased Ca2+ entry

2003 ◽  
Vol 285 (6) ◽  
pp. F1168-F1178 ◽  
Author(s):  
Scott S. Wildman ◽  
Kimberly M. Hooper ◽  
Clare M. Turner ◽  
James S. K. Sham ◽  
Edward G. Lakatta ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Fusion Protein ◽  
Polycystic Kidney Disease ◽  
Collecting Duct ◽  
Control Cell ◽  
Fluid Secretion ◽  
Cortical Collecting Duct ◽  
Polycystic Kidney ◽  
Pkd1 Gene ◽  
Autosomal Dominant Polycystic Kidney

The precise steps leading from mutation of the polycystic kidney disease ( PKD1) gene to the autosomal dominant polycystic kidney disease (ADPKD) phenotype remain to be established. Fluid accumulation is a requirement for cyst expansion in ADPKD, suggesting that abnormal fluid secretion into the cyst lumen might play a role in disease. In this study, we sought to establish a link between polycystin-1 (the PKD1 gene product) and ATP-stimulated Cl– secretion in renal tubule cells. To do this, we performed a whole cell patch-clamp analysis of the effects of expression of the isolated cytoplasmic COOH-terminus of polycystin-1 in stably transfected mouse cortical collecting duct cells. The truncated polycystin-1 fusion protein prolonged the duration of ATP-stimulated Cl– conductance and intracellular Ca2+ responses. Both effects were dependent on extracellular Ca2+. It was determined that expression of the truncated polycystin-1 fusion protein introduced, or activated, an ATP-induced Ca2+ entry pathway that was undetectable in transfection control cell lines. Our findings are concordant with increasing evidence for a role of polycystin-1 in cell Ca2+ homeostasis and indicate that dysregulated Ca2+ entry might promote Cl– secretion and cyst expansion in ADPKD.

scholarly journals Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca2+ Signals

2021 ◽  
Vol 22 (11) ◽  
pp. 6019
Author(s):  
Khaoula Talbi ◽  
Inês Cabrita ◽  
Rainer Schreiber ◽  
Karl Kunzelmann
Keyword(s):  
Cell Proliferation ◽  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Collecting Duct ◽  
Primary Cells ◽  
Loss Of Function ◽  
Polycystic Kidney ◽  
Chloride Currents ◽  
Duct Cells ◽  
Collecting Duct Cells

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl− channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1−/−, was larger in male when compared to female Pkd1−/− cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1−/− males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.

scholarly journals Effect of Trehalose Supplementation on Autophagy and Cystogenesis in a Mouse Model of Polycystic Kidney Disease

Nutrients ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 42 ◽  
Author(s):  
Li-Fang Chou ◽  
Ya-Lien Cheng ◽  
Chun-Yih Hsieh ◽  
Chan-Yu Lin ◽  
Huang-Yu Yang ◽  
...  
Keyword(s):  
Drinking Water ◽  
Kidney Disease ◽  
Mouse Model ◽  
Polycystic Kidney Disease ◽  
Renal Cyst ◽  
Pure Water ◽  
Immunohistochemical Analysis ◽  
Positive Staining ◽  
Wild Type ◽  
Polycystic Kidney

Autophagy impairment has been demonstrated in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) and could be a new target of treatment. Trehalose is a natural, nonreducing disaccharide that has been shown to enhance autophagy. Therefore, we investigated whether trehalose treatment reduces renal cyst formation in a Pkd1-hypomorphic mouse model. Pkd1 miRNA transgenic (Pkd1 miR Tg) mice and wild-type littermates were given drinking water supplemented with 2% trehalose from postnatal day 35 to postnatal day 91. The control groups received pure water or 2% sucrose for the control of hyperosmolarity. The effect on kidney weights, cystic indices, renal function, cell proliferation, and autophagic activities was determined. We found that Pkd1 miR Tg mice had a significantly lower renal mRNA expression of autophagy-related genes, including atg5, atg12, ulk1, beclin1, and p62, compared with wild-type control mice. Furthermore, immunohistochemical analysis showed that cystic lining cells had strong positive staining for the p62 protein, indicating impaired degradation of the protein by the autophagy-lysosome pathway. However, trehalose treatment did not improve reduced autophagy activities, nor did it reduce relative kidney weights, plasma blood urea nitrogen levels, or cystatin C levels in Pkd1 miR Tg mice. Histomorphological analysis revealed no significant differences in the renal cyst index, fibrosis score, or proliferative score among trehalose-, sucrose-, and water-treated groups. Our results demonstrate that adding trehalose to drinking water does not modulate autophagy activities and renal cystogenesis in Pkd1-deficient mice, suggesting that an oral supplement of trehalose may not affect the progression of ADPKD.

scholarly journals Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Edmund C. Lee ◽  
Tania Valencia ◽  
Charles Allerson ◽  
Annelie Schairer ◽  
Andrea Flaten ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Collecting Duct ◽  
Treatment Options ◽  
Subcutaneous Administration ◽  
Polycystic Kidney ◽  
Short Oligonucleotide ◽  
Mrna Targets ◽  
Stage Renal Disease

Abstract Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.

Deoxycorticosterone-stimulated bicarbonate secretion in rabbit cortical collecting ducts: effects of luminal chloride removal and in vivo acid loading

1985 ◽  
Vol 249 (2) ◽  
pp. F205-F212 ◽  
Author(s):  
J. Garcia-Austt ◽  
D. W. Good ◽  
M. B. Burg ◽  
M. A. Knepper
Keyword(s):  
Collecting Duct ◽  
High Rate ◽  
Bicarbonate Secretion ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Urinary Ph ◽  
Collecting Ducts ◽  
Acid Loading

To assess the role of cortical collecting duct bicarbonate secretion in the regulation of net acid excretion, we have sought to identify what factors influence the secretion rate. Net and unidirectional bicarbonate fluxes were measured in isolated perfused cortical collecting ducts from deoxycorticosterone-treated rabbits. The collecting ducts secreted bicarbonate at 11-24 pmol X mm-1 X min-1, confirming the high rate seen in earlier studies. Oral acid loading (50 mM NH4Cl drinking water) completely inhibited the net bicarbonate secretion. The bath-to-lumen flux was markedly reduced with acid loading, but the lumen-to-bath flux changed very little. In tubules from rabbits treated with deoxycorticosterone (but not NH4Cl), luminal chloride replacement with either sulfate or gluconate completely and reversibly inhibited the net bicarbonate secretion. The bath-to-lumen flux was greatly inhibited, but there was little change in the lumen-to-bath flux. We conclude: 1) High rates of bicarbonate secretion can be induced in rabbit cortical collecting ducts by chronic treatment of the animals with deoxycorticosterone. 2) When deoxycorticosterone-treated rabbits were made acidotic by oral administration of NH4Cl, the bicarbonate secretion was prevented, indicating that the systemic acid-base state of the animal may be an important factor regulating bicarbonate secretion. 3) Replacement of chloride in the lumen with sulfate inhibits bicarbonate secretion in the cortical collecting duct, an effect which may explain in part the decrease in urinary pH in response to sulfate infusions in mineralocorticoid-stimulated animals.

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