Transport, cilia, and PKD: must we in (cyst) on interrelationships? Focus on “Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease”

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.

Download Full-text

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

AJP Cell Physiology ◽

10.1152/ajpcell.00339.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. C952-C963 ◽

Cited By ~ 34

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.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.00171.2003 ◽

2003 ◽

Vol 285 (6) ◽

pp. F1168-F1178 ◽

Cited By ~ 28

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.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms22116019 ◽

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.

Download Full-text

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

Nature Communications ◽

10.1038/s41467-019-11918-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 19

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.

Download Full-text

Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.3.f387 ◽

1998 ◽

Vol 275 (3) ◽

pp. F387-F394 ◽

Cited By ~ 15

Author(s):

William E. Sweeney ◽

Ellis D. Avner

Keyword(s):

Growth Factor ◽

Kidney Disease ◽

Epidermal Growth Factor ◽

Polycystic Kidney Disease ◽

Autosomal Recessive ◽

Apical Surface ◽

Polycystic Kidney ◽

High Affinity ◽

Epidermal Growth

Evidence from a number of laboratories suggests a potential role for the epidermal growth factor (EGF)-transforming growth factor-α-epidermal growth factor receptor (EGF-R) axis in promoting epithelial hyperplasia and cyst formation in autosomal recessive polycystic kidney disease (ARPKD). As previously reported, in the C57BL-6Jcpk/cpk (CPK), BALB/c-bpk/bpk (BPK), and C3H-orpk/orpk (ORPK) murine models of ARPKD, as well as in human ARPKD and human ADPKD, the EGF-R is mislocated to the apical surface of cystic collecting tubule (CT) epithelial cells. The present studies demonstrate that cells from cystic and control CTs can be isolated and that these cells maintain their in vivo EGF-R phenotype in vitro. Domain-specific high-affinity ligand binding was assessed by standard Scatchard analysis, and selective ligand stimulation of apical vs. basolateral EGF-R in these cells was followed by measurement of receptor autophosphorylation and determination of cell proliferation. These studies demonstrate that in vitro apically expressed EGF-Rs exhibit high-affinity binding for EGF, autophosphorylate in response to EGF, and transmit a mitogenic signal when stimulated by the appropriate ligand.

Download Full-text

Collecting Duct Carcinoma Exhibiting Diastase-Resistant PAS-Positive Globular Cytoplasmic Inclusions and Rhabdoid Features Arising in Adult Polycystic Kidney Disease: A Case Report

International Journal of Surgical Pathology ◽

10.1177/106689690401200215 ◽

2004 ◽

Vol 12 (2) ◽

pp. 171-177 ◽

Cited By ~ 8

Author(s):

Naoto Kuroda ◽

Hirofumi Satake ◽

Eriko Miyazaki ◽

Yoshihiro Hayashi ◽

Makoto Hiroi ◽

...

Keyword(s):

Case Report ◽

Kidney Disease ◽

Polycystic Kidney Disease ◽

Collecting Duct ◽

Polycystic Kidney ◽

Adult Polycystic Kidney Disease ◽

Duct Carcinoma ◽

Cytoplasmic Inclusions ◽

Collecting Duct Carcinoma ◽

Rhabdoid Features

Download Full-text

Cystin gene mutations cause autosomal recessive polycystic kidney disease associated with altered Myc expression

10.1101/2020.02.18.946285 ◽

2020 ◽

Author(s):

Chaozhe Yang ◽

Amber K. O’Connor ◽

Robert A. Kesterson ◽

Jacob A. Watts ◽

Amar J. Majmundar ◽

...

Keyword(s):

Kidney Disease ◽

Fusion Protein ◽

Polycystic Kidney Disease ◽

Autosomal Recessive ◽

Collecting Duct ◽

Polycystic Kidney ◽

Specific Expression ◽

Human Patient ◽

Gfp Fusion Protein ◽

First Case

AbstractMutation of the Cys1 gene underlies the renal cystic disease in the Cys1cpk/cpk (cpk) mouse that phenocopies human autosomal recessive polycystic kidney disease (ARPKD). Cystin, the protein product of Cys1, is expressed in the primary apical cilia of renal ductal epithelial cells. In previous studies, we showed that cystin regulates Myc expression via interaction with the tumor suppressor, necdin. Here, we demonstrate rescue of the cpk renal phenotype by kidney-specific expression of a cystin-GFP fusion protein encoded by a transgene integrated into the Rosa26 locus. In addition, we show that expression of the cystin-GFP fusion protein in collecting duct cells down-regulates expression of Myc in cpk kidneys. Finally, we report the first human patient with an ARPKD phenotype due to homozygosity for a predicted deleterious splicing defect in CYS1. These findings suggest that mutations in the Cys1 mouse and CYS1 human orthologues cause an ARPKD phenotype that is driven by overexpression of the Myc proto-oncogene.Translational StatementThe cystin-deficient cpk mouse is a model for the study of autosomal recessive polycystic kidney disease (ARPKD). We show that the cpk mouse phenotype is associated with altered Myc expression. To date, the clinical relevance of cystin deficiency to human disease was unclear, due to the absence of ARPKD cases associated with CYS1 mutations. We report the first case of ARPKD linked to a CYS1 mutation disrupting normal splicing. These findings confirm the relevance of cystin deficiency to human ARPKD, implicate Myc in disease initiation or progression, and validate the cpk mouse as a translationally relevant disease model.

Download Full-text

MO007ANALYSIS OF CALCIUM SIGNALING IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD)

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab079.003 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Dorien Van Giel ◽

Jean-Paul Decuypere ◽

Djalila Mekahli ◽

Rudi Vennekens

Keyword(s):

Kidney Disease ◽

Epithelial Cells ◽

Polycystic Kidney Disease ◽

Molecular Mechanisms ◽

Autosomal Dominant ◽

Trp Channels ◽

Cell Model ◽

Healthy Controls ◽

Polycystic Kidney ◽

Autosomal Dominant Polycystic Kidney

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inheritable kidney disease characterized by the development of fluid-filled cysts in all nephron segments, leading to loss of renal function. Mutations in PKD1 or PKD2, which encode polycystin-1 and polycystin-2, are the most common cause of ADPKD. The molecular mechanisms underlying cystogenesis are poorly characterized but it is postulated that disturbed calcium homeostasis is a primary event in cystogenesis. The precise molecular players that cause this disturbance are still a poorly explored area, especially in relevant human cell types. We therefore aim to characterize the profile of calcium-coupled receptors and channels in a human renal epithelial cell model, to identify which receptors and channels are present and whether their function is affected in ADPKD. Method Human urine-derived conditionally immortalized proximal tubule epithelial cells (ciPTECs) of ADPKD patients and healthy controls were screened for calcium-coupled GPCRs, using a GPCR agonist library on Fura-2 loaded cell populations seeded in 96-well format using the Flexstation3 (Molecular Devices). Validation of specific hits was done using single-cell measurements with a fluorescence microscope and built-in perfusion system. The expression of TRP channels and STIM/Orai proteins was determined via qPCR. Results From a library of 418 GPCR agonists a selective amount of calcium-coupled GPCRs was found functionally active in ciPTECs. ciPTECs from both healthy controls and ADPKD patients were found to functionally express purinergic -, histamine -, serotonin and dopamine receptors. Through qPCR we found expression of various TRP channels, including TRPML1, TRPC1/3, TRPM3/4/7, TRPV4 and TRPA1, as well as high expression of STIM1/2 and Orai1/2/3. Conclusion We describe the first thorough characterization of molecular players involved in calcium signalling mechanisms in human renal epithelial cells, including the profile of calcium-coupled GPCRs and the expression of TRP channels and STIM/Orai proteins, of further interest to investigate disturbed calcium dynamics in ADPKD.

Download Full-text