scholarly journals Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts

2003 ◽  
Vol 285 (3) ◽  
pp. F397-F412 ◽  
Author(s):  
Mahmoud Loghman-Adham ◽  
Surya M. Nauli ◽  
Carlos E. Soto ◽  
Barbara Kariuki ◽  
Jing Zhou
Keyword(s):  
Cell Lines ◽  
Autosomal Dominant ◽  
Primary Cultures ◽  
Cyst Formation ◽  
T Antigen ◽  
Pcr Analysis ◽  
Phenotypic Change ◽  
Polycystic Kidney ◽  
Autosomal Dominant Polycystic Kidney

Autosomal dominant polycystic kidney disease (ADPKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by “second hit” somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with ADPKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37°C in the WT cell lines and at 33°C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39°C. The majority ( 29 ) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD.

A role for CFTR in human autosomal dominant polycystic kidney disease

1996 ◽  
Vol 270 (1) ◽  
pp. C389-C399 ◽  
Author(s):  
K. Hanaoka ◽  
O. Devuyst ◽  
E. M. Schwiebert ◽  
P. D. Wilson ◽  
W. B. Guggino
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Autosomal Dominant ◽  
Primary Cultures ◽  
Cyst Formation ◽  
Disulfonic Acid ◽  
Polycystic Kidney ◽  
Acid Sensitivity ◽  
Autosomal Dominant Polycystic Kidney

Human autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal dominant hereditary disorder characterized by enormous renal enlargement and the development of multiple cysts originating from nephrons. We investigated the pathogenesis of cyst formation in ADPKD by using patch-clamp and immunocytochemical techniques. Adenosine 3',5'-cyclic monophosphate-activated Cl- currents are present in primary cultures of ADPKD cells and have characteristics such as a linear current-voltage relation, insensitivity to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, sensitivity to glibenclamide and diphenylamine carboxylic acid, and an anion selectivity sequence of Br- > Cl- > I- > glutamate, all of which are identical to cystic fibrosis transmembrane conductance regulator (CFTR). With the use of CFTR antibodies raised against the regulatory and first nucleotide-binding domains, CFTR was detected in primary cultures of ADPKD cells. Similar results were obtained in vivo in cyst-lining epithelial cells in ADPKD kidneys, where staining was seen associated with the apical membrane regions. These data indicate that the CFTR Cl- channel exists in apical membranes of ADPKD cells and may play an important role in cyst formation or enlargement.

Autosomal dominant polycystic kidney disease decreases anion exchanger activity

1997 ◽  
Vol 272 (5) ◽  
pp. C1748-C1756 ◽  
Author(s):  
R. D. Perrone ◽  
S. A. Grubman ◽  
S. L. Murray ◽  
D. W. Lee ◽  
S. L. Alper ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Cell Line ◽  
Polycystic Kidney Disease ◽  
Cell Lines ◽  
Anion Exchanger ◽  
Autosomal Dominant ◽  
T Antigen ◽  
Polycystic Kidney ◽  
Liver Cysts ◽  
Autosomal Dominant Polycystic Kidney

Liver cysts, the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD), derive from the intrahepatic biliary epithelium (IBE) and are found in 60-75% of ADPKD patients on dialysis. Secretin-induced secretion by the normal IBE is rich in HCO3-, whereas intact ADPKD liver cysts secrete primarily Cl- in response to secretin. To evaluate the mechanisms of decreased HCO3- secretion by ADPKD liver cysts, we utilized SV40 large T antigen-immortalized normal IBE and ADPKD liver cyst-derived epithelia (LCDE) cell lines that we created. These cell lines express biliary but not hepatocyte markers. Anion exchanger (AE) function was assessed by the response of intracellular pH (pHi) to acute Cl- removal. 2',7'-Bis(carboxyethyl)-5-(6)-carboxyfluorescein-loaded monolayers were continuously perfused with physiological HCO3- buffer containing Cl- or gluconate. In IBE cell line H75 (n = 6), acute Cl- removal alkalinized pHi at a rate of 0.04 +/- 0.01 min-1. AE function was significantly decreased in LCDE cell line CL3 (n = 6) to a rate of 0.01 +/- 0.01 min-1 after Cl- removal. Northern blot analysis demonstrated equivalent levels of AE2 mRNA in both cell lines. AE1 mRNA was undetectable. Immunoblot analysis demonstrated the AE2 polypeptide in both cell lines, but the level of mature glycosylated AE2 polypeptide was reduced in LCDE cells. Immunofluorescence microscopy demonstrated decreased membrane-localized AE2 in LCDE cells. These findings suggest that decreased plasmalemmal AE2 may account for decreased AE function in LCDE cells and suggest a possible explanation for decreased secretion of HCO3- by ADPKD liver cysts.

scholarly journals Effect of Reducing Ataxia-Telangiectasia Mutated (ATM) in Experimental Autosomal Dominant Polycystic Kidney Disease

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 532
Author(s):  
Jennifer Q. J. Zhang ◽  
Sayanthooran Saravanabavan ◽  
Gopala K. Rangan
Keyword(s):  
Kidney Disease ◽  
Autosomal Dominant ◽  
Cyst Formation ◽  
Ataxia Telangiectasia Mutated ◽  
Polycystic Kidney ◽  
Kidney Cyst ◽  
Autosomal Dominant Polycystic Kidney ◽  
Cyst Growth

The DNA damage response (DDR) pathway is upregulated in autosomal dominant polycystic kidney disease (ADPKD) but its functional role is not known. The ataxia-telangiectasia mutated (ATM) and AT and Rad3-related (ATR) protein kinases are key proximal transducers of the DDR. This study hypothesized that reducing either ATM or ATR attenuates kidney cyst formation and growth in experimental ADPKD. In vitro, pharmacological ATM inhibition by AZD0156 reduced three-dimensional cyst growth in MDCK and human ADPKD cells by up to 4.4- and 4.1-fold, respectively. In contrast, the ATR inhibitor, VE-821, reduced in vitro MDCK cyst growth but caused dysplastic changes. In vivo, treatment with AZD0156 by oral gavage for 10 days reduced renal cell proliferation and increased p53 expression in Pkd1RC/RC mice (a murine genetic ortholog of ADPKD). However, the progression of cystic kidney disease in Pkd1RC/RC mice was not altered by genetic ablation of ATM from birth, in either heterozygous (Pkd1RC/RC/Atm+/−) or homozygous (Pkd1RC/RC/Atm−/−) mutant mice at 3 months. In conclusion, despite short-term effects on reducing renal cell proliferation, chronic progression was not altered by reducing ATM in vivo, suggesting that this DDR kinase is dispensable for kidney cyst formation in ADPKD.

scholarly journals Cell-Autonomous Hedgehog Signaling Is Not Required for Cyst Formation in Autosomal Dominant Polycystic Kidney Disease

2019 ◽  
Vol 30 (11) ◽  
pp. 2103-2111 ◽  
Author(s):  
Ming Ma ◽  
Emilie Legué ◽  
Xin Tian ◽  
Stefan Somlo ◽  
Karel F. Liem
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Mouse Models ◽  
Hedgehog Signaling ◽  
Autosomal Dominant ◽  
Cyst Formation ◽  
Hedgehog Pathway ◽  
Adult Onset ◽  
Polycystic Kidney ◽  
Autosomal Dominant Polycystic Kidney

BackgroundPKD1 or PKD2, the two main causal genes for autosomal dominant polycystic kidney disease (ADPKD), encode the multipass transmembrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Polycystins localize to the primary cilium, an organelle essential for cell signaling, including signal transduction of the Hedgehog pathway. Mutations in ciliary genes that build and maintain the cilium also cause renal cystic disease through unknown pathways. Although recent studies have found alterations in Hedgehog signaling in ADPKD-related models and tissues, the relationship between Hedgehog and polycystic kidney disease is not known.MethodsTo examine the potential role of cell-autonomous Hedgehog signaling in regulating kidney cyst formation in vivo in both early- and adult-onset mouse models of ADPKD, we used conditional inactivation of Pkd1 combined with conditional modulation of Hedgehog signaling components in renal epithelial cells, where mutations in Pkd1 initiate cyst formation. After increasing or decreasing levels of Hedgehog signaling in cells that underwent inactivation of Pkd1, we evaluated the effects of these genetic manipulations on quantitative parameters of polycystic kidney disease severity.ResultsWe found that in Pkd1 conditional mutant mouse kidneys, neither downregulation nor activation of the Hedgehog pathway in epithelial cells along the nephron significantly influenced the severity of the polycystic kidney phenotype in mouse models of developmental or adult-onset of ADPKD.ConclusionsThese data suggest that loss of Pkd1 function results in kidney cysts through pathways that are not affected by the activity of the Hedgehog pathway.

scholarly journals Somatostatin in renal physiology and autosomal dominant polycystic kidney disease

2019 ◽  
Vol 35 (8) ◽  
pp. 1306-1316 ◽  
Author(s):  
A Lianne Messchendorp ◽  
Niek F Casteleijn ◽  
Esther Meijer ◽  
Ron T Gansevoort
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Autosomal Dominant ◽  
Unmet Need ◽  
Cyst Formation ◽  
Renal Physiology ◽  
Intracellular Camp ◽  
Kidney Volume ◽  
Polycystic Kidney ◽  
Autosomal Dominant Polycystic Kidney

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst formation, leading to growth in kidney volume and renal function decline. Although therapies have emerged, there is still an important unmet need for slowing the rate of disease progression in ADPKD. High intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) are involved in cell proliferation and fluid secretion, resulting in cyst formation. Somatostatin (SST), a hormone that is involved in many cell processes, has the ability to inhibit intracellular cAMP production. However, SST itself has limited therapeutic potential since it is rapidly eliminated in vivo. Therefore analogues have been synthesized, which have a longer half-life and may be promising agents in the treatment of ADPKD. This review provides an overview of the complex physiological effects of SST, in particular renal, and the potential therapeutic role of SST analogues in ADPKD.

Pkd1-targeted mutation reveals a role for the Wolffian duct in autosomal dominant polycystic kidney disease

2019 ◽  
Vol 11 (1) ◽  
pp. 78-85 ◽  
Author(s):  
J. B. Tee ◽  
A. V. Dnyanmote ◽  
M. K. Lorenzo ◽  
O. R. Lee ◽  
S. Grisaru ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
High Throughput Sequencing ◽  
Autosomal Dominant ◽  
Cyst Formation ◽  
Wolffian Duct ◽  
Renal Tubules ◽  
Polycystic Kidney ◽  
Cystic Kidneys ◽  
Autosomal Dominant Polycystic Kidney

AbstractSeveral life-threatening diseases of the kidney have their origins in mutational events that occur during embryonic development. In this study, we investigate the role of the Wolffian duct (WD), the earliest embryonic epithelial progenitor of renal tubules, in the etiology of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with a germline mutation of one of the two Pkd1 alleles. For the disease to occur, a second event that disrupts the expression of the other inherited Pkd1 allele must occur. We postulated that this secondary event can occur in the pronephric WD. Using Cre-Lox recombination, mice with WD-specific deletion of one or both Pkd1 alleles were generated. Homozygous Pkd1-targeted deletion in WD-derived tissues resulted in mice with large cystic kidneys and serologic evidence of renal failure. In contrast, heterozygous deletion of Pkd1 in the WD led to kidneys that were phenotypically indistinguishable from control in the early postnatal period. High-throughput sequencing, however, revealed underlying gene and microRNA (miRNA) changes in these heterozygous mutant kidneys that suggest a strong predisposition toward developing ADPKD. Bioinformatic analysis of this data demonstrated an upregulation of several miRNAs that have been previously associated with PKD; pathway analysis further demonstrated that the differentially expressed genes in the heterozygous mutant kidneys were overrepresented in signaling pathways associated with maintenance and function of the renal tubular epithelium. These results suggest that the WD may be an early epithelial target for the genetic or molecular signals that can lead to cyst formation in ADPKD.

scholarly journals An Overview of In Vivo and In Vitro Models for Autosomal Dominant Polycystic Kidney Disease: A Journey from 3D-Cysts to Mini-Pigs

2020 ◽  
Vol 21 (12) ◽  
pp. 4537
Author(s):  
Svenja Koslowski ◽  
Camille Latapy ◽  
Pierrïck Auvray ◽  
Marc Blondel ◽  
Laurent Meijer
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Autosomal Dominant ◽  
In Vitro Models ◽  
Polycystic Kidney ◽  
Autosomal Dominant Polycystic Kidney ◽  
Stage Renal Disease ◽  
End Stage

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inheritable cause of end stage renal disease and, as of today, only a single moderately effective treatment is available for patients. Even though ADPKD research has made huge progress over the last decades, the precise disease mechanisms remain elusive. However, a wide variety of cellular and animal models have been developed to decipher the pathophysiological mechanisms and related pathways underlying the disease. As none of these models perfectly recapitulates the complexity of the human disease, the aim of this review is to give an overview of the main tools currently available to ADPKD researchers, as well as their main advantages and limitations.

scholarly journals Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3206-3217 ◽  
Author(s):  
N Dubois-Stringfellow ◽  
A Jonczyk ◽  
VL Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Basement Membrane ◽  
Organizational Behavior ◽  
Cell Lines ◽  
Primary Cultures ◽  
Cyst Formation ◽  
Fibrin Gels

Abstract Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

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