Epithelial-to-mesenchymal transition in cyst lining epithelial cells in an orthologous PCK rat model of autosomal-recessive polycystic kidney disease

2011 ◽  
Vol 300 (2) ◽  
pp. F511-F520 ◽  
Author(s):  
Hiroko Togawa ◽  
Koichi Nakanishi ◽  
Hironobu Mukaiyama ◽  
Taketsugu Hama ◽  
Yuko Shima ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Epithelial Cells ◽  
Polycystic Kidney Disease ◽  
Autosomal Recessive ◽  
Epithelial To Mesenchymal Transition ◽  
Cell Contact ◽  
Polycystic Kidney ◽  
Mesenchymal Transition ◽  
Cyst Lining ◽  
E Cadherin

In polycystic kidney disease (PKD), cyst lining cells show polarity abnormalities. Recent studies have demonstrated loss of cell contact in cyst cells, suggesting induction of epithelial-to-mesenchymal transition (EMT). Recently, EMT has been implicated in the pathogenesis of PKD. To explore further evidence of EMT in PKD, we examined age- and segment-specific expression of adhesion molecules and mesenchymal markers in PCK rats, an orthologous model of human autosomal-recessive PKD. Kidneys from 5 male PCK and 5 control rats each at 0 days, 1, 3, 10, and 14 wk, and 4 mo of age were serially sectioned and stained with segment-specific markers and antibodies against E-cadherin, Snail1, β-catenin, and N-cadherin. mRNAs for E-cadherin and Snail1 were quantified by real-time PCR. Vimentin, fibronectin, and α-smooth muscle actin (α-SMA) expressions were assessed as mesenchymal markers. E-cadherin expression pattern was correlated with the disease pathology in that tubule segments showing the highest expression in control had much severer cyst formation in PCK rats. In PCK rats, E-cadherin and β-catenin in cystic tubules was attenuated and localized to lateral areas of cell-cell contact, whereas nuclear expression of Snail1 increased in parallel with cyst enlargement. Some epithelial cells in large cysts derived from these segments, especially in adjacent fibrotic areas, showed positive immunoreactivity for vimentin and fibronectin. In conclusion, these findings suggest that epithelial cells in cysts acquire mesenchymal features in response to cyst enlargement and participate in progressive renal fibrosis. Our study clarified the nephron segment-specific cyst profile related to EMT in PCK rats. EMT may play a key role in polycystic kidney disease.

Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney disease cyst-lining renal epithelial cells

2008 ◽  
Vol 294 (4) ◽  
pp. F890-F899 ◽  
Author(s):  
Rajeev Rohatgi ◽  
Lorenzo Battini ◽  
Paul Kim ◽  
Sharon Israeli ◽  
Patricia D. Wilson ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Epithelial Cells ◽  
Polycystic Kidney Disease ◽  
Cell Lines ◽  
Autosomal Recessive ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
Cyst Lining

Mutations of cilia-expressed proteins are associated with an attenuated shear-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in renal epithelial cell lines derived from murine models of autosomal recessive polycystic kidney disease (ARPKD). We hypothesized that human ARPKD cyst-lining renal epithelial cells also exhibited dysregulated mechanosensation. To test this, conditionally immortalized cell lines derived from human fetal ARPKD cyst-lining (pool and clone 5E) cell lines with low levels of fibrocystin/polyductin expression and age-matched normal collecting tubule [human fetal collecting tubule (HFCT) pool and clone 2C] cell lines were grown in culture, loaded with a Ca2+ indicator dye, and subjected to laminar shear. Clonal cell lines were derived from single cells present in pools of cells from cyst-lining and collecting tubules, microdissected from human kidney. Resting and peak [Ca2+]i were similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flow-induced peak [Ca2+]i was greater in ARPKD 5E (700 ± 87 nM, n = 21) than in HFCT 2C (315 ± 58 nM, n = 12; P < 0.01) cells. ARPKD 5E cells treated with Gd3+, an inhibitor of nonselective cation channels, inhibited but did not abolish the shear-induced [Ca2+]i transient. Cilia were ∼20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory Gd3+-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular Ca2+ stores were similar between cells. In summary, human ARPKD cells exhibit an exaggerated Gd3+-sensitive mechano-induced Ca2+ response compared with controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored.

scholarly journals Abnormalities in focal adhesion complex formation, regulation, and function in human autosomal recessive polycystic kidney disease epithelial cells

2010 ◽  
Vol 298 (4) ◽  
pp. C831-C846 ◽  
Author(s):  
Sharon Israeli ◽  
Kurt Amsler ◽  
Nadezhda Zheleznova ◽  
Patricia D. Wilson
Keyword(s):  
Kidney Disease ◽  
Epithelial Cells ◽  
Complex Formation ◽  
Polycystic Kidney Disease ◽  
Focal Adhesion ◽  
Autosomal Recessive ◽  
Polycystic Kidney ◽  
Focal Adhesion Complex ◽  
Collecting Tubules ◽  
Adhesion Complex

Integrin-associated focal adhesion complex formation and turnover plays an essential role in directing interactions between epithelial cells and the extracellular matrix during organogenesis, leading to appropriate cell spreading, cell-matrix adhesion, and migration. Autosomal recessive polycystic kidney disease (ARPKD) is associated with loss of function of PKHD1-encoded protein fibrocystin-1 and is characterized by cystic dilation of renal collecting tubules (CT) in utero and loss of renal function in patients if they survive the perinatal period. Normal polycystin-1 (PC-1)/focal adhesion complex function is required for control of CT diameter during renal development, and abnormalities in these complexes have been demonstrated in human PC-1 mutant cystic cells. To determine whether loss of fibrocystin-1 was associated with focal adhesion abnormalities, ARPKD cells or normal age-matched human fetal (HF)CT cells in which fibrocystin-1 had been decreased by 85% by small interfering RNA inhibition were compared with normal HFCT. Accelerated attachment and spreading on collagen matrix and decreased motility of fibrocystin-1-deficient cells were associated with longer paxillin-containing focal adhesions, more complex actin-cytoskeletal rearrangements, and increased levels of total β1-integrin, c-Src, and paxillin. Immunoblot analysis of adhesive cells using site-specific phospho-antibodies demonstrated ARPKD-associated loss of activation of focal adhesion kinase (FAK) by phosphorylation at its autophosphorylation site (Y397); accelerated FAK inhibition by phosphorylation at Y407, S843, and S910; as well as increased activation of c-Src at pY418. Paxillin coimmunoprecipitation analyses suggested that fibrocystin-1 was a component of the normal focal adhesion complex and that actin and fibrocystin-1 were lost from ARPKD complexes.

scholarly journals Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease

2017 ◽  
Vol 313 (6) ◽  
pp. F1223-F1231 ◽  
Author(s):  
Taketsugu Hama ◽  
Koichi Nakanishi ◽  
Masashi Sato ◽  
Hironobu Mukaiyama ◽  
Hiroko Togawa ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Epithelial Cells ◽  
Polycystic Kidney Disease ◽  
Autosomal Recessive ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Specific Cell ◽  
Polycystic Kidney ◽  
Phenotypic Alteration ◽  
Cpk Mouse

Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/ cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention.

scholarly journals Autosomal Recessive Polycystic Kidney Disease Epithelial Cell Model Reveals Multiple Basolateral Epidermal Growth Factor Receptor Sorting Pathways

2010 ◽  
Vol 21 (15) ◽  
pp. 2732-2745 ◽  
Author(s):  
Sean Ryan ◽  
Susamma Verghese ◽  
Nicholas L. Cianciola ◽  
Calvin U. Cotton ◽  
Cathleen R. Carlin
Keyword(s):  
Kidney Disease ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mouse Model ◽  
Polycystic Kidney Disease ◽  
Autosomal Recessive ◽  
Egf Receptor ◽  
Cell Model ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells

Sorting and maintenance of the EGF receptor on the basolateral surface of renal epithelial cells is perturbed in polycystic kidney disease and apical expression of receptors contributes to severity of disease. The goal of these studies was to understand the molecular basis for EGF receptor missorting using a well-established mouse model for the autosomal recessive form of the disease. We have discovered that multiple basolateral pathways mediate EGF receptor sorting in renal epithelial cells. The polycystic kidney disease allele in this model, Bicc1, interferes with one specific EGF receptor pathway without affecting overall cell polarity. Furthermore one of the pathways is regulated by a latent basolateral sorting signal that restores EGF receptor polarity in cystic renal epithelial cells via passage through a Rab11-positive subapical compartment. These studies give new insights to possible therapies to reconstitute EGF receptor polarity and function in order to curb disease progression. They also indicate for the first time that the Bicc1 gene that is defective in the mouse model used in these studies regulates cargo-specific protein sorting mediated by the epithelial cell specific clathrin adaptor AP-1B.

scholarly journals Identification of pathological transcription in autosomal dominant polycystic kidney disease epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sebastian Friedrich ◽  
Hannah Müller ◽  
Caroline Riesterer ◽  
Hannah Schüller ◽  
Katja Friedrich ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Epithelial Cells ◽  
Polycystic Kidney Disease ◽  
Protein Modification ◽  
Autosomal Dominant ◽  
Polycystic Kidney ◽  
Mesenchymal Transition ◽  
Cystic Kidneys ◽  
Kidney Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney

AbstractAutosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.

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