Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells

2008 ◽  
Vol 295 (1) ◽  
pp. F82-F90 ◽  
Author(s):  
Min Li ◽  
Saravanan Balamuthusamy ◽  
Eric E. Simon ◽  
Vecihi Batuman
Keyword(s):  
Epithelial Cells ◽  
Proximal Tubule ◽  
Light Chain ◽  
Epithelial To Mesenchymal Transition ◽  
Human Kidney ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Monocyte Chemoattractant Protein 1 ◽  
Mesenchymal Transition ◽  
Morphological Alterations

Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to κ-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring κ-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled κ-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

scholarly journals Regulation of PDZ domain-containing 1 (PDZK1) expression by hepatocyte nuclear factor-1α (HNF1α) in human kidney

2017 ◽  
Vol 313 (4) ◽  
pp. F973-F983 ◽  
Author(s):  
Katharina Prestin ◽  
Janine Hussner ◽  
Celio Ferreira ◽  
Isabell Seibert ◽  
Vivien Breitung ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proximal Tubule ◽  
Pdz Domain ◽  
Human Kidney ◽  
Renal Proximal Tubule ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Hepatocyte Nuclear Factor 1Α ◽  
Adenoviral Transfer ◽  
Sirna Knockdown

In the renal proximal tubule the secretion and reabsorption of glomerularly filtrated compounds is realized by a functional network of uptake and efflux transporters. The activity and localization of several transporters expressed at the apical tubular membrane are regulated by the membrane-associated protein PDZ domain-containing 1 (PDZK1). We aimed to characterize the transcriptional regulation of this modulator of renal transport. Coexpression analyses of PDZK1 and putative regulators were performed using human kidney samples. Protein and mRNA expression of PDZK1 in renal proximal tubule epithelial cells after adenoviral transfer and siRNA knockdown of transcription factor hepatocyte nuclear factor-1α (HNF1α) was assessed by quantitative real-time PCR and Western blot analysis. Transactivation of the PDZK1 promoter was quantified in cell-based reporter gene assays. Subsequently, the binding of HNF1α to the PDZK1 promoter was verified by in silico analyses and chromatin immunoprecipitation assay. HNF1α positively regulated the promoter activity of PDZK1. Adenoviral overexpression of HNF1α in renal proximal tubule epithelial cells (RPTEC) increased PDZK1 mRNA and protein expression, whereas siRNA knockdown of HNF1α resulted in decreased expression of PDZK1. Our results show that HNF1α, which has previously been described as a modulator of several transporters of the renal transportosome, is also a key determinant of PDZK1 transcription.

scholarly journals c-Jun-N-Terminal Kinase Signaling Is Involved in Cyclosporine-Induced Epithelial Phenotypic Changes

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Nicolas Pallet ◽  
Eric Thervet ◽  
Dany Anglicheau
Keyword(s):  
Epithelial Cells ◽  
Er Stress ◽  
Epithelial To Mesenchymal Transition ◽  
Primary Cultures ◽  
Independent Manner ◽  
Jnk Signaling ◽  
Mesenchymal Transition ◽  
Morphological Alterations ◽  
Phenotypic Changes

Tubular epithelial cells play a central role in the pathogenesis of chronic nephropathies. Previous toxicogenomic studies have demonstrated that cyclosporine- (CsA-) induced epithelial phenotypic changes (EPCs) are reminiscent of an incomplete epithelial to mesenchymal transition (EMT) in a TGF-β-independent manner. Furthermore, we identified endoplasmic reticulum (ER) stress as a potential mechanism that may participate in the modulation of tubular cell plasticity during CsA exposure. Because c-jun-N-terminal kinase (JNK), which is activated during ER stress, is implicated in kidney fibrogenesis, we undertook the current study to identify the role of JNK signaling in EPCs induced by CsA. In primary cultures of human renal epithelial cells, CsA activates JNK signaling, and the treatment with a JNK inhibitor reduces the occurrence of cell shape changes, E-cadherin downregulation, cell migration, and Snail-1 expression. Our results suggest that CsA activates JNK signaling, which, in turn, may participate in the morphological alterations through the regulation of Snail-1 expression.

scholarly journals Regulation of P-Glycoprotein in Renal Proximal Tubule Epithelial Cells by LPS and TNF-α

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Suzanne Heemskerk ◽  
Janny G. P. Peters ◽  
Jochem Louisse ◽  
Seil Sagar ◽  
Frans G. M. Russel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proximal Tubule ◽  
De Novo ◽  
Rat Kidney ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Kidney Proximal Tubule ◽  
P Glycoprotein ◽  
Gene And Protein Expression ◽  
Tubule Cells

During endotoxemia, the ATP-dependent drug efflux pump P-glycoprotein (Abcb1/P-gp) is upregulated in kidney proximal tubule epithelial cells. The signaling pathway through which lipopolysaccharide (LPS) or tumor necrosis factor-α(TNF-α) regulates P-gp expression and activity was investigated further in the present study. Exposure of rat kidney proximal tubule cells to TNF-αalone or TNF-αand LPS increased P-gp gene and protein expression levels and efflux activity, suggesting de novo P-gp synthesis. Upon exposure to TNF-αin combination with LPS, P-gp activity in renal proximal tubule cells is increased under influence of nitric oxide (NO) produced by inducible NO synthase. Upon exposure to TNF-αalone, P-gp upregulation seems to involve TLR4 activation and nuclear factor kappaB (NF-κB) translocation, a pathway that is likely independent of NO. These findings indicate that at least two pathways regulate P-gp expression in the kidney during endotoxemia.

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