Hepatocyte Nuclear Factor-1β Induces Redifferentiation of Dedifferentiated Tubular Epithelial Cells

<div><p>Tubular epithelial cells (TECs) can be dedifferentiated by repetitive insults, which activate scar-producing cells generated from interstitial cells such as fibroblasts, leading to the accumulation and deposition of extracellular matrix molecules. The dedifferentiated TECs play a crucial role in the development of renal fibrosis. Therefore, renal fibrosis may be attenuated if dedifferentiated TECs are converted back to their normal state (re-epithelialization). However, the mechanism underlying the re-epithelialization remains to be elucidated. In the present study, TGF-β1, a profibrotic cytokine, induced dedifferentiation of cultured TECs, and the dedifferentiated TECs were re-epithelialized by the removal of TGF-β1 stimulation. In the re-epithelialization process, transcription factor hepatocyte nuclear factor 1, beta (HNF-1β) was identified as a candidate molecule involved in inducing re-epithelialization by means of DNA microarray and biological network analysis. In functional validation studies, the re-epithelialization by TGF-β1 removal was abolished by HNF-1β knockdown. Furthermore, the ectopic expression of HNF-1β in the dedifferentiated TECs induced the re-epithelialization without the inhibition of TGF-β/Smad signaling, even in the presence of TGF-β1 stimulation. In mouse renal fibrosis model, unilateral ureteral obstruction model, HNF-1β expression in the TECs of the kidney was suppressed with fibrosis progression. Furthermore, the HNF-1β downregulated TECs resulted in dedifferentiation, which was characterized by expression of nestin. In conclusion, HNF-1β suppression in TECs is a crucial event for the dedifferentiation of TECs, and the upregulation of HNF-1β in TECs has a potential to restore the dedifferentiated TECs into their normal state, leading to the attenuation of renal fibrosis.</p></div

Time course of expression of proximal tubular epithelial marker genes in dedifferentiated hRPTECs after Ad-HNF1B infection.

<p>Human RPTECs were stimulated with 3 ng/ml TGF-β1 for 48 h, followed by re-stimulation with fresh TGF-β. After replacement with fresh TGF-β1, the hRPTECs were infected with 2.0 MOI Ad-HNF1B or Ad-LacZ. The levels of mRNA encoding γ-GT1 (A), claudin-2 (B), and SLC6A13 (C) in differentiated hRPTECs were determined by real-time RT-PCR analyses. Each column shows the data from medium incubation for 48 h followed by treatment with medium and Ad-LacZ (white), TGF-β1 stimulation for 48 h followed by treatment with TGF-β1 and Ad-LacZ (hatched line), and TGF-β1 and Ad-HNF1B for 24 h (dot). Each column and bar presents the means ± SD of three independent experiments. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding TGF-β1 and Ad-LacZ-treated groups by t-tests.</p

Effect of HNF1B-targeting siRNA on re-epithelialization (gene expression and morphological changes) by TGF-β1 removal.

<p>Human RPTECs were cultivated with medium or 3 ng/ml TGF-β1 for 48 h, followed by incubation in fresh medium with or without TGF-β1 for 48 h (gene expression experiment: A–D) and 96 h (morphology experiment: E–H). Cells were treated with two types of HNF1B-targeting siRNA (15 nM) and negative control siRNA (Control) (15 nM) for 24 h after the first TGF-β1 stimulation. The levels of mRNA encoding proximal tubular epithelial marker genes (A: γ-GT1 and B: claudin-2) and mesenchymal marker genes (C: type I collagen and D: fibronectin) in differentiated hRPTECs were determined by real-time RT-PCR. Each column shows data from siRNA-1 (white) and siRNA-2 (black). Each column and bar presents the means ± SD of three independent experiments. Statistical significance: # P < 0.05, ## P < 0.01, ### P < 0.001 vs. TGF-β1 (0-48h) + TGF-β1 (48-96h) with corresponding Control siRNA; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. TGF-β1 (0-48h) + Medium (48-96h) with corresponding Control siRNA by t-tests. Representative phase-contrast microscopy photographs of the hRPTECs show non-stimulation (E), TGF-β1 stimulation for 48 h (F), TGF-β1 stimulation for 48 h followed by incubation with TGF-β1-free fresh medium with control siRNA (G) and HNF1B siRNA (H) for 96 h. Scale bar = 100 μm.</p

Time course of morphological changes and gene expression in re-epithelialized hRPTECs induced by TGF-β1 removal.

<p>Human RPTECs were cultivated with medium or 3 ng/ml TGF-β1 for 48 h, followed by incubation in fresh medium with or without TGF-β1 for 48 h or 96 h. Representative phase-contrast microscopy photographs of hRPTECs show non-stimulation (A), TGF-β1 stimulation for 48 h (B), TGF-β1 stimulation for 48 h followed by TGF-β1 re-stimulation for 48 (C) or 96 h (E), and TGF-β1 stimulation for 48 h followed by incubation with TGF-β1-free fresh medium for 48 (D) or 96 h (F). Scale bar = 100 μm. The levels of mRNA encoding proximal tubular epithelial marker genes (G: γ-glutamyl transferase [γ-GT1] and H: claudin-2) and mesenchymal marker genes (I: type I collagen and J: fibronectin) in differentiated hRPTECs were determined by real-time RT-PCR analyses. Each column shows data from non-stimulation (white), TGF-β1 stimulation for 48 h (gray), TGF-β1 stimulation for 48 h followed by TGF-β1 re-stimulation (hatched line), and TGF-β1 stimulation for 48 h followed by incubation with TGF-β1-free fresh medium (dot). Each column and bar presents the means ± SD of three independent experiments. Statistical significance: ## <i>P</i> < 0.01, ### <i>P</i> < 0.001 vs. medium group (white); * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 vs. TGF-β1 re-stimulation group (hatched line) at each time point by <i>t</i>-tests.</p

Effect of HNF-1β ectopic expression on TGF-β1-induced phosphorylation of Smad3 in hRPTECs.

<p>Human RPTECs were stimulated with 3 ng/ml TGF-β1 for 48 h followed by re-stimulation with fresh TGF-β1 for 12 h or 24 h. After replacement with fresh TGF-β1, the hRPTECs were infected with 1.0 MOI and 2.0 MOI Ad-HNF1B or Ad-LacZ. Representative western blotting shows the expression of phosphorylated Smad3 and total Smad3 at 48 kD in Ad-HNF1B-transfected hRPTECs 12 (A) and 24 h (B) after TGF-β1 re-stimulation. Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to total Smad3 expression. Each column presents the means of twice independent experiments from non-stimulation with Ad-LacZ (white), TGF-β1 stimulation for 48 h followed by incubation with TGF-β1 and Ad-LacZ (hatched line), and TGF-β1 and Ad-HNF1B (dot) for 12 (C) and 24 h (D). Each dot symbol shows an individual value.</p

Expression of HNF-1β in mouse kidneys after ureteral obstruction.

<p>Representative western blotting shows the expression of HNF-1β at 61 kD, with β-actin as a control, in contralateral and obstructed kidneys (A). Densitometric quantification of the corresponding bands was performed using an image analyzer (B). The data are presented after normalization to β-actin expression. Each column and bar presents the mean ± SEM of four or five kidneys from UUO mice. Statistical significance was based on Wilcoxon’s rank-sum test; * <i>P</i> < 0.05 vs. contralateral kidney. Representative fluorescence microscopy photographs of renal sections represent HNF-1β and nestin protein expression in the contralateral non-obstructed kidney (C) and 7 days (D) after ureteral obstruction. Double-labeled immunofluorescence shows the expression of HNF1B (red) and nestin (green) using each specific antibody. The nuclei were counterstained with DAPI (blue). Scale bar = 20μm.</p

Time course of HNF1B gene and protein expression in differentiated hRPTECs after Ad-HNF1B infection.

<p>Human RPTECs were cultivated with medium or 3 ng/ml TGF-β1 for 48 h, followed by re-stimulation with TGF-β1 for 24 h. After TGF-β1 stimulation for 48 h, the hRPTECs were infected with Ad-HNF1B or Ad-LacZ. The expression level of HNF1B mRNA was determined by real-time RT-PCR analysis (A). Each column shows data from non-stimulation (white), TGF-β1 stimulation for 48 h (gray), TGF-β1 stimulation for 48 h followed by TGF-β1 re-stimulation with Ad-LacZ (hatched line), and with Ad-HNF1B (dot) for 3, 6, 9 and 24 h. Each column and bar presents the means ± SD of three independent experiments. Statistical significance: *** P < 0.001 vs. corresponding TGF-β1 and Ad-LacZ-treated groups (hatched column) by t-tests. The expression of HNF1B protein (HNF-1β) 6, 9, 12, and 24 h after adenovirus infection was analyzed using the western blot method. A representative photograph shows the expression of HNF-1β at 61 kD, with β-actin at 43 kD as a control, in adenovirus vector-infected hRPTECs (B). Densitometric quantification of the corresponding bands was performed using an image analyzer. The data were presented after normalization to β-actin expression (C). Each column shows data from non-stimulation with Ad-LacZ (white), TGF-β1 stimulation for 48 h followed by incubation with TGF-β1 and Ad-LacZ for 6 h (hatched line), and TGF-β1 stimulation for 48 h followed by incubation with TGF-β1 and Ad-HNF1B for 6, 9, 12, and 24 h (dot). Each column and bar presents the means ± SD of three independent experiments. The dotted line indicates the protein expression at 6 h in the non-stimulation with Ad-LacZ group. Statistical significance: # <i>P</i> < 0.05 vs. non-stimulation with Ad-LacZ group (white); * P < 0.05 vs. TGF-β1 stimulation with Ad-LacZ group (hatched line) by t-tests.</p