Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation
Chelsea C. Estrada, Praharshasai Paladugu, Yiqing Guo, Jesse Pace, Monica P. Revelo, David J. Salant, Stuart J. Shankland, Vivette D. D’Agati, Anita Mehrotra, Stephanie Cardona, Agnieszka B. Bialkowska, Vincent W. Yang, John C. He, Sandeep K. Mallipattu
Chelsea C. Estrada, Praharshasai Paladugu, Yiqing Guo, Jesse Pace, Monica P. Revelo, David J. Salant, Stuart J. Shankland, Vivette D. D’Agati, Anita Mehrotra, Stephanie Cardona, Agnieszka B. Bialkowska, Vincent W. Yang, John C. He, Sandeep K. Mallipattu
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Research Article Nephrology

Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

Abstract

Pathologic glomerular epithelial cell (GEC) hyperplasia is characteristic of both rapidly progressive glomerulonephritis (RPGN) and subtypes of focal segmental glomerulosclerosis (FSGS). Although initial podocyte injury resulting in activation of STAT3 signals GEC proliferation in both diseases, mechanisms regulating this are unknown. Here, we show that the loss of Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, enhances GEC proliferation in both RPGN and FSGS due to dysregulated STAT3 signaling. We observed that podocyte-specific knockdown of Klf4 (C57BL/6J) increased STAT3 signaling and exacerbated crescent formation after nephrotoxic serum treatment. Interestingly, podocyte-specific knockdown of Klf4 in the FVB/N background alone was sufficient to activate STAT3 signaling, resulting in FSGS with extracapillary proliferation, as well as renal failure and reduced survival. In cultured podocytes, loss of KLF4 resulted in STAT3 activation and cell-cycle reentry, leading to mitotic catastrophe. This triggered IL-6 release into the supernatant, which activated STAT3 signaling in parietal epithelial cells. Conversely, either restoration of KLF4 expression or inhibition of STAT3 signaling improved survival in KLF4-knockdown podocytes. Finally, human kidney biopsy specimens with RPGN exhibited reduced KLF4 expression with a concomitant increase in phospho-STAT3 expression as compared with controls. Collectively, these results suggest the essential role of KLF4/STAT3 signaling in podocyte injury and its regulation of aberrant GEC proliferation.

Authors

Chelsea C. Estrada, Praharshasai Paladugu, Yiqing Guo, Jesse Pace, Monica P. Revelo, David J. Salant, Stuart J. Shankland, Vivette D. D’Agati, Anita Mehrotra, Stephanie Cardona, Agnieszka B. Bialkowska, Vincent W. Yang, John C. He, Sandeep K. Mallipattu

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Figure 7

shRNA-mediated KLF4 knockdown increased the susceptibility to mitotic catastrophe.

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shRNA-mediated KLF4 knockdown increased the susceptibility to mitotic ca...
Control (Scr-shRNA) and KLF4 knockdown (KLF4-shRNA21 and KLF4-shRNA22) human podocytes were transferred from 33°C (permissive) to 37°C (nonpermissive) to induce differentiation. (A) Cell survival (counting) at 3, 7, 10, and 14 days in 37°C (n = 6; ***P < 0.001 as compared with Scr-shRNA, #P < 0.05, ##P < 0.01, and ###P < 0.001 as compared with KLF4-shRNA21; 2-way ANOVA with Tukey’s multiple comparisons post hoc test). (B) Cell viability (MTS assay) at day 3, 7, and 10 in 37°C (n = 6–8; ***P < 0.001; Mann-Whitney U test). (C) Cell cycle analysis at day 3 in 37°C. Percentages of human podocytes in sub-G0, G0/G1, S, and G2/M phases in left panel (n = 3; *P < 0.05, ***P < 0.001; Mann-Whitney U test). Histograms generated with ModFit software to evaluate the relative cell count in each cell cycle stage. (D) CCNA2, CCNB1, CCNE1, CCND1, and p57 mRNA expression are shown relative to Scr-shRNA podocytes at day 3 in 37°C (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney U test). (E) Representative images of Phalloidin and Hoechst staining in surviving Scr-shRNA and KLF4-shRNA22 podocytes at 5 days in 37°C to evaluate for actin stress fiber formation and apoptotic bodies (n = 3, ×20). Inset shows higher magnification (×60). Arrowheads show apoptotic bodies. (F) Annexin V/propidium iodide staining in combination with FACS at 3 days in 37°C (n = 3; *P < 0.05; Mann-Whitney U test). Scale bars: 100 µm.