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Krüppel-like factor 6 regulates mitochondrial function in the kidney
Sandeep K. Mallipattu, … , Vincent W. Yang, John C. He
Sandeep K. Mallipattu, … , Vincent W. Yang, John C. He
Published February 17, 2015
Citation Information: J Clin Invest. 2015;125(3):1347-1361. https://doi.org/10.1172/JCI77084.
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Research Article

Krüppel-like factor 6 regulates mitochondrial function in the kidney

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Abstract

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.

Authors

Sandeep K. Mallipattu, Sylvia J. Horne, Vivette D’Agati, Goutham Narla, Ruijie Liu, Michael A. Frohman, Kathleen Dickman, Edward Y. Chen, Avi Ma’ayan, Agnieszka B. Bialkowska, Amr M. Ghaleb, Mandayam O. Nandan, Mukesh K. Jain, Ilse Daehn, Peter Y. Chuang, Vincent W. Yang, John C. He

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Figure 9

shRNA-mediated KLF6 knockdown resulted in activation of the intrinsic apoptotic pathway.

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shRNA-mediated KLF6 knockdown resulted in activation of the intrinsic ap...
Cultured human podocytes (EV-shRNA and KLF6-shRNA) were treated with and without ADR for 24 hours. (A) Immunofluorescence images using Hoechst staining was performed to assess for apoptotic bodies. Representative images of six independent experiments are shown (original magnification, ×20). Arrows show apoptotic bodies. (B) To quantify apoptosis, annexin V/propidium iodide staining in combination with FACS was performed (n = 3). Kruskal-Wallis test with Dunn’s post-hoc test, *P < 0.05. (C) Immunofluorescence images of cytochrome c staining in EV-shRNA and KLF6-shRNA podocytes treated with and without ADR are shown. Representative images of six independent experiments are shown to demonstrate the distribution of cytochrome c staining (original magnification, ×20). Arrows show mitochondrial cytochrome c distribution. Arrowhead shows cytosolic distribution of cytochrome c. (D) Activation of the intrinsic apoptotic pathway was assessed using Western blot analysis for cleaved caspase-9, pro–caspase-3, and cleaved caspase-3 and is shown with β-actin as a loading marker. The representative images of six independent experiments are shown in the top panel. Quantification by densitometry (n = 6) is shown in the bottom panel. Kruskal-Wallis test with Dunn’s post-hoc test, *P < 0.05 vs. treated and untreated EV-shRNA, #P < 0.05 vs. all groups, †P < 0.01 vs. all groups. (E) To confirm whether the preservation of KLF6 prevents apoptosis, Western blot analysis was performed on human podocyte lysates from ADR-treated podocytes with (LentiORF-KLF6) and without (LentiORF-control) KLF6 overexpression. Representative images of three independent experiments are shown in the top panel (cleaved caspase-3 and pro–caspase-3 are from the same samples run on parallel gels). Quantification by densitometry (n = 3) is shown in the bottom panel. Mann-Whitney U test, ***P < 0.001, **P < 0.01.
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