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. 2012 Jun 1;287(23):19122-35.
doi: 10.1074/jbc.M112.345983. Epub 2012 Apr 9.

Kruppel-like factor 15 (KLF15) is a key regulator of podocyte differentiation

Sandeep K Mallipattu  1 , Ruijie Liu, Feng Zheng, Goutham Narla, Avi Ma'ayan, Steven Dikman, Mukesh K Jain, Moin Saleem, Vivette D'Agati, Paul Klotman, Peter Y Chuang, John C He
Affiliations

Kruppel-like factor 15 (KLF15) is a key regulator of podocyte differentiation

Sandeep K Mallipattu et al. J Biol Chem. .

Abstract

Podocyte injury resulting from a loss of differentiation is the hallmark of many glomerular diseases. We previously showed that retinoic acid (RA) induces podocyte differentiation via stimulation of the cAMP pathway. However, many podocyte maturity markers lack binding sites for RA-response element or cAMP-response element (CREB) in their promoter regions. We hypothesized that transcription factors induced by RA and downstream of CREB mediate podocyte differentiation. We performed microarray gene expression studies in human podocytes treated with and without RA to identify differentially regulated genes. In comparison with known CREB target genes, we identified Krüppel-like factor 15 (KLF15), a kidney-enriched nuclear transcription factor, that has been previously shown to mediate cell differentiation. We confirmed that RA increased KLF15 expression in both murine and human podocytes. Overexpression of KLF15 stimulated expression of differentiation markers in both wild-type and HIV-1-infected podocytes. Also, KLF15 binding to the promoter regions of nephrin and podocin was increased in RA-treated podocytes. Although KLF15(-/-) mice at base line had minimal phenotype, lipopolysaccharide- or adriamycin-treated KLF15(-/-) mice had a significant increase in proteinuria and podocyte foot process effacement with a reduction in the expression of podocyte differentiation markers as compared with the wild-type treated mice. Finally, KLF15 expression was reduced in glomeruli isolated from HIV transgenic mice as well as in kidney biopsies from patients with HIV-associated nephropathy and idiopathic focal segmental glomerulosclerosis. These results indicate a critical role of KLF15 in mediating podocyte differentiation and in protecting podocytes against injury.

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Figures

FIGURE 1.
FIGURE 1.
RA stimulates KLF15 mRNA expression by real time PCR. KLF15 mRNA expression was measured in atRA (1 μm) treated and untreated wild-type (WT) and HIV-1-infected murine (A) and human (B) podocytes in culture (n = 3, **, p < 0.05 versus control cells without RA treatment). C, KLF15 protein expression was measured in atRA (1 μm)-treated and untreated wild-type (WT) and HIV-1-infected human podocytes in culture. The representative blots of three independent experiments are shown. The densitometry analyses of these blots are shown in the lower panel (n = 3, *, p < 0.05 versus cells without atRA treatment). Human podocytes were treated with atRA (1 μm) for the indicated time intervals, and cells were collected for KLF15 expression by real time PCR (D) and Western (E) (n = 3, *, p < 0.05 versus control cells without atRA treatment). The representative blots of three independent experiments are shown. The densitometry analyses of these blots are shown in the lower panel (n = 3, *, p < 0.05 versus cells without atRA treatment).
FIGURE 2.
FIGURE 2.
Exposure to atRA increased binding of KLF15 in the promoter region of nephrin and podocin. Differentiated wild-type (WT) podocytes were stimulated with or without atRA for 4 h, and nuclear proteins were extracted for ChIP assay as described. Binding of KLF15 to the putative KLF15-binding sites in the promoter of nephrin (A) and podocin (B) was measured in atRA-treated and untreated WT murine podocytes. Primary podocytes were isolated from wild-type and KLF15−/− mice and then stimulated with or without atRA for 4 h, and nuclear proteins were extracted for ChIP assay (n = 3, *, p < 0.05 versus atRA 0 μm). Binding of KLF15 to the putative KLF15-binding sites in the promoter of nephrin (C) and podocin (D) was measured in atRA-treated and untreated primary podocytes from WT mice and KLF15−/− mice.
FIGURE 3.
FIGURE 3.
KLF15 mRNA and protein expression is increased in differentiated human podocytes. Immortalized human podocytes in culture were either incubated and 33 or 37 °C. Cells were collected 1 week after incubation for real time PCR and Western blot. KLF15 mRNA (A) and protein expression (B) were measured in human podocytes in culture at 37 °C compared with 33 °C (n = 3, *, p < 0.01 versus 33 °C; representative blot of three independent experiments is shown). The densitometry analysis is shown in the lower panel (n = 3, *, p < 0.01 versus cells at 33 °C).
FIGURE 4.
FIGURE 4.
Overexpression of KLF15 increases expression of podocyte differentiation markers in both control and HIV-1-infected cells. A, murine podocytes were transiently transfected with control vector or KLF15 construct, and protein was extracted for Western blot. Cells were collected 24 h after transfection for determining mRNA levels of podocyte differentiation markers by real time PCR. Synaptopodin (B), podocin (C), and nephrin (D) were measured in control and HIV-1-infected cells with and without KLF15 overexpression (n = 3, *, p < 0.05 compared with the control cells; n = 3, **, p < 0.05 compared between cells with or without KLF15 overexpression).
FIGURE 5.
FIGURE 5.
LPS-treated KLF15−/− mice had increased albuminuria with podocyte effacement. Urine was collected at 0 h (prior to LPS injection) and subsequently collected at 12-h intervals. All mice were sacrificed and renal cortex fixed for histology at 48 h. A, proteinuria (urine protein/creatinine) was measured in LPS-treated wild-type (WT) and KLF15−/− mice (n = 6, *, p < 0.01 versus LPS-treated WT mice). B, Coomassie stain revealed that the change in proteinuria was mainly albuminuria. The representative gel of three mice in each group is shown. C, podocyte foot process effacement was compared between LPS-treated WT and KLF15−/− mice (×5000). The representative images are shown. D, quantification of foot process effacement is shown (n = 6, *, p < 0.01).
FIGURE 6.
FIGURE 6.
LPS-treated KLF15−/− mice had a reduction in markers of podocyte differentiation. Synaptopodin (A), nephrin (B), and WT-1 (C) mRNA expressions were compared between LPS-treated wild-type (WT) and KLF15−/− mice (n = 6, *, p < 0.01 versus all other groups.) This was confirmed by immunofluorescence as shown in the middle panel. The representative pictures of three mice in each group are shown. The glomerular region was selected, and optical density (OD) was measured and quantified as a relative fold change to wild-type in the lower panel (n = 4, *, p < 0.01).
FIGURE 7.
FIGURE 7.
Adriamycin (AD)-treated KLF15−/− mice had increased albuminuria with podocyte effacement. Urine was collected prior to treatment and 4 weeks after treatment. All mice were sacrificed and renal cortex fixed for histology at 4 weeks. A, proteinuria (urine protein/creatinine) was measured in adriamycin-treated wild-type (WT) and KLF15−/− mice (n = 6, *, p < 0.01 versus all other groups). B, Coomassie stain revealed that the change in proteinuria was mainly albuminuria. The representative gel from two mice in each group is shown. C, podocyte foot process effacement was compared between AD-treated WT and KLF15−/− mice (×5000). The representative images are shown. D, quantification of foot process effacement is shown (n = 6, *, p < 0.01).
FIGURE 8.
FIGURE 8.
Adriamycin (AD)-treated KLF15−/− mice had a reduction in markers of podocyte differentiation. Synaptopodin (A), nephrin (B), and Wilms tumor 1 (WT-1) (C) mRNA expressions were compared between adriamycin-treated wild-type (WT) and KLF15−/− mice. (n = 6, *, p < 0.01 versus all other groups). This was confirmed by immunofluorescence as shown in the middle panel. The representative pictures of three mice in each group are shown. The glomerular region was selected, and optical density (OD) was measured and quantified as a relative fold change to wild type in the lower panel (n = 4, *, p < 0.001).
FIGURE 9.
FIGURE 9.
RA does not increase podocyte differentiation markers in podocytes lacking KLF15 expression. A, primary mouse podocytes were initially isolated from wild-type (WT) and KLF15−/− mice, and protein was extracted for Western. These isolated primary podocytes were also stimulated with or without atRA for 8 h, and RNA was extracted for real time PCR. Synaptopodin (B), podocin (C), and nephrin (D) mRNA expressions were measured in atRA-treated and untreated WT and KLF15−/− podocytes (n = 3, *, p < 0.01 versus atRA 0 μm).
FIGURE 10.
FIGURE 10.
RA did not attenuate proteinuria in LPS-treated KLF15−/− mice. First dose of atRA (16 mg/kg) was administered 12 h prior to first dose of LPS with the subsequent two doses administered at 24-h time intervals of the initial dose. Urine was collected at 24 h, and proteinuria was measured in LPS-treated wild-type (WT) and KLF15−/− mice co-treated with and without atRA (n = 4, *, p < 0.0001).
FIGURE 11.
FIGURE 11.
KLF15 expression is reduced in HIV transgenic (Tg26) mice. Glomeruli were isolated, and RNA was extracted for real time PCR. A, KLF15 mRNA expression was measured in wild-type (WT) and Tg26 mice (n = 4, *, p < 0.05 versus control). B, Western blot analysis was performed in glomerular lysates from WT and Tg26 mice for KLF15 and GAPDH. The representative blot of three independent experiments is shown. The densitometry analysis of these blots is shown in the lower panel (n = 3, *, p < 0.01). C, representative images from the immunostaining of KLF15 in kidney sections from WT and Tg26 mice are shown.
FIGURE 12.
FIGURE 12.
Reduced KLF15 expression in human glomerular disease. A, immunostaining for KLF15 performed on healthy donor nephrectomy specimens shows a nuclear distribution in normal podocytes, parietal cells, and tubular cells. In comparison with biopsy specimen from healthy donor subjects (A), KLF15 expression in the podocytes is shown in biopsy specimens from patients with diagnosed HIVAN (B) and idiopathic FSGS (C). The representative images of three subjects in each group are shown. D, glomerular region was selected, and optical density (OD) was measured and quantified as a relative fold change to healthy donor specimens (n = 3, *, p < 0.0001).
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