Modulation of apolipoprotein L1-microRNA-193a axis prevents podocyte dedifferentiation in high-glucose milieu

Abstract

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms' tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.

Keywords: APOL1; diabetic podocytopathy; high glucose; miR193a; podocyte dedifferentiation.

Fig. 1.

High glucose causes dedifferentiation of podocytes. A: differentiated podocytes [preincubated in RPMI 1640 media containing glucose (11 mM) at 37°C; DPDs] were incubated in media containing normal glucose (C; 5 mM) or high glucose (HG; 30 mM) for 48 h (n = 3). Proteins were extracted. Protein blots were probed for PAX2 and reprobed for actin. Gels from 3 different lysates are displayed (top). Cumulative densitometric data are shown in bar graphs (bottom). *P < 0.05 compared with C. B: protein blots from the lysate preparations of A were probed for WT1 and reprobed for actin. Gels from 3 different lysates are displayed. Cumulative data are shown in a bar diagram. *P < 0.05 compared with C. C: RNAs were extracted from the lysates of A. cDNAs were amplified with a specific primer for PAX2. Cumulative data on mRNA expression of PAX2 are shown. *P < 0.05 compared with C. D: RNAs were extracted from the lysates of A. cDNAs were amplified with a specific primer for WT1. Cumulative data on WT1 mRNA expression are shown. *P < 0.05 compared with C. E: DPDs were transfected with scrambled (SCR; 25 nM) or WT1 (25 nM) siRNAs with Lipofectamine RNAiMAX transfection reagent according to manufacturer’s protocol and left in Opti-MEM for 48 h (n = 3). Subsequently, proteins were extracted, and protein blots from control and transfected cells were probed for WT1 and reprobed for PAX2 and GAPDH. Gels from 3 different lysates are displayed. F: cumulative densitometric data from the gels of E are shown in a bar diagram. *P < 0.05 compared with respective control and SCR.

Fig. 2.

High glucose induces PD dedifferentiation through upregulation of miR193a. A: DPDs were incubated in media containing either normal glucose (control; 5 mM) or high glucose (30 mM) for 48 h (n = 4). RNAs were extracted and assayed for miR193a. Cumulative data are shown in a bar diagram. *P < 0.05 compared with C. B: DPDs were incubated in media containing normal glucose (5 mM; control), high glucose (30 mM), or empty vector (25 nM; pCMV-MIR; using Lipofectamine as a carrier) with or without miR193a inhibitor (25 nM; plasmid-based inhibitor using Lipofectamine as a carrier) for 48 h (n = 3). RNAs were extracted and assayed for miR193a. **P < 0.01 with other variables. C: DPDs were incubated in media containing normal glucose (control; 5 mM), high glucose (30 mM), or empty vector (EV; 25 nM) with/without a specific inhibitor (inh) of miR193a (25 nM; n = 3). After 48 h, proteins were extracted. Protein blots were probed for WT1 and reprobed for PAX2 and actin. Gels are displayed. D: cumulative densitometric data from the protein blots of C. *P < 0.05 compared with other WT1/PAX2 variables; ^aP < 0.05 compared with respective C.

Fig. 3.

High glucose downregulates DPD expression of apolipoprotein (APO) L1 through upregulation of miR193a. A: DPDs were incubated in media containing different concentrations of glucose (5, 10, 20, 30, and 35 mM) for 48 h (n = 3). Protein blots were probed for APOL1 and reprobed for GAPDH. Representative gels are displayed. B: DPDs were incubated in media containing either conventional glucose (11 mM) or HG (30 mM) for 48 h. Proteins were extracted from UNDPDs and experimental DPDs (n = 4). Protein blots were probed for APOL1 and reprobed for WT1, PAX2, and GAPDH. Gels of 3 different lysates are displayed. C: cumulative densitometric data of protein blots of B are shown in a bar diagram. *P < 0.05 compared with respective UNDPD and DPD/HG; **P < 0.01 compared with respective UNDPD and DPD/HG. D: DPDs were incubated in media containing normal glucose (5 mM), high glucose (30 mM), or empty vector (25 nM) with or without miR193a inhibitor (25 nM; miR-Inh) for 48 h (n = 3). Proteins were extracted. Protein blots were probed for APOL1 and reprobed for GAPDH. Gels are displayed. E: cumulative densitometric data are shown in bar graphs. *P < 0.05 compared with C and empty vector (EV); ^aP < 0.05 compared with HG alone. F: RNAs were extracted from the lysate preparations of D, and cDNAs were amplified for APOL1 mRNA. Cumulative data are shown in a bar diagram. *P < 0.05 compared with respective C and EV; **P < 0.01 compared with C, EV, and HG alone; ***P < 0.001 compared with C, EV, and HG alone; ^aP < 0.05 compared with miR-Inh alone. G: DPDs grown on coverslips were incubated in media containing either normal glucose (C) or high glucose with or without a miR193a inhibitor (miR; 25 nM) for 48 h (n = 3) followed by immunolabeling for APOL1. Subsequently, cells were examined under a confocal microscope. Representative fluoromicrographs are shown.

Fig. 4.

Overexpression of miR193a downregulates APOL1. A: DPDs were transfected with either empty vector (EV) or miR193a plasmid (n = 3). Proteins were extracted. Protein blots were probed for WT1, PAX2, and APOL1 and reprobed for GAPDH. Gels from 3 different lysates are displayed. B: cumulative densitometric data from the lysates of A. *P < 0.05 compared with control and EV; **P < 0.01 compared with control and EV. C: RNAs were extracted from the lysates of A. cDNAs were amplified with a specific primer for APOL1. *P < 0.05 compared with other variables.

Fig. 5.

WT1 repressor complex preserves DPD molecular phenotype. A: protein blots of UNDPDs (0-day incubation) and DPDs (10-day incubation) were probed for PD (nephrin, WT1, and podocalyxin) and PEC (PAX2) markers, APOL1, and actin. Gels from 3 different lysates are displayed. B: protein blots from A were reprobed for the components of WT1 repressor complex. Gels from 3 different lysates are displaced. C: cumulative densitometric data from the lysates of A are shown as a bar diagram. *P < 0.05 compared with respective 0-day. D: cumulative densitometric data from the lysates of B are shown as a bar diagram. *P < 0.05 compared with respective 0-day. E: lysates from A were immunoprecipitated (IP) with the anti-WT1 antibody. IP fractions were probed for WT1, RBBP4 (Polycomb group protein), menin, H3K27me3, DNMT1, and IgG. Gels from 3 different IP fractions are displayed. F: cumulative densitometric data from the lysates of E are shown as bar graphs. *P < 0.05 compared with respective 0-day. G: DPDs were transfected with scrambled (SCR), WT1 siRNA (25 nM), DNMT1 (25 nM), or WT1 + DNMT1 siRNAs with Lipofectamine RNAiMAX transfection reagent according to manufacturer’s protocol and left in Opti-MEM for 48 h (in WT1 + DNMT1 experiments, cells were exposed to WT1 siRNA for 48 h and DNMT1 siRNA for 24 h). Subsequently, proteins were extracted. Protein blots were probed for PAX2, WT1, nephrin, podocalyxin (PDX), and DNMT1 and reprobed for actin. Gels from 3 different lysates are displayed. H: cumulative densitometric data from the lysates of G are shown as a bar diagram. *P < 0.05 compared with C, SCR, and DNMT1 siRNA in PAX2 variables; *P < 0.05 compared with C, SCR, siRNA WT1 and siRNA DNMT1/WT1 in PDX and DNMT1 variables; **P < 0.01 compared with C, SCR, and DNMT1 siRNA in PAX2 variables; ^aP < 0.05 compared with C and SCR in nephrin variables; ^bP < 0.0.05 compared with C and SCR in PDX variables; ^cP < 0.01 compared C and SCR in WT1 variables; ^dP < 0.01 compared with DNMT1 siRNA, C, and SCR in DNMT1 variables.

Fig. 6.

Role of APOL1 in preservation of DPD molecular phenotype. A: DPDs were transfected with either control (scrambled, SCR) or APOL1 siRNA. Proteins were extracted from control and transfected cells (n = 3). Protein blots were probed for APOL1 and reprobed for PAX2, WT1, and GAPDH. Gels from 3 different lysates are displayed. B: cumulative densitometric data of protein blots displayed in A. *P < 0.05 compared with respective APOL1, WT1, and PAX2 in control and SCR variables. C: DPDs were transfected with either control (scrambled, SCR) or APOL1 siRNA. RNAs were extracted from control and transfected cells (n = 3) and assayed for miR193a. Cumulative data are shown in a bar diagram. **P < 0.01 compared with other variables. D: DPDs were transfected with either control (scrambled, SCR) or APOL1 siRNA and incubated in media with or without miR193a inhibitor for 48 h (n = 3). Protein blots were probed for APOL1, WT1, PAX2, and GAPDH. Gels from 3 different lysates are displayed. E: RNAs were extracted from the lysate preparations of D and assayed for miR193a. Cumulative data are shown in a bar diagram. *P < 0.05 compared with control and SCR; **P < 0.01 compared with control, miR193a inh alone, and SCR; ^aP < 0.05 compared with all other variables.

Fig. 7.

APOL1 negatively regulates miR193a expression in DPDs. A: UNDPDs stably expressing vector and overexpressing APOL1G0 were incubated in RPMI containing 11 mM glucose and 10% serum for 10 days at 37°C. APOL1G0-expressing DPDs were transfected with either scrambled or APOL1 siRNAs (n = 6). After 48 h, proteins were extracted from control (vector) and siRNA-transfected cells. Protein blots were probed for APOL1 and reprobed for WT1, PAX2, and actin. Representative gels from 3 different lysates are displayed. B: cumulative densitometric data (n = 6) from the protein blots of A are shown in a bar diagram. *P < 0.05 compared with vector (V) and G0 APOL1 siRNA in WT1 and all other variables in PAX2 proteins; **P < 0.01 compared with V and G0 APOL1 siRNA in APOL1 protein. C: RNAs were extracted from the lysates of A. RNAs were assayed for miR193a, and cumulative data are shown in a bar diagram. *P < 0.05 compared with vector; **P < 0.01 compared with vector; ***P < 0.001 compared with G0 and G0/SCR.

Fig. 8.

Vitamin D receptor (VDR) agonist (VDA) preserves DPD phenotype through modulation of miR193a-APOL1 axis in high-glucose milieu. A: UNDPDs were incubated in media containing either vehicle (0.1% DMSO) alone or different concentrations of VDA (EB 1089; 0, 1, 10, and 100 nM) for 48 h (n = 3). RNAs were extracted and assayed for miR193a. Cumulative data are shown in a bar diagram. *P < 0.05 compared with vehicle (VDA; 0 nM), VDA, 0 and 1.0 nM; **P < 0.01 compared with vehicle (VDA; 0 nM), VDA, 0 and 1.0 nM; ^aP < 0.05 compared with VDA, 10 nM. B: DPDs were incubated in media containing normal glucose (C; 5 mM), high glucose (HG; 30 mM), or vehicle (0.1% DMSO) with or without VDA (EB 1089; 10 nM) for 48 h (n = 3). RNAs were extracted and assayed for miR193a. Cumulative data are shown in a bar diagram. **P < 0.01 compared with other variables. C: DPDs were incubated in media containing either normal glucose (C; 5 mM) or high glucose (HG; 30 mM) with or without VDA (EB 1089; 10 nM) for 48 h (n = 3). Protein blots were probed for APOL1 and reprobed for GAPDH. Representative gels are displayed. D: cumulative densitometric data from the lysates of C are shown in a bar diagram. *P < 0.05 compared with C; ^aP < 0.05 compared with HG alone. E: DPDs were incubated in media containing normal glucose (C; 5 mM), vehicle (Veh; 0.1% DMSO), or high glucose (HG; 30 mM) with or without VDA (EB 1089; 10 nM) for 48 h (n = 3). Protein blots were probed for WT1 and PAX2 and reprobed for GAPDH. Representative gels are displayed. F: cumulative densitometric data from the protein blots of E are shown in a bar diagram. *P < 0.05 compared with respective all other variables.

Fig. 9.

Proposed mechanistic schemes. A: composition of WT1 repressor complex is shown in a cartoon. WT1 repressor complex binding to PAX2 promoter represses its transcription. Disruption of this complex would derepress the expression of PAX2. B: high glucose enhanced the expression of miR193a, which led to downregulation of APOL1 expression in DPDs. These alterations in miR193a-APOL1 axis induced DPD dedifferentiation. VDA provided protection against this effect of high glucose through the reversal of miR193a-APOL1 axis alterations. PcG, Polycomb group.