MicroRNA-29a promotion of nephrin acetylation ameliorates hyperglycemia-induced podocyte dysfunction

Abstract

Podocyte dysfunction is a detrimental feature in diabetic nephropathy, with loss of nephrin integrity contributing to diabetic podocytopathy. MicroRNAs (miRs) reportedly modulate the hyperglycemia-induced perturbation of renal tissue homeostasis. This study investigated whether regulation of histone deacetylase (HDAC) actions and nephrin acetylation by miR-29 contributes to podocyte homeostasis and renal function in diabetic kidneys. Hyperglycemia accelerated podocyte injury and reduced nephrin, acetylated nephrin, and miR-29a levels in primary renal glomeruli from streptozotocin-induced diabetic mice. Diabetic miR-29a transgenic mice had better nephrin levels, podocyte viability, and renal function and less glomerular fibrosis and inflammation reaction compared with diabetic wild-type mice. Overexpression of miR-29a attenuated the promotion of HDAC4 signaling, nephrin ubiquitination, and urinary nephrin excretion associated with diabetes and restored nephrin acetylation. Knockdown of miR-29a by antisense oligonucleotides promoted HDAC4 action, nephrin loss, podocyte apoptosis, and proteinuria in nondiabetic mice. In vitro, interruption of HDAC4 signaling alleviated the high glucose-induced apoptosis and inhibition of nephrin acetylation in podocyte cultures. Furthermore, HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), the enrichment of H3K9Ac in miR-29a proximal promoter, and miR-29a transcription in high glucose-stressed podocytes. In conclusion, hyperglycemia impairs miR-29a signaling to intensify HDAC4 actions that contribute to podocyte protein deacetylation and degradation as well as renal dysfunction. HDAC4, via epigenetic H3K9 hypoacetylation, reduces miR-29a transcription. The renoprotective effects of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis highlights the importance of post-translational acetylation reactions in podocyte microenvironments. Increasing miR-29a action may protect against diabetic podocytopathy.

Figure 1.

Effects of hyperglycemia on podocyte integrity and miR-29 expression in kidneys. (A) Diabetes increases urinary protein levels. (B and C) Podocytes in diabetic kidneys strongly display desmin immunoreactivity and TUNEL stain (arrows) (B) and weakly express nephrin and WT-1 (arrows) immunofluorescence (C). (D and E) Diabetes reduces the levels of nephrin, acetylated protein, and acetylated nephrin (D) and the expression of miR-29a (E). (F) miR-29a, miR-29b, and miR-29c are detectable in podocytes (arrows) and tubular cells (arrowheads) of normal kidneys. Few podocytes and tubular cells in diabetic kidney weakly display miR-29a transcripts. Data are expressed as the mean±SEM calculated from eight mice at each time point. *P<0.05, significant difference from the normal control group. Ac-lysine, acetylated lysine; DM, diabetic mice; IB, immunoblotting; IP, immunoprecipitates; NC, normal control.

Figure 2.

Effect of miR-29a overexpression on miR-29a expression and renal function. (A) hPGK promoter-coded full-length 506 bp miR-29a precursors are constructed. Transgenic mice with or without diabetes have higher miR-29a expression in renal glomeruli than that of wild-type mice. (B) Podocytes (arrows) and tubular cells in transgenic mice strongly display the miR-29a transcript. (C–E) There is no significant difference in body weight (C), blood glucose (D), or HbA1c (E) levels between the transgenic mice and wild-type mice with diabetes. (F–H) Overexpression of miR-29a reduces diabetes-induced hyperfiltration (F), kidney weight loss (G), and urinary protein secretion (H). Data are expressed as the mean±SEM calculated from six to eight mice at each time point. *P<0.05, significant difference versus the NC group; ^#P<0.05, significant difference versus wild-type group. CCR, creatinine clearance; DM, diabetic mice; HbA1c, hemoglobin A1c; NC, normal control; PC, positive control; STZ, streptozotocin; Tg, transgenic mice; WT, wild-type mice.

Figure 3.

Effect of miR-29a overexpression on glomerular fibrosis and podocyte integrity of diabetic kidneys. (A) Overexpression of miR-29a reduces the diabetes promotion of PAS-stained renal glomeruli. (B–E) Podocytes in diabetic kidneys of transgenic mice weakly express desmin immunoreactivity (B) and TUNEL stain (arrows) (C), and strongly express nephrin (D) and WT-1 immunofluorescence (E). (F and G) Overexpression of miR-29a attenuates diabetes-induced nephrin and WT-1 loss (arrows) (F) and the expression of TGF-β1, fibronectin, and IL-1β in renal glomeruli (G). *P<0.05, significant difference versus the NC group; ^#P<0.05, significant difference versus the wild-type group. Mean glomerular volume (GV; μm³/10⁶) was calculated according to the Weibel and Gomez formula. DM, diabetic mice; NC, normal control; PAS, periodic acid–Schiff; Tg, transgenic mice; WT, wild-type mice.

Figure 4.

Effect miR-29a overexpression on HDAC level and nephrin acetylation in renal glomeruli. (A) Electrophoretography and liquid chromatography–tandem mass spectrometry spectrum of HDAC4. (B) miR-29a overexpression reduces the diabetes promotion of HDAC4 levels but not the HDAC7A or PCAF level. (C–E) Overexpression of miR-29a increased acetylated nephrin levels (C) and reduced ubiquitinated nephrin levels (D) in renal glomeruli and urinary nephrin excretion in diabetic kidneys (E). Data are expressed as the mean±SEM calculated from six to eight mice in each group. *P<0.05, significant difference versus the NC group; ^#P<0.05 indicates significant difference versus the wild-type group. Ac-lysine, acetylated lysine; Ac-nephrin, acetylated nephrin; DM, diabetic mice; IB, immunoblotting; IP, immunoprecipitates; MM, molecular mass; NC, normal control; PCAF, P300/CBP-associated factor; PI, isoelectric point; Tg, transgenic mice; Ub-nephrin, ubiquitinated nephrin; WT, wild-type mice.

Figure 5.

Representative immunofluorescence images of nephrin and acetylated lysine in renal glomeruli of miR-29a transgenic mice and wild-type mice. Podocytes strongly display nephrin immunofluorescence (green fluorescence) and acetylated-lysin immunoreactions (red fluorescence) in transgenic mice. DAPI, 4′,6-diamidino-2-phenylindole; DM, diabetic mice; NC, normal control; Tg, transgenic mice; WT, wild-type mice.

Figure 6.

Effect of exogenous miR-29a antisense oligonucleotide on podocyte survival and renal function. (A) The miR-29a antisense oligonucleotide decreases miR-29a expression in renal glomeruli. (B and C) Podocytes (arrows) and tubular cells weakly express miR-29a transcripts (B) and strongly express desmin immunoreactivity and TUNEL stain (arrows) (C). (D) Knockdown of miR-29a reduces nephrin and acetylated nephrin and increases the levels of HDAC4 and ubiquitinated nephrin. (E) Knockdown of miR-29a increases the excretion of urinary nephrin and protein. Data are expressed as the mean±SEM calculated from six mice in each group. *P<0.05, significant difference versus empty vector group. Ac-nephrin, acetylated nephrin; AS, miR-29a antisense oligonucleotide; IB, immunoblotting; IP, immunoprecipitates; NC, normal control; Ub-nephrin, ubiquitinated nephrin; Vector, empty vector.

Figure 7.

Effects of miR-29a precursor and HDAC4 siRNA on nephrin acetylation in high glucose–stressed podocyte cultures. (A and B) The miR-29a precursor and HDAC4 RNAi attenuates the high glucose promotion of HDAC4 and ubiquitinated nephrin levels and increases the levels of nephrin and acetylated nephrin in primary podocytes (A) and immortalized podocyte cultures (B). (C and D) Representative images of ligation between HDAC4 and nephrin (C) and TUNEL fluorescence stain in podocytes (D). Podocytes in the miR-29a precursor and HDAC4 RNAi group weakly display HADC4 and nephrin ligation and TUNEL stain. Data are expressed as the mean±SEM calculated from at least three repeated experiments. *P<0.05, significant difference versus scrambled control group; ^#P<0.05, significant difference versus high glucose group. Ac-lysine, acetylated lysine; Ac-nephrin, acetylated nephrin; HG, high glucose; IB, immunoblotting; IP, immunoprecipitates; NC, normal control; PLA, proximity ligation assay; siRNA, small interfering RNA; Ub-nephrin, ubiquitinated nephrin.

Figure 8.

Effect of miR-29 and HDAC4 signaling on H3K9 acetylation and miR-29a transcription in podocyte cultures. (A and B) The miR-29a precursor and HDAC4 RNAi reduces the high glucose inhibition of H3K9Ac levels in primary podocytes (A) and immortalized podocyte cultures (B). (C and D) The miR-29a precursor and HDAC4 RNAi increases the enrichment of H3K9AC on the miR-29a proximal promoter region in primary podocytes (C) and immortalized podocyte cultures (D). (E and F) The miR-29a precursor and HDAC4 RNAi increases miR-29a transcription in high glucose–stressed primary podocytes (E) and immortalized podocyte cultures (F). Data are expressed as the mean±SEM calculated from at least three repeated experiments. *P<0.05, significant difference versus scrambled control group; ^#P<0.05, significant difference versus high glucose group. GADPH, glyceraldehyde 3-phosphate dehydrogenase; HG, high glucose; siRNA, small interfering RNA.

Figure 9.

Scheme of miR-29a signaling protection against diabetes-induced podocyte injury and renal dysfunction. miR-29a signaling attenuates the promoting effects of hyperglycemia on HDAC4-dependent nephrin deacetylation and ubiquitination and WT-1 integrity loss that accelerates podocyte apoptosis, proteinuria, and renal fibrosis. HDAC4 modulation of H3K9 hypoacetylation impedes miR-29a transcription in high glucose–stressed podocytes.