Downregulation of microRNA-30 facilitates podocyte injury and is prevented by glucocorticoids

Abstract

MicroRNAs (miRNAs) are essential for podocyte homeostasis, and the miR-30 family may be responsible for this action. However, the exact roles and clinical relevance of miR-30s remain unknown. In this study, we examined the expression of the miR-30 family in the podocytes of patients with FSGS and found that all members are downregulated. Treating cultured human podocytes with TGF-β, LPS, or puromycin aminonucleoside (PAN) also downregulated the miR-30 family. Podocyte cytoskeletal damage and apoptosis caused by treatment with TGF-β or PAN were ameliorated by exogenous miR-30 expression and aggravated by miR-30 knockdown. Moreover, we found that miR-30s exert their protective roles by direct inhibition of Notch1 and p53, which mediate podocyte injury. In rats, treatment with PAN substantially downregulated podocyte miR-30s and induced proteinuria and podocyte injury; however, transfer of exogenous miR-30a to podocytes of PAN-treated rats ameliorated proteinuria and podocyte injury and reduced Notch1 activation. Finally, we demonstrated that glucocorticoid treatment maintains miR-30 expression in cultured podocytes treated with TGF-β, LPS, or PAN and in the podocytes of PAN-treated rats. Glucocorticoid-sustained miR-30 expression associated with reduced Notch1 activation and alleviated podocyte damage. Taken together, these findings demonstrate that miR-30s protect podocytes by targeting Notch1 and p53 and that the loss of miR-30s facilitates podocyte injury. In addition, sustained miR-30 expression may be a novel mechanism underlying the therapeutic effectiveness of glucocorticoids in treating podocytopathy.

Figure 1.

miR-30s are expressed in human glomerular podocytes and downregulated in patients with FSGS. (A) qRT-PCR quantifications of the miR-30s in the microdissected glomeruli from 16 patients with FSGS and 6 normal controls. Black triangles and red dots represent normal controls (NC) and patients with FSGS, respectively, and indicate their relative miR-30 abundance in the glomeruli. The black and red lines represent the means of miR-30 abundance in the NC and FSGS groups, respectively. Statistical significance (P<0.05) is present between the control and FSGS groups for all miR-30 family members. (B) In situ hybridization of miR-30a and miR-30d with renal biopsies of patients with FSGS and normal controls (CTL). Black arrows indicate normal podocytes with abundant miR-30 expression, white arrows indicate sclerotic areas with a reduced miR-30 expression, and yellow arrows indicate parietal epithelial cells that also express miR-30. (C) Quantification of in situ hybridization results in B. *P<0.05.

Figure 2.

miR-30 family members are downregulated in cultured human podocytes treated with TGF-β, PAN, and LPS, respectively. (A) qRT-PCR analyses of miR-30a through miR-30e in podocytes treated with TGF-β (5 ng/ml) for 1, 6, 12, and 24 hours, respectively, demonstrating that all miR-30 members are downregulated by TGF-β. (B and C) qRT-PCR analyses of miR-30s in podocytes treated with PAN (50 µg/ml) (B) or LPS (10 µg/ml) (C) for 12 or 24 hours. The data are expressed as the mean ± SD of three independent experiments. *P<0.05 versus untreated control.

Figure 3.

Exogenous miR-30a protects podocytes from cytoskeletal damage and apoptosis induced by TGF-β or PAN. (A) Fluorescein-conjugated phalloidin staining of stably transfected Scram or miR-30a podocytes after treatment with TGF-β (5 ng/ml). (B) Quantification of results in A. (C) Annexin V flow cytometry of Scram or miR-30a podocytes after TGF-β treatment for 12 or 24 hours. (D) Quantification of results in C. (E) TUNEL assay of the cells as in C. Each bar represents the mean ± SD of the percentages of apoptotic cells in 30 high-power fields. (F) Annexin V flow cytometry of Scram or miR-30a podocytes after PAN treatment (50 µg/ml) for 12 or 24 hours. (G–I) Immunoblotting of cleaved caspase 3 (G), BAX and BCL2 (H), and p38 (t-p38) and phosphorylated p38 (p-p38) (I) in the Scram and miR-30a podocytes after TGF-β treatment for the indicated time. All data are presented as the mean ± SD of three independent experiments. *P<0.05 versus untreated control; ^#P<0.05.

Figure 4.

miR-30 knockdown by Sponge damages the podocytes directly or aggravated the damage induced by TGF-β or PAN. (A) F-actin staining with phalloidin in the podocytes transiently transfected with or without miR-30 Sponge after TGF-β treatment for 12 and 24 hours. (B) Quantification of the stress fibers in A. (C and D) Annexin V flow cytometry of podocytes transiently transfected with vector (Mock) or miR-30 Sponge and treated with PAN (50 µg/ml) (C) or TGF-β (5 ng/ml) (D) for 12 or 24 hours. The percentages of Annexin V-positive cells are shown in the graph on the right, and each bar represents the mean ± SD (expressed in arbitrary units of stress fiber intensity) of triplicate experiments. *P<0.05 versus untreated control; ^#P<0.05.

Figure 5.

Notch1 is a direct target of miR-30s. (A) The predicted miR-30 binding site in the Notch1 3′UTR (Targetscan) and the point mutations (G-T, U-T, and A-C) made in the region corresponding to the miR-30 seed sequence to create a mutant Notch1 3′-UTR for the luciferase reporter construction. (B) Immunoblotting of Notch1 intracellular domain (NICD) in Scram or miR-30a podocytes after TGF-β treatment for the time indicated. (C) Immunoblotting of the full-length (FL) or the transmembrane domain (TM) of Notch1 with Scram or miR-30a podocytes after TGF-β treatment. (D) Immunoblotting of FL and TM Notch1 with podocytes transiently transfected with Mock or miR-30 Sponge after TGF-β treatment for 24 hours. (E) Immunoblotting of FL and TM Notch1 with the podocytes transfected with the Scramble miR- (Scram.) or miR-30c to miR-30d (miR-30cd)–expressing construct after TGF-β treatment for 24 hours. (F) Luciferase assays of the podocytes cotransfected with each luciferase reporter and Scramble miR or miR-30 precursors. (G) The luciferase assay demonstrates that the wild-type reporter has increased activity in the cells transiently transfected with miR-30 Sponge but not in those treated with the mutant reporter. All data are presented as the mean ± SD of three independent experiments. In A–E and G, *P<0.05 versus untreated control. ^#P<0.05; in F, *P<0.05 versus Scramble miR-transfected cells.

Figure 6.

miR-30s inhibits p53 in human podocytes. (A) Immunoblotting of p53 and phosphorylated p53 with Scram and miR-30a podocytes after TGF-β treatment for the time indicated. Quantifications of the blots are shown on the right. (B) Immunoblotting of p53 and phosphorylated p53 (p-p53) with podocytes transiently transfected with empty vector (Mock) or miR-30 Sponge followed by TGF-β treatment. Quantifications of the results are shown on the right. *P<0.05 versus untreated control; ^#P<0.05.

Figure 7.

Glucocorticoid (DEX) prevents downregulation of miR-30s and ameliorated podocyte injury. (A and B) qRT-PCR analysis of miR-30s in the podocytes treated with TGF-β (A) and PAN (B) in the presence or absence of DEX. (C and D) Annexin V flow cytometry analyses of the podocytes transfected with miR-30 Sponge or vehicle, followed by treatment of TGF-β (C) or PAN (D) in the absence or presence of DEX. The results show that DEX reduces podocyte apoptosis induced by TGF-β or PAN through sustaining miR-30 expression. (E) qRT-PCR analysis of BAX, BCL2, or CD2AP expression in cells treated with PAN in the presence or absence of DEX. All data are expressed as the mean ± SD of three independent experiments. *P<0.05 versus untreated control; ^#P<0.05. DEX, dexamethasone.

Figure 8.

Characterization of PAN-treated rats with either Scramble miR or miR-30a transfer. Scramble miR or miR-30 transfer is performed every 3 days. Day 0 is defined as the day when PAN injection and the first miR-30a transfer are conducted. (A) qPCR analysis of miR-30a in the glomeruli of untreated rats (CTL) or PAN-treated rats, immediately followed by Scramble miR (PAN + Scram) or miR-30a transfer (PAN + miR-30a) (n=9 in each group) at day 9, demonstrating that miR-30a is downregulated by PAN, but exogenous miR-30a transfer maintains the level of miR-30a in the podocytes/glomeruli of the rats. (B) Twenty-four hour urinary protein levels in rats in A. *P<0.05 versus the PAN + Scram group. (C) Periodic acid–Schiff staining of kidney sections of the rats at day 9, showing sclerotic lesions (arrows) in PAN + Scram rats, which were ameliorated in PAN + miR-30a rats. One hundred glomeruli of each rat were examined for sclerotic lesions, and the percentage of sclerotic glomeruli of each group of rats is shown on the right (n=9). #P<0.05. (D–G) Representative electron microscopy images (D), immunofluorescence staining of podocin (E), immunohistochemical staining of Notch1 intracellular domain (NICD) (F), and TUNEL assay (G) of the glomeruli in PAN + Scram and PAN + miR-30a rats, demonstrating an ameliorated foot process effacement (yellow asterisks), less affected podocin expression, reduced Notch1 activation, and less severe apoptosis (arrows) in the latter. **P<0.05.

Figure 9.

Characterization of PAN-treated rats with or without glucocorticoid (MP) treatment. (A) qPCR analyses of miR-30s with isolated glomeruli from rats that are treated with vehicle (CTL), PAN, or PAN followed by MP (PAN + MP) from day 0 through day 9 (d0), or from day 3 through day 9 (d3) (n=6 in each group). The glomeruli are isolated at day 9 after PAN injection. *P<0.05 versus CTL; ^#P<0.05 versus PAN. (B) Twenty-four hour urinary protein levels of rats at days 3, 5, 7, and 9, demonstrating that MP prevents PAN-induced proteinuria. *P<0.05 versus CTL; ^#P<0.05 versus PAN. (C) Periodic acid–Schiff staining of rat kidneys at day 9 after PAN injection. Arrows denote sclerotic lesions in the glomeruli. Quantification of glomerular lesions of these rats is shown on the right. **P<0.05. MP, methylprednisolone.