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. 2015 Nov 2;125(11):4091-106.
doi: 10.1172/JCI81061. Epub 2015 Oct 5.

MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes

Junnan Wu, Chunxia Zheng, Xiao Wang, Shifeng Yun, Yue Zhao, Lin Liu, Yuqiu Lu, Yuting Ye, Xiaodong Zhu, Changming Zhang, Shaolin Shi, Zhihong Liu

MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes

Junnan Wu et al. J Clin Invest. .

Abstract

Calcium/calcineurin signaling is critical for normal cellular physiology. Abnormalities in this pathway cause many diseases, including podocytopathy; therefore, understanding the mechanisms that underlie the regulation of calcium/calcineurin signaling is essential. Here, we showed that critical components of calcium/calcineurin signaling, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, are the targets of the microRNA-30 family (miR-30s). We found that these 5 genes are highly expressed as mRNA, but the level of the proteins is low in normal podocytes. Conversely, protein levels were markedly elevated in podocytes from rats treated with puromycin aminonucleoside (PAN) and from patients with focal segmental glomerulosclerosis (FSGS). In both FSGS patients and PAN-treated rats, miR-30s were downregulated in podocytes. In cultured podocytes, PAN or a miR-30 sponge increased TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 expression; calcium influx; intracellular Ca2+ concentration; and calcineurin activity. Moreover, NFATC3 nuclear translocation, synaptopodin degradation, integrin β3 (ITGB3) activation, and actin fiber loss, which are downstream of calcium/calcineurin signaling, were induced by miR-30 reduction but blocked by the calcineurin inhibitor FK506. Podocyte-specific expression of the miR-30 sponge in mice increased calcium/calcineurin pathway component protein expression and calcineurin activity. The mice developed podocyte foot process effacement and proteinuria, which were prevented by FK506. miR-30s also regulated calcium/calcineurin signaling in cardiomyocytes. Together, our results identify miR-30s as essential regulators of calcium/calcineurin signaling.

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Figures

Figure 11
Figure 11. Working model of the relationship between miR-30s, calcium/calcineurin signaling, and actin stability in podocytes.
Figure 10
Figure 10. Characterization of the miR-30 sponge–expressing transgenic (SP+) mice.
(A) The SP+ mice developed significant proteinuria (n = 10). Two-tailed Student’s t test, *P < 0.05. (B) Electron microscopic examination of the kidneys of SP+ mice revealed massive foot process effacement of the podocytes. Representative images from 6 mice in each group are shown. Scale bars: 1 μm. (C) The calcineurin phosphatase assay of the isolated glomeruli demonstrated increased calcineurin activity in the glomeruli of SP+ mice compared with that in control mice (n = 5 each). Two-tailed Student’s t test, *P < 0.05. (D) Immunoblotting of the isolated glomeruli revealed increased expression of calcineurin and decreased expression of SYNPO in the glomeruli of SP+ mice. Parallel gels were run for calcineurin or SYNPO and GAPDH. (E) IHC showing that TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 protein expression levels were upregulated in the glomeruli of SP+ mice. Original magnification, ×40. Representative images from 6 mice in each group are shown. (F) FK506 reduced proteinuria in SP+ mice (n = 8). Two-way ANOVA, *P < 0.05. (G) Electron microscopic image showing that FK506 abolished podocyte foot process effacement in SP+ mice (n = 4). Quantification of the results is shown on the right. FPW, foot processes width. Scale bars: 1 μm. Two-way ANOVA, *P < 0.05. (H) CsA treatment of SP+ mice significantly reduced proteinuria (n = 8). Two-way ANOVA, *P < 0.05.
Figure 9
Figure 9. The generation of miR-30 sponge transgenic mice.
(A) Schematic of the structure of the conditional miR-30 sponge transgene and the resulting product of recombination induced by Cre. (B) Sequence of the miR-30 sponge transgene. The 11 miR-30 cognate sequences are indicated. (C) Identification by PCR of the founders carrying the transgene. The “MW” lane, which was originally on the right, has been moved to the left. (D) Red fluorescence of the body of a transgenic mouse. (E) Crossing the conditional miR-30 sponge transgenic mice with NPHS2-Cre transgenic mice resulted in the loss of RFP expression in podocytes, presumably resulting in expression of the miR-30 sponge. Original magnification, ×40. The failure to observe EGFP expression may be due to the following 2 reasons: 1) the miR-30 sponge sequence at the 3′UTR of EGFP may have recruited the miR-30–guided Ago complex that inhibits EGFP translation; and 2) the CAG promoter may have been inhibited in the injured podocytes (66). RFP appeared to be expressed only in podocytes but not in other cell types in the glomeruli. A similar phenomenon has been observed for a transgene carrying a ubiquitous CMV promoter (7). (F) qPCR analysis of sponge expression in the glomeruli from conditional miR-30 sponge transgenic mice (n = 3) and double-transgenic mice (SP+) (n = 3) demonstrated the successful induction of miR-30 sponge expression in the glomeruli of SP+ mice. Two-tailed Student’s t test, *P < 0.05.
Figure 8
Figure 8. miR-30s do not inhibit ITGB3 protein expression but suppress its activity.
(A) Neither exogenous miR-30a nor sponge transfection changed the protein levels of ITGB3 in cultured podocytes in the absence or presence of PAN or Ang II. (B) AP5 assays showed that ITGB3 activity was enhanced by PAN or by the miR-30 sponge, but the enhancement could be blocked with miR-30a, FK506, or 11R-VIVIT. Original magnification, ×20. Representative images from 3 independent experiments are shown. (C) Representative immunoblots showing that uPAR could be upregulated by PAN or the miR-30 sponge, but the upregulation could be blocked by exogenous miR-30a, FK506, or 11R-VIVIT. Parallel gels were run for ITGB3 or uPAR and the loading control (GAPDH) in all immunoblotting experiments. Quantification was performed on the basis of 3 independent experiments. Two-way ANOVA, *P < 0.05.
Figure 7
Figure 7. miR-30 silencing using the miR-30 sponge induced podocyte apoptosis, which was prevented by FK506 treatment.
(A) Flow cytometric analysis of the annexin V–stained cells treated as indicated. (B) Normalized quantification of the results in A. (C) Calcineurin phosphatase activity assays of the cells treated as indicated. All experiments were repeated at least 3 times, and the results are expressed as the mean ± SD. Two-way ANOVA, *P < 0.05.
Figure 6
Figure 6. miR-30s maintain SYNPO protein expression in podocytes.
(A) Immunoblotting showing that miR-30a overexpression prevented the PAN-induced loss of SYNPO expression. (B) The miR-30 sponge was sufficient to reduce SYNPO expression and augment the PAN-induced decrease in SYNPO expression. (C) FK506 prevented the effect of the miR-30 sponge on SYNPO expression. (D–F) Quantification of the results in A (D), B (E), and C (F). Parallel gels were run for SYNPO and GAPDH in each experiment. The results are expressed as the mean ± SD of at least 3 experiments. Two-way ANOVA, *P < 0.05.
Figure 5
Figure 5. miR-30 knockdown causes cytoskeletal damage to podocytes.
(A) Representative results of phalloidin staining showing that transfection with the miR-30 sponge resulted in loss of F-actin fibers from the cells and that this effect was prevented by FK506. Original magnification, ×20. (B) Quantification of the results in A (n = 20 cells). Two-way ANOVA, *P < 0.05. (C) Transfection of LNA–anti–miR-30s into cells similarly disrupted the F-actin fibers, an effect that was prevented by FK506. Original magnification, ×20. (D) Quantification of the results in C (n = 20 cells). Two-way ANOVA, *P < 0.05. LNA-Scr, scrambled control.
Figure 4
Figure 4. miR-30s inhibit Ca2+ entry and calcineurin signaling in cultured human podocytes.
(A) The Ca2+ influx assay demonstrated that miR-30a overexpression attenuated the PAN-induced increase in Ca2+ influx (n = 15–20 cells). (B) miR-30a prevented PAN-induced intracellular Ca2+ accumulation (n = 5). Two-way ANOVA, *P < 0.05. (C) The calcineurin phosphatase activity assay showed that miR-30a overexpression prevented a PAN-induced increase in calcineurin activity (left panel), but miR-30 silencing using a miR-30 sponge was sufficient to increase calcineurin activity in the cultured podocytes (right panel) (n = 6). (D) IF staining revealed that the nuclear translocation of NFATC3 was induced by PAN (arrow) but was prevented by FK506 or glucocorticoids (DEX) (arrowheads). Original magnification, ×20. Representative images from 3 independent experiments are shown.
Figure 3
Figure 3. miR-30s regulate the expression of TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 proteins in cultured human podocytes.
(A and B) Immunoblots showing that miR-30a overexpression (A) or DEX (B) blocked the PAN-induced upregulation of these proteins. (C) A miR-30 sponge increased the expression of these proteins in the absence or presence of PAN. (D–F) Quantification of the results from A (D), B (E), and C (F). Parallel gels were run in each experiment for the indicated protein and GAPDH. Results are expressed as the mean ± SD of at least 3 experiments. One-way ANOVA, #P < 0.05 versus scrambled, PBS, or mock; *P < 0.05.
Figure 2
Figure 2. TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 protein expression in human podocytes from control subjects and patients with FSGS.
(A) IF staining demonstrated that the protein levels of these genes are low in normal podocytes but are significantly upregulated in the podocytes of patients with FSGS. Original magnification, ×20. Representative images of 4 subjects are shown for each group. (B) IF staining for NFATC3 in the same tissues revealed upregulation and nuclear accumulation of NFATC3 (original magnification, ×20, as shown by colocalization with WT1; insets, original magnification, ×100)in the podocytes of FSGS patients. Representative images of 4 subjects are shown for each group. (C) Calcineurin activity in the glomeruli of controls and FSGS patients (n = 12 in each group). Two-tailed Student’s t test, *P < 0.05. (D) Immunoblotting for calcineurin in glomeruli isolated from controls or FSGS patients (n = 8 in each group). Parallel gels were run for calcineurin and GAPDH. Quantification of the gels is shown on the right. Two-tailed Student’s t test, *P < 0.05. (E) qPCR analysis showing that the mRNA levels of these genes in the glomeruli were not different between FSGS patients and controls (n = 6 in each group).
Figure 1
Figure 1. Trpc6, Ppp3ca, Ppp3cb, Ppp3r1, and Nfatc3 are the family members expressed at high mRNA levels in mouse podocytes.
(A) qPCR analysis of purified mouse podocytes demonstrating the relative abundances of all family members in mouse podocytes and showing that 5 genes are the predominantly expressed members of these families at the mRNA level (n = 6). (B) Comparison of the mRNA abundance of these 5 genes with that of other genes known to be highly expressed or to function in podocytes (n = 6).
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