MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes
Junnan Wu, … , Shaolin Shi, Zhihong Liu
Junnan Wu, … , Shaolin Shi, Zhihong Liu
Published October 5, 2015
Citation Information: J Clin Invest. 2015;125(11):4091-4106. https://doi.org/10.1172/JCI81061.
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Research Article Nephrology

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

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.

Authors

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

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Figure 2

TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 protein expression in human podocytes from control subjects and patients with FSGS.

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TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 protein expression in human po...
(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).