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 9

The generation of miR-30 sponge transgenic mice.

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The generation of miR-30 sponge transgenic mice. (A) Schematic of the st...
(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.