A MicroRNA Cluster miR‐23–24–27 Is Upregulated by Aldosterone in the Distal Kidney Nephron Where it Alters Sodium Transport

Abstract

The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23–24–27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3′-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC’s function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone’s action via a novel target.

Aldosterone‐induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia

“…Inserts were mounted in modified Ussing chambers (P2300; Physiologic Instruments, San Diego, CA) and continuously short circuited with an automatic voltage clamp (VCC MC8; Physiologic Instruments) as described previously. [60][61][62] The apical and basolateral chambers each contained 4 mL of Ringer solution (120 mM of NaCl, 25 mM of NaHCO 3 , 3.3 mM of KH 2 PO 4 , 0.8 mM of K 2 HPO 4 , 1.2 mM of MgCl 2 , 1.2 mM of CaCl 2 , and 10 mM of glucose). Chambers were constantly gassed with a mixture of 95% of O 2 , 5% of CO 2 at 37°C, which maintained the pH at 7.…”

“…4 DNA plasmids and RNA oligonucleotide constructs were transiently transfected into the mCCD and HEK293 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and as described previously. [60][61][62] The sequences for siRNA and all primers are listed in Table 1. Double-stranded RNA miR mimics (miRIDIAN microRNA Mimics) were obtained from Thermo Fisher Scientific.…”

“…Forward primer sequence Reverse primer sequence mouse For all miRNA qPCRs, the miRNA-specific forward primers were paired to a universal reverse primer as described before. 60,62 Real-time PCR was carried out using an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Life Technologies, Grand Island, NY). Detected signals from miR amplifications were normalized to the relative expression of small nucleolar RNA (SNO-202 and SNO-135) with each reaction/sample run in triplicate.…”

“…Distal kidney nephron principal epithelial cells were isolated from a crude kidney tubule preparation (1 kidney from each animal) using a lectin binding and magnetic bead isolation technique as described before. 60,62 The isolated cells were immediately processed for RNA isolation; isolated RNA was used immediately or stored at −80°C until needed.…”

“…Distal nephron epithelial cells were isolated from kidneys of mice on control and sodium-deficient diets following enzymatic digestion and magnetic bead isolation as we have described previously. 60,62 The abundance of miRs-466a/e and miR-466b/c from isolated CCD cells in mice on a 72-hours low sodium diet was significantly greater relative to mice on normal sodium diets ( Figure 1C). Expression of control miR-10a which is abundant in the CCD epithelial cells, and not regulated by aldosterone, related family member miR-466d, and unrelated miR-466g were not significantly altered in response to the low Na + diet.…”