The protease corin regulates electrolyte homeostasis in eccrine sweat glands

Abstract

Sweating is a basic skin function in body temperature control. In sweat glands, salt excretion and reabsorption are regulated to avoid electrolyte imbalance. To date, the mechanism underlying such regulation is not fully understood. Corin is a transmembrane protease that activates atrial natriuretic peptide (ANP), a cardiac hormone essential for normal blood volume and pressure. Here, we report an unexpected role of corin in sweat glands to promote sweat and salt excretion in regulating electrolyte homeostasis. In human and mouse eccrine sweat glands, corin and ANP are expressed in the luminal epithelial cells. In corin-deficient mice on normal- and high-salt diets, sweat and salt excretion is reduced. This phenotype is associated with enhanced epithelial sodium channel (ENaC) activity that mediates Na+ and water reabsorption. Treatment of amiloride, an ENaC inhibitor, normalizes sweat and salt excretion in corin-deficient mice. Moreover, treatment of aldosterone decreases sweat and salt excretion in wild-type (WT), but not corin-deficient, mice. These results reveal an important regulatory function of corin in eccrine sweat glands to promote sweat and salt excretion.

Fig 1. Corin, ANP, and NPR-A expression in human and mouse eccrine sweat glands.

(A) Immunohistochemical staining of corin (brown) in human eccrine sweat glands. IgG was used in negative controls. (B) Co-staining of corin (red) and keratin (an epithelial marker) (green) in ductal and secretory epithelial cells of human eccrine sweat glands. (C) Co-staining of corin (green) and SMA (red) in the secretory epithelial cells. (D) Immunohistochemical staining of ANP and NPR-A (brown) in human eccrine sweat glands. (E) HE staining of a mouse paw skin section with eccrine sweat glands. (F and G) RT-PCR (F) and western blotting (G) analysis of corin expression in WT and corin KO mouse footpads. Hearts (positive) and livers (negative) were controls. (H–J) Co-staining of corin (green) and SMA (H), ANP (I), or NPR-A (J) (red) in eccrine sweat glands from WT and corin KO mouse footpads. Scale bars are indicated. Data are representative of at least 3 experiments in each set of the studies. ANP, atrial natriuretic peptide; HE, hematoxylin–eosin; IgG, immunoglobulin G; KO, knockout; NPR-A, natriuretic peptide receptor-A; RT-PCR, reverse transcription PCR; SMA, smooth muscle actin; WT, wild-type.

Fig 2. Sweat response in WT, corin KO, and corin hcKO mice.

(A and B) An iodine–starch assay was used to measure sweat response in WT and corin KO mice. Photos were taken before (0 min) and 2 min after pilocarpine (pilocar) injection. Sweat excretion is indicated by black staining. Areas of black staining were analyzed by computer software. Quantitative data are presented. (C and D) To calculate sweat volume, photos were taken by a digital camera 10 min after pilocarpine injection. The number and diameter of sweat droplets were analyzed by computer software. Representative photos and quantitative data are presented. (E) RT-PCR analysis of corin expression in hearts, livers, and footpads (skin) from WT and hcKO mice. (F and G) Sweat excretion in WT and corin hcKO mice was analyzed by the iodine–starch assay. Representative photos and quantitative data are shown. (H) Sweat excretion in WT and corin KO mice on 0.3% or 4% salt diet before (−) and 2 min after (+) pilocarpine (pilocar) injection. (I and J) Sweat Na⁺ (I) and Cl⁻ (J) levels in WT and corin KO mice on 0.3% or 4% salt diet and with pilocarpine injection. In bar graphs, data are mean ± SEM. Each dot represents data from 1 mouse, except for sweat Na⁺ and Cl⁻ data, in which each dot represents data from 8 paws of 2 mice. P values were analyzed by Student t test or 1-way ANOVA, and the original numerical values are in S1 Data. hcKO, heart-conditional corin KO; KO, knockout; RT-PCR, reverse transcription PCR; WT, wild-type.

Fig 3. Effects of ENaC and CFTR inhibitors on sweat excretion.

(A) Immune staining of β-ENaC (brown) in eccrine sweat glands from WT and corin KO mouse footpads. IgG was a negative control. (B) Co-staining of corin (green) and β-ENaC (red) in WT and corin KO eccrine sweat glands. (C and D) Sweat excretion, indicated by black-staining areas, was measured in WT and corin KO mice on 0.3% (C) or 4% (D) salt diet, with (+) or without (−) amiloride treatment, before (−) and 2 min after (+) pilocarpine (pilocar) injection. (E and F) Sweat Na⁺ (E) and Cl⁻ (F) levels in WT and corin KO mice on 0.3% or 4% salt diet and treated with amiloride. (G) Immune staining of CFTR (brown) in eccrine sweat glands in WT and corin KO mouse footpads. (H) Co-staining of corin (red) and CFTR (green) in eccrine sweat glands in WT and corin KO mouse footpads. (I and J) Sweat excretion, indicated by black-staining areas, was measured in WT and corin KO mice on 0.3% (I) or 4% (J) salt diet, with (+) or without (−) CFTR(inh)-172 (CFTR-Inh) treatment, before (−) and 2 min after (+) pilocarpine (pilocar) injection. (K and L) Sweat Cl⁻ (K) and Na⁺ (L) levels in WT and corin KO mice on 0.3% or 4% salt diet and treated with (+) CFTR-Inh. In bar graphs, data are mean ± SEM. Each dot represents data from 1 mouse, except for sweat Na⁺ and Cl⁻ data, in which each dot represents data from 8 paws of 2 mice. P values were analyzed by 1-way ANOVA, and the original numerical values are in S1 Data. CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel; IgG, immunoglobulin G; KO, knockout; WT, wild-type.

Fig 4. Effects of aldosterone on sweat and salt excretion.

(A and B) Quantitative data (mean ± SEM) of black-staining areas in the iodine–starch test in WT and corin KO mice on 0.3% (A) or 4% (B) salt diet without (−) or with (+) aldosterone treatment and pilocarpine (pilocar) injection. (C and D) Sweat Na⁺ (C) and Cl⁻ (D) levels in WT and corin KO mice on 0.3% or 4% salt diet and treated with (+) aldosterone. Data are mean ± SEM. Each dot represents data from 1 mouse, except for sweat Na⁺ and Cl⁻ data, in which each dot represents data from 8 paws of 2 mice. P values were analyzed by 1-way ANOVA, and the original numerical values are in S1 Data. KO, knockout; WT, wild-type.