Localization of corin and atrial natriuretic peptide expression in human renal segments

Abstract

Atrial natriuretic peptide (ANP)-mediated natriuretic response is a well-established cardiac endocrine function. Corin is a transmembrane protease that activates ANP in the heart. Corin expression has been detected in non-cardiac tissues including the kidney. Here we examined corin, pro-ANP/ANP and natriuretic peptide receptor-A (NPR-A) expression in human renal segments. By immunostaining and in situ hybridization, we found similar corin, pro-ANP/ANP and NPR-A protein and mRNA expression in human renal segments. The expression was most abundant in the proximal convoluted tubules and the medullary connecting ducts. In the proximal tubules, corin protein was present in the apical membrane region underneath the brush border where the ANP-degrading protease neprilysin was abundant. These results suggest that corin-mediated pro-ANP activation may occur in renal segments and that locally produced ANP may act in an autocrine manner to regulate sodium and water reabsorption in situ Our results also point to the proximal convoluted tubules as a major site for local ANP action. Such a renal corin/ANP autocrine mechanism may differ from the cardiac corin/ANP endocrine mechanism in regulating sodium homoeostasis under physiological and pathological conditions.

Keywords: atrial natriuretic peptide; corin; natriuretic peptide receptor; neprilysin.

Figure 1. Corin protein expression in renal segments

Human kidney sections were stained by H&E. Immunohistochemistry was performed using an anti-corin antibody or control antibodies against AQP1 and CK7, respectively. Proximal and distal convoluted tubules around glomeruli (g) are indicated by red and black arrows, respectively. Asterisks indicate collecting ducts. Black arrowheads indicate thick ascending limbs. Open arrowheads indicate descending thin limbs. Green arrowheads indicate ascending thin limbs. Scale bars: 40 μm.

Figure 2. ANP protein expression in renal segments

Human pro-ANP/ANP protein in the renal cortex and medullary sections was stained by immunohistochemistry. AQP1 staining was included as a control. Black arrows indicate distal convoluted tubules near glomeruli (g) (top panels). Asterisks indicate collecting ducts (middle and lower panels). Black arrowheads indicate thick ascending limbs (middle panels). Open arrowheads indicate descending thin limbs. Green arrowheads indicate ascending thin limbs. Scale bars: 40 μm.

Figure 3. NPR-A protein expression in renal segments

Human NPR-A protein in the renal cortex and medullary sections was stained by immunohistochemistry. AQP1 staining was included as a control. Black arrows indicate distal convoluted tubules near glomeruli (g) (top panels). Asterisks indicate collecting ducts (middle and lower panels). Black arrowheads indicate thick ascending limbs (middle panels). Open arrowheads indicate descending thin limbs. Green arrowheads indicate ascending thin limbs. Scale bars: 40 μm.

Figure 4. Corin, NPPA and NPR-A mRNA expression in human renal segments

In situ hybridization was performed to detect corin, NPPA and NPR-A mRNAs in human renal cortex (top panels) and medulla (lower panels). Proximal and distal convoluted tubules around glomeruli (g) are indicated by arrows and arrowheads, respectively (top panels). Medullary collecting ducts and thin limbs are indicated by asterisks and open arrowheads, respectively (lower panels). Scale bars: 20 μm.

Figure 5. Neprilysin expression in renal segments

Neprilysin protein in human renal cortex (A) and medullary (B) sections was stained by immunohistochemistry. AQP1 and CK7 staining was included as controls. Black arrows indicated distal convoluted tubules near glomeruli (g). Yellow arrows indicate the brush border. Asterisks indicate collecting ducts. Black arrowheads indicate thick ascending limbs. Green arrowheads indicate ascending thin limbs. Scale bars: 40 μm.

Figure 6. Corin and neprilysin expression in proximal tubules and transfected HEK293 cells

(A) Human renal cortex sections were co-stained for corin (red), neprilysin (green), and nuclei (DAPI). Scale bar: 20 μm. (B) Apical corin expression in proximal tubular epithelial cells of mouse kidneys was verified by EM. Red arrows indicate antibody-conjugated gold particles. Original power magnifications of the top panel and lower panels were x34003 and x91568, respectively. (C and D) Plasmid expressing human neprilysin or a control vector was transfected in HEK293 cells. Neprilysin protein in cell lysates from the transfected cells was examined by Western analysis (C). Neprilysin activity in the transfected cells was verified by a fluogenic assay (D). Additional controls in the neprilysin activity assay included parental HEK293 cell lysate (no transfection), buffer (negative control), and purified neprilysin protein. (E) Corin protein fragments in the conditioned medium (top panel) and cell lysates (lower panel) from the transfected HEK293 cells were analyzed by Western blotting.

Figure 7. ADAM10 expression in renal segments

(A) Human ADAM10 protein in renal cortex and medullary sections was immune-stained. AQP1 staining was used as a control. Black arrows indicate distal convoluted tubules near glomeruli (g) (top panels). Asterisks indicate collecting ducts (lower panels). Open arrowheads indicate descending thin limbs. Green arrowheads indicate ascending thin limbs. Scale bar: 40 μm. (B) Immunofluorescent staining of corin (red), ADAM10 (green) and nuclei (DAPI) in proximal and distal tubules. Yellow dashed circles indicate glomeruli. Scale bars: 40 μm.