Cell adhesion molecule-1 shedding induces apoptosis of renal epithelial cells and exacerbates human nephropathies

Abstract

Chronic kidney disease (CKD) is an important problem throughout the world, associated with the increase of blood urea nitrogen (BUN) and serum creatinine (sCre) and with renal tubular injuries. It is crucial to elucidate the molecular mechanisms of renal injuries to identify the new therapeutics and early diagnostic methods. We focused on cell adhesion molecule-1 (CADM1) protein. CADM1, its isoform SP4, is expressed in the epithelial cells of various tissues, including renal distal tubules, localized on the lateral cell membrane, mediates cell-cell adhesion via trans-homophilic binding, and interacts with various proteins. We previously reported that its expression was downregulated by post-proteolytic cleavage (α- and β-shedding) in pulmonary diseases. To investigate whether CADM1 α-shedding occurs in human nephropathies, we performed Western blotting and immunohistochemical analysis of specimens with arterionephrosclerosis (AS) and diabetic nephropathy (DN) from autopsied kidneys. CADM1 α-shedding was induced in AS and DN kidneys and derived from the decrease in full-length CADM1 (FL-CADM1) and increase of the COOH-terminal fragment (α-CTF). In particular, the reduced FL-CADM1 level was correlated with tubular and tubulointerstitial injuries and the increases in BUN and sCre levels. Apoptosis of renal tubular epithelial cells (TECs) was promoted in both nephropathies, and it was significantly correlated with the decrease in the FL-CADM1. Furthermore, FL-CADM1 knockdown by small interfering RNA downregulated anti-apoptotic Bcl-2 protein and promoted apoptosis of cultured renal TECs. The present study suggests that the reduction of FL-CADM1 leads to renal TEC apoptosis and could exacerbate renal tubular and tubulointerstitial injuries, which contribute to the development of CKD.

Keywords: CADM1; DM nephropathy; apoptosis; arterionephrosclerotic nephropathy; shedding.

Fig. 1.

Representative images of renal tissues stained with HE stain. A: noninjured (NI). B: diabetes mellitus nephropathy (DN); C and D: nephropathy with arterionephrosclerosis (AS). White arrows, interstitial fibrosis; yellow arrows, inflammation; black arrowheads, tubular degeneration. Scale bar = 100 μm. E: pathological grade in NI and each nephropathy (DN and AS). **P < 0.01.

Fig. 2.

Apoptotic cells in nephropathies. A: representative images of immunohistochemical staining of ssDNA against NI and DN kidney. The sections were counterstained with hematoxylin. The arrows indicate ssDNA-positive TECs. Scale bar = 100 μm. B: the numbers of apoptotic cells among human renal tubular cells per section (×400). Values are means ± SD. NI, n = 13; DN, n = 9; AS, n = 8. *P < 0.05.

Fig. 3.

Western blot analysis of CADM1 in nephropathies. A: Western blot analysis using anti-COOH-terminal-CADM1 antibody. Arrowheads indicate bands corresponding to the FL, β-CTF, and α-CTF forms of CADM1. B: CADM1 ectodomain shedding rates (amount of α-CTFs relative to FL-CADM1). *P < 0.05. **P < 0.01. C and D: FL- and α-CTF-CADM1 levels, respectively, relative to β-actin are plotted as dots in each patient sample. Significance between two groups was analyzed using the t-test. Values are means ± SD. NI, n = 13; DN, n = 9; AS, n = 8. P values ≤ 0.05 are shown. E: total RNAs from renal sections of human NI, AS, and DN were analyzed by RT-PCR using a primer set encompassing the CADM1 extracellular juxtamembrane region. The PCR products were electrophoresed together with CADM1 isoform (SP1–4) size markers. L, 100-base pair (bp) ladder. RNAs were also PCR-amplified using a primer set for β-actin to indicate RNA loading per lane. *FFPE samples that did not have the sufficient RNA quantity.

Fig. 4.

Representative localizations of CADM1 in tubular epithelial cells. NI (left) and DN kidneys (right). Immunohistochemical stain was performed with a COOH-terminal CADM1 antibody (top row: ×200). The arrows and arrowheads indicate the CADM1 stain at the lateral and/or basal membrane and at the intracytoplasm, respectively. Scale bar = 100 μm.

Fig. 5.

Correlation between CADM1 ectodomain shedding and tubular injuries or TEC apoptosis. A–C: correlation of CADM1 shedding rate (A), FL-CADM1 (B), or α-CTF (C) with tubular injuries. D–F: correlation of CADM1 shedding rate (D), FL-CADM1 (E), or α-CTF (F) with tubular epithelial cell apoptosis. n = 28. P values ≤ 0.05 are shown.

Fig. 6.

A decreased FL-CADM1 level, not increased α-CTF, induces the apoptosis of CNT cells. A: CNT cells were left untreated or transfected with mock or α-CTF or α-CTFmut. After 48 h, CADM1 expression was examined by Western blot analyses. B: the numbers of apoptotic cells in CNT cells/section. C: CNT cells were left untreated or transfected with control or CADM1-targeting siRNA. After 48 h, CADM1 expression was examined by Western blot analyses, compared with β-actin levels. The CADM1 levels were further normalized by the value in untreated cells. FL-CADM1 levels were calculated from triplicate experiments for each cell type. D: after 48 h, CNT cells untreated and transfected with either control or the siRNA vector were analyzed by TUNEL assays. TUNEL (green) and DAPI (blue) fluorescent images were merged. White arrowheads indicate TUNEL-positive cells. E: the numbers of apoptotic cells in CNT cells/section (n = 3). The rates of TUNEL-positive cells are presented as means ± SD. **P values ≤ 0.05. F: after 48 h, CNT cells untreated and transfected with either control or the siRNA vector were analyzed by ssDNA immunofluorescence assays. ssDNA (green) and DAPI (blue) fluorescent images were merged. White arrowheads indicate ssDNA-positive cells. G: the numbers of apoptotic cells in CNT cells/section (n = 3). The rates of ssDNA-positive cells are presented as means ± SD. **P values ≤ 0.05.

Fig. 7.

Decreased FL-CADM1 level effects on the decrease of Bcl-2, but not caspase-3 and Bax. A: after 48 h, CNT cells untreated and transfected with either control or the siRNA vector were analyzed by Western blot assays. Capase-3 (B), Bax (C), and Bcl-2 (D) levels relative to β-actin are shown. The rates of each are presented as means ± SD. Significance between two groups was analyzed using the t-test. *P values ≤ 0.05.