Epithelial-Fibroblast Crosstalk Protects against Acidosis-Induced Inflammatory and Fibrotic Alterations

Abstract

Pathogenesis of chronic kidney disease (CKD) is accompanied by extracellular acidosis inflammation, fibrosis and epithelial-to-mesenchymal transition (EMT). The aim of this study was to assess the influence of acidosis on tubule epithelial cells (NRK-52E) and fibroblasts (NRK-49F) in dependence of cellular crosstalk. NRK-52E and NRK-49F were used in mono- and co-cultures, and were treated with acidic media (pH 6.0) for 48 h. The intracellular proteins were measured by Western blot. Secreted proteins were measured by ELISA. Distribution of E-cadherin was assessed by immunofluorescence and epithelial barrier function by FITC-dextran diffusion. Inflammation: Acidosis led to an increase in COX-2 in NRK-52E and TNF in NRK-49F in monoculture. In co-culture, this effect was reversed. EMT: Acidosis led to an increase in vimentin protein in both cell lines, whereas in co-culture, the effect was abolished. In NRK-52E, the E-cadherin expression was unchanged, but subcellular E-cadherin showed a disturbed distribution, and cellular barrier function was decreased. Fibrosis: Monoculture acidosis led to an increased secretion of collagen I and fibronectin in NRK-52E and collagen I in NRK-49F. In co-culture, the total collagen I secretion was unchanged, and fibronectin secretion was decreased. Intercellular crosstalk between epithelial cells and fibroblasts has a protective function regarding the development of acidosis-induced damage.

Keywords: EMT; cellular crosstalk; chronic kidney diseases; extracellular acidosis; fibrosis; inflammation.

Figure 1

Acidosis effect on EMT markers in NRK-52E cells in mono- and co-cultures. Impact of acidosis on vimentin (a,d), E-cadherin (g,j), N-cadherin (h,k) and α-SMA (i,l) protein expression changes, vimentin degradation (b,e) and relative changes of vimentin mRNA expression (c,f). Representative Western blots of proteins isolated from cells exposed to acidosis (m). n − (8 − 9); exposure time = 48 h. p < 0.05 at significant difference compared with the control group.

Figure 2

Impact of acidosis on E-cadherin distribution in NRK-52E in monoculture. (a–d) n − (24). Acidosis effect on barrier function of NRK-52E cells in mono-and co-culture (e–h). n − (9); exposure time = 48 h. p < 0.05 at significant difference compared with the control group.

Figure 3

Acidosis effect on inflammation markers in NRK-52E cells in mono-and co-culture. Impact of acidosis on TGF-β (a,d), TNF (b,e), and COX-2 (c,f); representative Western blots of proteins isolated from cells exposed to acidosis (g). p < 0.05 at significant difference compared with the control group; n − (7 − 18). Exposure time = 48 h.

Figure 4

Acidosis effect on fibrosis markers in NRK-52E cells in mono- and co-cultures. Impact of acidosis on the expression of secreted collagen I (a,d) and fibronectin (g,j). Intracellular protein expression changes of collagen I (b,e) and fibronectin (h,k) as well as relative changes of collagen I (c,f) and fibronectin (i,l) mRNA expression. (m) Representative Western blots of proteins isolated from cells exposed to acidosis. p < 0.05 at significant difference compared with the control group; n − (6 − 16). Exposure time = 48 h.

Figure 5

Acidosis effect on EMT markers in NRK-49F cells in mono- and co-cultures. Impact of acidosis on vimentin (a,d), E-cadherin (g,j), N-cadherin (h,k) and α-SMA (i,l) protein expression changes, vimentin degradation (b,e) and relative changes of vimentin mRNA expression (c,f). Representative Western blots of proteins isolated from cells exposed to acidosis. (m) p < 0.05 at significant difference compared with the control group; n − (5 − 11). Exposure time = 48 h.

Figure 6

Acidosis effect on inflammation markers in NRK-49F cells in mono- and co-cultures. Impact of acidosis on TGF-β (a,d), TNF (b,e), COX-2 (c,f). Representative Western blots of proteins isolated from cells exposed to acidic media (g). p < 0.05 at significant difference compared with the control group; n − (8 − 19). exposure time = 48 h.

Figure 7

Acidosis effect on fibrosis markers in NRK-49F cells in mono- and co-cultures. Impact of acidosis on the expression of extracellular collagen I (a,d) and fibronectin (g,j). Intracellular protein expression changes of collagen I (b,e) and fibronectin (h,k) as well as relative changes of collagen I (c,f) and fibronectin (i,l) mRNA expression. Representative Western blots of proteins isolated from cells exposed to acidosis (m). p < 0.05 at significant difference compared with the control group; n − (8 − 24). Exposure time = 48 h.