Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells

Abstract

Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells

“…A heat map of all 8 PTEC samples shows a clear distinction between fluid shear stress treated samples and static controls (Figure b). Furthermore, our genome wide RNA sequencing analysis confirmed genes known to be altered by fluid shear stress in renal epithelial cells, including Ptgs2 ( Cox2 ), Ccl2 ( Mcp1 ), Edn1 , Egr1, Snai1 , Cdh1 , and Tgfb1 (Flores, Battini, Gusella, & Rohatgi, ; Flores, Liu, Liu, Satlin, & Rohatgi, ; Grabias & Konstantopoulos, , ; Maggiorani et al, ; Pandit et al, ; Schwachtgen, Houston, Campbell, Sukhatme, & Braddock, ).…”

Keratinocytes are mechanoresponsive to the microflow‐induced shear stress

“…Till date, the research conducted in epithelial mechanobiology has emphasized the application of low fluid flow‐induced shear stress to trigger the mechanoresponse in the cells. A higher shear stresses of magnitude >4 dyne/cm 2 has been associated with the damage of cellular actin cytoskeleton, thereby, resulting in the loss of their viability (Maggiorani et al, ; Wang et al, ). Moreover, the mechanoresponsive behavior of the epithelial cells at a magnitude of shear as low as 0.02 dyne/cm 2 has also been reported previously (Kim et al, ; Kim et al, ).…”

“…Fluid flow‐induced shear stress has been recognized as mechanostimuli for various epithelial cell types including corneal epithelial cells (Kang et al, ; Molladavoodi, Robichaud, Wulff, & Gorbet, ), renal epithelial cell (Duan et al, ; Maggiorani et al, ; Verma, Ye, & Hua, ; Wang, Heo, & Hua, ), alveolar epithelial cell (Flitney, Kuczmarski, Adam, & Goldman, ), and intestinal epithelial cells (Chi et al, ; Kim et al, ; Kim, Huh, Hamilton, & Ingber, ). However, the impact of the flow‐induced shear stress on the skin epithelial cells (keratinocytes) has not been explored yet.…”

Reconstructing the Human Renal Vascular–Tubular Unit In Vitro

“…Finally the gravity-driven perfusion protocol we used provides a decaying flow profile with an average shear stress of ~ 0.1 dyne cm −2 , different from the more continuous ~ 1 dyne cm −2 experienced by the renal tubules in vivo. [30] While we noted appropriate cellular flow alignment, future work could explore the effect of different flow profiles on hRVTU function.…”