The Role of Renal Epithelial Cell Crosstalk in The Regulation of Cyclosporine A‐Induced MMP‐9 Production in Calcineurin Isoform‐Specific Renal Fibroblasts
Authors: Francis Cynthia E. , Nguyen Christina , Mata Laurice , Bai Yun 
Published: June 2021

Background

It's been shown that activated renal fibroblasts are considered to be major contributors to renal fibrosis through the production of excessive extracellular matrix (ECM). More specifically, several studies have shown that matrix metalloproteinases (MMPs) produced by fibroblasts are implicated in the initiation and progression of calcineurin inhibitor (CNI)‐ induced nephrotoxicity. Renal tubular epithelial cells have been shown to also produce ECM when stimulated with cyclosporine A (CsA). Some in vitro studies have demonstrated that epithelial/fibroblast crosstalk through secreted factors may play a role in MMP‐9 production in renal fibroblasts. Previously, we demonstrated the usefulness of Human Embryonic Kidney (HEK) 293 cell line as an in vitro model in the study of CsA‐induced renal fibrosis. Additionally, we demonstrated that upregulated CsA‐induced MMP‐9 secretion is calcineurin isoform specific in vitro. The loss of the calcineurin A alpha (CnAα) isoform (or presence of the calcineurin A beta (CnAβ) isoform) is associated with enhanced CsA‐induced MMP‐9 secretion (4 to 5‐fold) vs. in wild‐type renal fibroblasts. Considering that renal fibrosis involves several cell types within the microenvironment of the kidney, it stands to reason that epithelial cells may also contribute to this complex process.

Objective

This study evaluates the role MMPs play in crosstalk between renal epithelial cells and fibroblasts during calcineurin inhibitor‐induced kidney fibrosis and whether their roles are calcineurin A isoform‐specific.

Methods and Results

HEK293 cells, Wild‐type (WT), CnAα knockout (CnAα−/−) and CnAβ knockout (CnAβ−/−) kidney fibroblast cell lines (an in vitro innovative tool that was previously created in our lab) were treated with CsA at 10 ng/ml for 24 and 48 hrs. To characterize the CsA‐induced MMP‐9 and MMP‐2 profile of HEK293 cells, their supernatants were studied using the enzyme‐linked immunosorbent assay (ELISA) technique. After CsA treatment, significantly higher levels of total MMP‐2 (4 to 6‐fold) were detected compared to levels of total MMP‐9. To study the effect of secreted factors from CsA‐treated HEK293 cells on total MMP‐9 production by renal fibroblasts, 48 hr HEK293 supernatants were added to WT, CnAα−/− and CnAβ−/− in culture. Crosstalk between secreted epithelial factors and fibroblasts was assessed using total MMP‐9 ELISAs. There was no significant difference in the extracellular levels of MMP‐9 secreted by WT renal fibroblasts. Surprisingly, HEK293 supernatants significantly reduced MMP‐9 levels (2 to 2.5‐fold) in CnAα−/− and increased it (~8‐fold) in CnAβ−/− fibroblasts vs CsA‐treated fibroblasts. To evaluate intracellular MMPs, cells were lysed in TNESV buffer with protease inhibitor cocktail and protein levels evaluated using Western blots. Western blot analysis demonstrated slightly elevated levels of total MMP‐9 in WT and CnAα−/− fibroblasts. However, MMP‐9 levels were significantly elevated (~8‐fold) for CnAβ−/− cell lysates.

Conclusions

Our results demonstrate that MMPs play important roles in epithelial/fibroblast cell crosstalk in CNI‐induced kidney fibrosis. Factors secreted by HEK293 play a regulatory role in renal fibroblast MMP‐9 secretion and that role is CnA isoform‐specific. In the future, we will further study the molecular mechanism of CnA isoform‐specific crosstalk through MMP secretion.

Support or Funding Information

NIH R15GM113120 and PCOM CRSO

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.