MXRA5 is a TGF-β1-regulated human protein with anti-inflammatory and anti-fibrotic properties

Abstract

Current therapy for chronic kidney disease (CKD) is unsatisfactory because of an insufficient understanding of its pathogenesis. Matrix remodelling-associated protein 5 (MXRA5, adlican) is a human protein of unknown function with high kidney tissue expression, not present in rodents. Given the increased expression of MXRA5 in injured tissues, including the kidneys, we have suggested that MXRA5 may modulate kidney injury. MXRA5 immunoreactivity was observed in tubular cells in human renal biopsies and in urine from CKD patients. We then explored factors regulating MXRA5 expression and MXRA5 function in cultured human proximal tubular epithelial cells and explored MXRA5 expression in kidney cancer cells and kidney tissue. The fibrogenic cytokine transforming growth factor-β1 (TGFβ1) up-regulated MXRA5 mRNA and protein expression. TGFβ1-induced MXRA5 up-regulation was prevented by either interference with TGFβ1 activation of the TGFβ receptor 1 (TGFBR1, ALK5) or by the vitamin D receptor agonist paricalcitol. By contrast, the pro-inflammatory cytokine TWEAK did not modulate MXRA5 expression. MXRA5 siRNA-induced down-regulation of constitutive MXRA5 expression resulted in higher TWEAK-induced expression of chemokines. In addition, MXRA5 down-regulation resulted in a magnified expression of genes encoding extracellular matrix proteins in response to TGFβ1. Furthermore, in clear cell renal cancer, von Hippel-Lindau (VHL) regulated MXRA5 expression. In conclusion, MXRA5 is a TGFβ1- and VHL-regulated protein and, for the first time, we identify MXRA5 functions as an anti-inflammatory and anti-fibrotic molecule. This information may yield clues to design novel therapeutic strategies in diseases characterized by inflammation and fibrosis.

Keywords: adlican; chronic kidney disease; fibrosis; inflammation; kidney; perlecan; polycystic kidney disease.

Figure 1

The MXRA5 gene. (A) Gene tree representing MXRA5 orthologues. The display shows the maximum likelihood phylogenetic tree representing the evolutionary history of gene. Note the existence of a human gene (Ensembl: accessed on January 25th). (B) MXRA5 gene expression in diverse human organs according to publicly accessible databases. Note high kidney MXRA5 mRNA levels as compared to other organs (n = 10, *P < 0.001 versus Universal RNA; source: GEO 17426686, 4689086). (C) Correlation between MXRA5 and Fn14 (TNFRSF12A) mRNA expression in human focal segmental glomerulosclerosis according to publicly accessible databases. Source: Nephromine 21.

Figure 2

MXRA5 is present in CKD patients. (A) Urine supernatant. (B) Urine sediment. Western blot shows bands recognized by anti‐MXRA5 antibody in urine of CKD patients but not in healthy controls (N = 2 healthy controls and 3 CKD patients). (C) MXRA5 mRNA expression in ADPKD cysts or normal kidneys assessed by RT‐qPCR (N = 3 controls and 6 ADPKD patients, *P < 0.001 versus control). (D) MXRA5 immunohistochemistry in ADPKD cysts or normal kidneys (N = 6 controls and 6 ADPKD patients). Representative image. (E) MXRA5 immunohistochemistry in renal fibrosis and control kidney (N = 2 controls and 1 fibrotic, non‐ADPKD kidney).

Figure 3

TGFβ1 increases MXRA5 in cultured proximal tubular cells. (A) Human proximal tubular cells were exposed to 0.1, 1 and 10 ng/ml TGFβ1 for 3 and 6 hr and MXRA5 mRNA expression was assessed by RT‐qPCR (N = 3, *P < 0.001 versus control, #P < 0.01 versus control). (B) Cells were exposed to 1 ng/ml TGFβ1 for 3, 6 and 24 hr and MXRA5 protein expression was assessed by Western blot. Tubulin was used as loading control (N = 3, *P < 0.025 versus control, #P < 0.05 versus control). (C) Cells were pre‐treated with 10⁻⁵ M TGFβ1 receptor 1 inhibitor SB431542 for 1 hr and then exposed to 1 ng/ml TGFβ1 for 3 and 6 hr, MXRA5 mRNA expression was assessed by RT‐qPCR (N = 3, *P < 0.001 versus control, #P < 0.006 versus control). (D) Cells were pre‐treated with 10⁻⁵ M SB431542 for 1 hr and then exposed to 1 ng/ml TGFβ1 for 6 hr, MXRA5 protein levels were assessed by Western blot (N = 3, *P < 0.005 versus control). (E) Cells were pre‐treated with 1 ng/ml neutralizing anti‐TGFβ1 antibody ab100NA for 1 hr and then exposed to 1 ng/ml TGFβ1 for 3 and 6 hr, MXRA5 mRNA expression was assessed by RT‐qPCR (N = 3, *P < 0.001 versus control). (F) Cells were pre‐treated with 1 ng/ml neutralizing anti‐TGFβ1 antibody ab100NA for 1 hr and then exposed to 1 ng/ml TGFβ1 for 6 hr, MXRA5 protein levels were assessed by Western blot (N = 3, *P < 0.005 versus control, #P < 0.018 versus control).

Figure 4

Paricalcitol prevents TGFβ1‐induced MXRA5 up‐regulation. Cells were pre‐treated with 1 μg/ml paricalcitol for 90 min. and then exposed to 1 ng/ml TGFβ1 for 6 hr. (A) MXRA5 mRNA expression was assessed by RT‐qPCR (N = 3, *P < 0.001 versus control, #P < 0.018 versus control). (B) MXRA5 protein expression was assessed by Western blot (N = 3, *P < 0.02 versus control, #P < 0.05 versus control).

Figure 5

MXRA5 targeting has no effect on cell viability or proliferation. MXRA5 was successfully knocked down by means of a specific siRNA. (A) MXRA5 protein expression was assessed by Western blot (B) and RT‐qPCR (*P < 0.001 versus control). (C) No morphological changes were observed in MXRA5 knocked down cells. Contrast phase microscopy. (D) No changes in cell death or proliferation were observed in MXRA5‐silenced cells. DNA was stained with propidium iodide in permeabilized cells and DNA content quantified by flow cytometry.

Figure 6

Endogenous constitutive MXRA5 has an anti‐inflammatory role in cultured proximal tubular cells. MXRA5 was knocked down and then cells were treated with 100 ng/ml TWEAK for 3 hr. TWEAK did not modify MXRA5 expression (supplemental figure). (A) MCP1 mRNA expression assessed by RT‐qPCR (N = 3, *P < 0.002 versus control). (B) MCP1 secretion assessed by ELISA in cell supernatants (N = 3, *P < 0.025). (C) Rantes mRNA expression assessed by RT‐qPCR (N = 3, *P < 0.001 versus control). (D) Cxcl16 mRNA expression assessed by RT‐qPCR (N = 3, *P < 0.001 versus control).

Figure 7

MXRA5 has an anti‐fibrotic role in cultured proximal tubular cells. MXRA5 was knocked down and then cells were treated with 1 ng/ml TGFβ1 for 6 hr. (A) Fibronectin mRNA expression assessed by RT‐qPCR (N = 3, *P < 0.001 versus control, #P < 0.005 versus control). (B) Type IV collagen mRNA expression assessed by RT‐qPCR (N = 3, *P < 0.002 versus control).

Figure 8

MXRA5 in cancer. (A) Human proximal tubular cells and VHL ^−/− and VHL ^+/+ clear cell carcinoma cells were serum‐depleted and collected at 1, 3, 6 and 24 hr. MXRA5 mRNA expression was assessed by RT‐qPCR (N = 3). (B) MXRA5 and VHL mRNA expression assessed in biopsies from renal carcinoma tissue (N = 10) and healthy kidney tissue (n = 9) from the same kidney (*P < 0.05).