Background: Renal fibrosis is the consequence of excessive accumulation of extracellular matrix that occurs in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we identified a member of Src family of tyrosine kinases, HCK, up-regulated in allografts with CAN and studied its role in renal fibrosis.

Method: We used an integrative bio-informatics approach incorporating publically available data as well as data from the Genomics of allograft rejection study (GoCAR) to identify that the HCK network is up-regulated in CAN. In vitro over-expression/knock-down as well as HCK-inhibitor (Dasatinib) studies were performed to examine pro-fibrosis markers in tubular/fibroblast cell lines. To study the in vivo role of HCK in fibrosis, we performed unilateral-ureteric obstruction (UUO) on both PBS- or Dasatinib-treated mice.

Results: From bio-informatic metadata and GoCAR, HCK was identified as a key driver for allograft injury and CAN. In vitro, HCK over-expression activated SMAD-signaling in HEK-Blue TGF-b 293-T cells (which report SMAD-activity) while shRNA-mediated knock down inhibited TGF-b/Smad signaling. Next, HK2-cells treated with Dasatinib showed reduced COL1A1-expression, while rat fibroblasts (NRK-49F) showed reduced proliferation along with reduced α-SMA transcripts suggesting inhibited activation to myofibroblasts. In mice, Dasatinib-gavaged animals showed substantial down-regulation of inflammatory markers, like CCL3, CCL5 and TNFa at 3 days after UUO, compared to PBS-controls. At 7-days UUO, Col1A1, Fibronectin and Snail mRNA levels were inhibited and phosphorylation of SMAD3 was reduced in the UUO kidneys of dasatinib animals. By immunofluorescence, Col1A1 production was inhibited in UUO-kidneys upon Dasatinib-treatment. Renal interstitial fibrosis, measured by picrosirius red staining, was also significantly abrogated with dasatinib treatment (P<0.05).

Conclusion: HCK may be a potential mediator of kidney fibrosis in CAN via effects on inflammation, fibroblast cell proliferation, as well as direct effects on TGF-b signaling.

CITATION INFORMATION: Wei C, Menon M, Li L, Yi Z, Keung K, Woytovich C, Greene I, Zhang W, He J, Murphy B. HCK Is a Potential Mediator of Renal Fibrosis. Am J Transplant. 2016;16 (suppl 3).

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