Genomic Analysis of Kidney Allograft Injury Identifies Hematopoietic Cell Kinase as a Key Driver of Renal Fibrosis.

Renal fibrosis is the common pathway of progression for patients with CKD and chronic renal allograft injury (CAI), but the underlying mechanisms remain obscure. We performed a meta-analysis in human kidney biopsy specimens with CAI, incorporating data available publicly and from our Genomics of Chronic Renal Allograft Rejection study. We identified an Src family tyrosine kinase, hematopoietic cell kinase (Hck), as upregulated in allografts in CAI. Querying the Kinase Inhibitor Resource database revealed that dasatinib, a Food and Drug Administration-approved drug, potently binds Hck with high selectivity. In vitro, Hck overexpression activated the TGF-ß/Smad3 pathway, whereas HCK knockdown inhibited it. Treatment of tubular cells with dasatinib reduced the expression of Col1a1 Dasatinib also reduced proliferation and α-SMA expression in fibroblasts. In a murine model with unilateral ureteric obstruction, pretreatment with dasatinib significantly reduced the upregulation of profibrotic markers, phosphorylation of Smad3, and renal fibrosis observed in kidneys pretreated with vehicle alone. Dasatinib treatment also improved renal function, reduced albuminuria, and inhibited expression of profibrotic markers in animal models with lupus nephritis and folic acid nephropathy. These data suggest that Hck is a key mediator of renal fibrosis and dasatinib could be developed as an antifibrotic drug.

Biopsy transcriptome expression profiling to identify kidney transplants at risk of chronic injury: a multicentre, prospective study.

BACKGROUND: Chronic injury in kidney transplants remains a major cause of allograft loss. The aim of this study was to identify a gene set capable of predicting renal allografts at risk of progressive injury due to fibrosis. METHODS: This Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicentre study. We prospectively collected biopsies from renal allograft recipients (n=204) with stable renal function 3 months after transplantation. We used microarray analysis to investigate gene expression in 159 of these tissue samples. We aimed to identify genes that correlated with the Chronic Allograft Damage Index (CADI) score at 12 months, but not fibrosis at the time of the biopsy. We applied a penalised regression model in combination with permutation-based approach to derive an optimal gene set to predict allograft fibrosis. The GoCAR study is registered with ClinicalTrials.gov, number NCT00611702. FINDINGS: We identified a set of 13 genes that was independently predictive for the development of fibrosis at 1 year (ie, CADI-12 ≥2). The gene set had high predictive capacity (area under the curve [AUC] 0·967), which was superior to that of baseline clinical variables (AUC 0·706) and clinical and pathological variables (AUC 0·806). Furthermore routine pathological variables were unable to identify which histologically normal allografts would progress to fibrosis (AUC 0·754), whereas the predictive gene set accurately discriminated between transplants at high and low risk of progression (AUC 0·916). The 13 genes also accurately predicted early allograft loss (AUC 0·842 at 2 years and 0·844 at 3 years). We validated the predictive value of this gene set in an independent cohort from the GoCAR study (n=45, AUC 0·866) and two independent, publically available expression datasets (n=282, AUC 0·831 and n=24, AUC 0·972). INTERPRETATION: Our results suggest that this set of 13 genes could be used to identify kidney transplant recipients at risk of allograft loss before the development of irreversible damage, thus allowing therapy to be modified to prevent progression to fibrosis. FUNDING: National Institutes of Health.

Novel Therapeutics Identification for Fibrosis in Renal Allograft Using Integrative Informatics Approach.

Chronic allograft damage, defined by interstitial fibrosis and tubular atrophy (IF/TA), is a leading cause of allograft failure. Few effective therapeutic options are available to prevent the progression of IF/TA. We applied a meta-analysis approach on IF/TA molecular datasets in Gene Expression Omnibus to identify a robust 85-gene signature, which was used for computational drug repurposing analysis. Among the top ranked compounds predicted to be therapeutic for IF/TA were azathioprine, a drug to prevent acute rejection in renal transplantation, and kaempferol and esculetin, two drugs not previously described to have efficacy for IF/TA. We experimentally validated the anti-fibrosis effects of kaempferol and esculetin using renal tubular cells in vitro and in vivo in a mouse Unilateral Ureteric Obstruction (UUO) model. Kaempferol significantly attenuated TGF-ß1-mediated profibrotic pathways in vitro and in vivo, while esculetin significantly inhibited Wnt/ß-catenin pathway in vitro and in vivo. Histology confirmed significantly abrogated fibrosis by kaempferol and esculetin in vivo. We developed an integrative computational framework to identify kaempferol and esculetin as putatively novel therapies for IF/TA and provided experimental evidence for their therapeutic activities in vitro and in vivo using preclinical models. The findings suggest that both drugs might serve as therapeutic options for IF/TA.

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis.

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7-like 2-dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-ß1 administration upregulated SHROOM3 expression in a ß-catenin/TCF7L2-mediated manner, while SHROOM3 in turn facilitated canonical TGF-ß1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell-specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-ß1 signaling and contributes to allograft injury.