Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor β1

Abstract

BackgroundChronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1.ResultsCortical fibroblast cultures were successfully established from the kidney tissue of cats with normal kidney function (FCF) and cats with chronic kidney disease (CKD-FCF). Both cell types expressed the mesenchymal markers vimentin, CD44 and CD29, and were negative for the epithelial marker cytokeratin, mesangial cell marker desmin and endothelial cell marker vWF. Only CKD-FCF expressed VCAM-1, a cell marker associated with inflammation. Incubation with TGF-β1 (0–10 ng/ml) induced a concentration dependent change in cell morphology, and upregulation of myofibroblast marker gene α-SMA expression alongside collagen 1α1, fibronectin, TGF-β1 and CTGF mRNA. These changes were blocked by the TGF-β1 receptor 1 antagonist SB431542 (5 μM).ConclusionsFCF and CKD-FCF can be cultured via a simple method and represent a model for the investigation of the progression of fibrosis in feline CKD. The findings of this study suggest TGF-β1 may be involved in fibroblast-myofibroblast transition in feline CKD, as in other species.

Profibrotic effects of angiotensin II and transforming growth factor beta on feline kidney epithelial cells

“…Recently published data from feline renal cortical fibroblast cultures confirm our results, as incubation of these cells with TGF-β1 also increased the expression of COL1 , α-SMA and CTGF . 26 Although the CRFK cells could have changed over time from more of an epithelial to a somewhat more fibroblastic phenotype, the results of the present study clearly demonstrated the formation of myofibroblasts by TGF-β1 via increased expression of COL1 , α-SMA and CTGF .…”

“…These results further support the profibrotic effect of TGF-β1 in the kidney demonstrated by earlier (clinical) studies in cats. 26–28 The CRFK cells showed almost no expression of the AT 1 receptor, precluding induction of these EMT marker genes by AT-II incubation. As AT-II contributes to renal fibrosis by various mechanisms, of which one is by TGF-β1 gene induction, it can be hypothesised that AT-II would show similar results to TGF-β1 if the AT 1 receptor was expressed more in CRFK cells.…”

Relationship between transforming growth factor-β1 and type 2 diabetic nephropathy risk in Chinese population

“…Transforming growth factor beta1 (TGF-β1) is one of the pro-fibrotic cytokines and is thought to be the primary mediator driving the progression of fibrosis, glomerulosclerosis and especially mesangial cell phenotype transformation in diabetic nephropathy (DN) [ 1 , 2 ]. TGF-β1 directly stimulates the transcription of extracellular matrix (ECM).…”

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype

“…Cryopreserved CRFK cells were purchased at passage 185 (CCL-94, ATCC) and cultured according to the manufacturer's protocol 1. FPTEC, FCF and human umbilical vein endothelial cells (HUVEC) were cultured for comparison as previously described (Lawson et al, 2018a; Lawson et al, 2018b). Cells were assessed for the expression of the marker proteins cytokeratin AE1/AE3 (Dako), vimentin (Dako), desmin (Dako), von Willebrand factor (vWF)(Dako), CD44 (ABD Serotec) and CD29 (Bio-rad) by immunofluorescence, and cytokeratin AE1/AE3, vimentin and vWF by western blotting as previously described (Lawson et al, 2018a).…”

“…RNA was extracted from CRFK, FPTEC and FCF using a column based kit (Genelute™ Mammalian Total RNA Miniprep Kit, Sigma-Aldrich). Expression of S100A4 was quantified by RT-qPCR and normalised to GAPDH/RPS7 using previously published primers (Lawson et al, 2018b). Data are expressed as mean fold change relative to untreated control and statistical significance was evaluated by one-way analysis of variance (ANOVA) with post-hoc Dunnet's test.…”