Kidneys of Alb/TGF-beta1 transgenic mice are deficient in retinoic acid and exogenous retinoic acid shows dose-dependent toxicity

Abstract

Background: Alb/TGF-beta(1) transgenic mice overexpress active transforming growth factor-beta(1) (TGF-beta(1)) in the liver, leading to increased circulating levels of the cytokine and progressive renal fibrosis. This study was designed to explore if exogenous all-trans retinoic acid (tRA) prevents renal fibrosis in this animal model.

Methods: The retinoid profile in kidney and liver of wild-type and Alb/TGF-beta(1) transgenic mice was examined by high-performance liquid chromatography and slow-release pellets containing different amounts of tRA were implanted subcutaneously to treat the Alb/TGF-beta(1) transgenic mice, starting at 1 week of age; mice were sacrificed 2 weeks later.

Results: Kidneys of 3-week-old wild-type mice had abundant tRA, which was completely absent in kidneys of the transgenic mice. Low doses of tRA (6-10.7 mg/kg/day) failed to affect renal fibrosis although it tended to suppress the mRNA expression of some molecular markers of fibrosis and retinal dehydrogenase 2 (RALDH2), a gene encoding a key tRA-synthesising enzyme. These tendencies disappeared, mortality tended to increase and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased in the medium-dose group (12.7-18.8 mg/kg/day). High doses (20.1-27.4 mg/kg/day) showed even higher toxicity with increased renal fibrosis and significant mortality.

Conclusions: Alb/TGF-beta(1) transgenic mice are characterised by depletion of endogenous renal tRA. Exogenous tRA dose-dependently increases mortality and kidney fibrosis, which is associated with dose-dependent regulation of renal RALDH2 and CTGF mRNA expression.

Fig. 1

HPLC analysis of retinoids in kidney and liver of 3-week-old WT and Alb/TGF-β₁ TG mice. Extracts of kidney (a) and liver (b) tissues were subjected to HPLC assay to measure retinoid concentrations. a n = 4 for WT and n = 5 for TG. b n = 8 for WT and n = 7 for TG.

Fig. 2

Dose-dependent effects of tRA on survival, renal pathology, Scr and albuminuria in Alb/TGF-β₁ mice. Survival curve of the different groups (a), end-of-study interstitial fibrosis (IF) score (b), end-of-study glomerulosclerosis (GS) score (c), end-of-study Scr (d) and urine albumin:creatinine ratio (e). Only 8 out of 9 samples were analysed in the G2 group and only 2 in 6 samples were analysed in the G3 group due to animal death and loss of samples in these groups.

Fig. 3

End-of-study renal mRNA expression of CTGF, RALDH2 and ECM genes. The mRNA expression of CTGF (a), RALDH2 (b), FN (c), COL1 A1 (d), COL1 A2 (e) and COL4 A1 (f) was examined by Taqman RT-qPCR and GAPDH was used as a ‘house-keeping’ gene to normalise gene expression. The average mRNA expression level of 3 WT mice was set as 1; G0 is the vehicle-treated group; G1, G2 and G3 are low, medium and high doses of tRA-treated groups, respectively. Only 8 out of 9 samples were analysed in the G2 group and only 2 in 6 samples were analysed in the G3 group due to animal death and loss of samples in these groups.