Abstract
Purpose :
Alport syndrome (AS) is a genetic condition caused by a dysfunctional collagen (IV) α3α4α5 heterotrimer, leading to basement membrane instability and abnormalities in the kidney, inner ear, and eyes. The ocular pathogenesis of AS is currently not well understood. We investigated the inflammatory and fibrotic changes in the Col4a3 knockout (KO) mouse model to better characterize the ocular pathology of AS.
Methods :
Col4a3tm1Dec mice were euthanized at 25 weeks, and eyes were processed for paraffin sectioning. Immunohistochemical staining (IHC) was performed to evaluate the localization of collagen (IV) α3 and to assess inflammation using the markers transforming growth factor-β2 (TGF-β2), connective tissue growth factor (CTGF), and β-catenin. Photomicrographs visualizing protein distribution were captured under a confocal microscope. mRNA levels of the pro-fibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, Twist1, and Wfdc2 were assessed using real-time RT-qPCR; Actb and Tbp were used as housekeeping genes.
Results :
There was positive staining of collagen (IV) α3 in Descemet’s membrane of the corneal subendothelium, but was absent in the retina. Immunofluorescence studies revealed an upregulation of TGF-β2, CTGF, and β-catenin in the cornea but no observable difference in the retina. In the retinas of Col4a3 KO mice, there was an increase in the transcription of S100a4, the gene encoding fibroblast-specific protein-1 (FSP1) (p=.0212, 6.7-fold increase normalized to β-actin; p=.0059, 12.3-fold increase normalized to Tbp) and Acta2, the gene encoding α-smooth muscle actin (αSMA) (p=.0075, 3.6 fold increase normalized to β-actin; p=.0012, 6.6-fold increase normalized to Tbp). However, there was no significant difference in the levels of any of the evaluated pro-fibrotic genes in the cornea (nWT=4, nKO=3).
Conclusions :
The increase of inflammatory markers in the corneas of Col4a3 KO mice may be due to the degradation of corneal endothelial integrity without apparent changes in profibrotic targets. Interestingly, the retina reflects a profibrotic phenotype despite the absence of α3 (IV) on immunostaining. In conclusion, the Col4a3 KO mice exhibit a differential inflammatory pro-fibrotic response in the cornea and retina, which may play a role in the ocular pathology of AS.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.