Date Published: June 20, 2019
Publisher: Public Library of Science
Author(s): Tiago Giulianni Lopes, Maysa Lucena de Souza, Vinicius Duval da Silva, Mariane dos Santos, William Israel Cardoso da Silva, Thiago Pereira Itaquy, Henrique Iahnke Garbin, Francisco Veríssimo Veronese, Xu-jie Zhou.
Renal fibrosis is the result of the interaction of cellular and molecular pathways, which is induced by sustained glomerular injury and involves the podocytes and multiple profibrotic factors. In this study, we investigated the correlation of the mRNA expression of podocyte proteins and profibrotic factors with renal fibrosis measured in renal biopsies of patients with primary and secondary glomerulopathies.
Eighty-four adult patients with primary or secondary glomerular diseases and 12 controls were included. Demographic and clinical data were collected. Seventy-two percent of the renal biopsies were done less than one year from clinical disease manifestation. The quantification of the podocyte-associated mRNAs of alpha-actinin-4, podocin, and podocalyxin, as well as of the profibrotic factors TGF-β1, CTGF, and VEGF-A were quantified by real-time polymerase chain reaction. The percent positive area of renal fibrosis was measured by immunohistochemistry staining, using anti-CTGF and anti-HHF35 antibodies and unpolarized Sirius Red. Correlations between the expression of tissue mRNAs and the positive area of fibrosis for the measured markers were made by Spearman’s rank correlation coefficient.
In relation to control biopsies, podocyte-specific proteins were downregulated in podocytopathies, in proliferative nephritis, in diabetic kidney disease (DRD), and in IgA nephropathy (IgAN). Messenger RNA of TGF-β1, CTGF, and VEGF-A was upregulated in patients with podocytopathies and in DRD but not in proliferative nephritis and IgAN. Tissue mRNA expression of TGF-β1, CTGF, and VEGF-A were strongly correlated with renal fibrosis, as measured by HHF35; however, the correlation, albeit significant, was moderate for Sirius Red and weak for CTGF. The percent positive area of renal fibrosis measured by Sirius Red was similar between podocytopathies and DRD and significantly higher in podocytopathies compared to IgAN or proliferative nephritis.
In patients with glomerular diseases, the mRNA of TGF-β1, CTGF, and VEGF-A correlated positively with the extent of renal fibrosis, and the positive area of fibrosis was larger in the podocytopathies and in DRD as measured by Sirius Red. The pathways connecting podocyte damage and activation of profibrotic factors to kidney tissue fibrosis need to be better investigated.
Chronic glomerular diseases result in the accumulation of extracellular matrix in the interstitium, referred to as renal tissue fibrosis, that correlates with the loss of kidney function and progressive renal failure [1,2]. Renal fibrosis and global glomerulosclerosis are the histological expressions of chronic damage related to various etiopathogenic mechanisms. It is not entirely clear how these mechanisms develop to induce nephron damage in proteinuric glomerulopathies, but circulating auto-antibodies, pro-inflammatory cytokines and immuno-complex deposition in the initial phase , as well as later cellular events and molecular mediators, such as fibrogenic growth factors, pericyte-to-myofibroblast transdifferentiation and apoptosis, are certainly involved [4,5]. Metabolic disorders, oxidative stress, hemodynamic intra-glomerular factors resulting from stimulation of vasoactive molecules, and renal tissue hypoxia are other pathways that also contribute to glomerular and tubulointerstitial fibrosis and the progression of kidney impairment .
In this study, we analysed the correlation between the mRNA expression of podocyte proteins, profibrotic and angiogenic factors and the percentage of renal tissue fibrosis, as quantified by three different stains in kidney biopsies of patients with glomerular diseases. In relation to the expression of TGF-β1, CTGF and VEGF-A, and the extent of renal fibrosis, we found a positive and significant correlation that was strong for HHF35, moderate for Sirius Red, and weak for CTGF. In addition to that, an inverse correlation, which was significant for CTGF, was observed between alpha-actinin-4, podocin, and podocalyxin mRNA expression and the percentage of renal fibrosis. Renal fibrosis, as measured by Sirius Red, was more extensive in podocytopathies and DRD, probably reflecting the progressive podocyte injury and podocytopenia of glomerular diseases in whom podocytes are most affected, which triggers several mechanisms of tissue fibrosis in the glomeruli, tubulointerstitium, and the vasculature.
In these patients with glomerular diseases of different etiologies, the expression of profibrotic and angiogenic factors correlated positively with the percentage of renal fibrosis as quantified by HHF35, Sirius Red, and CTGF staining. Despite significant advances in the understanding of the mechanisms and molecular pathways of renal fibrosis, the translation to clinical trials is still scarce. As recently reviewed [48,49], the use of effective antifibrotic treatments to slow progression or even reverse the chronic kidney injury in glomerular diseases is a great challenge to be explored in future clinical trials.