Expression of the collagen iv 345molecule along the renal tubule in health and disease

Abstract Background and Aims Patients suffering from Alport Syndrome (AS) have a high life time risk of kidney failure and hearing loss. The underlying molecular cause is a germline mutation in one of the COL4A3/4/5 genes, whose gene products collectively form an important component of the glomerular basement membrane and the inner ear. Recent genetic studies have shown that the AS is much more prevalent than clinically recognized, suggesting that atypical clinical cases are frequent. Thus, patients with AS may phenocopy other kidney diseases. To date, pathomechanistic studies of the AS have focused exclusively on the glomerular membrane, yet equally strong expression of collagen IV can be found along the distal renal tubule. Structural disintegration of the distal tubular membrane may induce inflammation and fibrosis, thus progression of kidney disease. We hypothesize that genetic loss of the tubular collagen IV molecule contributes to kidney failure and may drive atypical phenotypes. Method Immunohistochemistry (IHC) and in situ hybridization (ISH, RNAscope) for COL IV α5, where IHC was performed on healthy human tissues as well as biopsies from patients with AS. Tubular marker staining for localization of COL IV α5 expression as well as spatial changes on consecutive sections. Human urinary primary tubular cells (huPTC) from healthy probands and patients with AS were generated and cultivated in cell culture, with analysis of COL IV mRNA by qPCR and detection of specifically expressed proteins by immunoblotting. Results COL IV α5 was detected within the tubular basement membrane (TBM) of the distal segments of renal tubules by IHC, including the thick ascending limb (TAL) of Henle (Tamm-Horsfall Protein), the distal convoluted tubule (11ß-Hydroxysteroid-Dehydrogenase) and the collecting duct (aquaporin 2). Spacial vicinity of these areas showed a predominance of interstitial fibrosis and inflammation in patient samples with AS, but not control samples with primary proteinuric diseases. Collagen IV protein is lost along the TBM of patients with AS, even in the hemizygous male and heterozygous female carriers of the hypomorphic but frequent COL4A5 mutation G624D. By ISH, COL4A5 expression was mainly observed in the glomerulus (most likely podocytes) and broadly in tubular epithelial cells, with only few tubulointerstitial cells showing mRNA expression. Likewise, the huPTC showed COL IV expression in qPCR, as well as COL IV α5 protein in immunoblotting. Conclusion Our studies show that the COL IV molecule is situated within the TBM of the complete distal tubule, starting from the TAL throughout the distant collecting duct. Although the epithelial cells have no detectable protein, in situ hybridization and primary cell cultures clearly suggest that tubular cells are a major source of the COL IV α345 molecule within the TBM. Renal biopsies of AS patients with diagnostic genetic COL IV variants indicate that tubulointerstitial fibrosis and inflammation starts in the vicinity of the distal tubule. Therefore, we postulate that the pathogenesis of AS may in part stem from the (distal) tubular apparatus, which is already known from other diseases such as ADTKD and nephronophthisis.