Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.

Collagen IV nephropathies (COL4Ns) comprise benign familial microscopic hematuria, thin basement membrane nephropathy (TBMN), X-linked Alport syndrome (AS) and also autosomal recessive and dominant AS. Apart from the X-linked form of AS, which is caused by hemizygous mutations in the COL4A5 gene, the other entities are caused by mutations in the COL4A3 or COL4A4 genes. The diagnosis of these conditions used to be based on clinical and/or histological findings of renal biopsies, but it is the new molecular genetics approach that revolutionised their investigation and proved particularly instrumental, especially, in many not so clear-cut cases. More recently, the spectrum of COL4N has expanded to include late onset focal segmental glomerulosclerosis (FSGS) that develops on top of TBMN in later life. Also, other reports showed that some patients with a primary diagnosis of familial FSGS proved to have variants in COL4 genes. In the presence of a renal biopsy picture of FSGS and in the absence of either electron microscopy studies or molecular genetic studies that point to TBMN and COL4N, the patient and his family may be mistakenly diagnosed with hereditary FSGS leading to unnecessary further investigations, erroneous family counselling and improper corticosteroid treatment. TBMN is a frequent finding in the general population, and according to several recent reports, it may be the underlying cause and the explanation for many familial and sporadic cases of late-onset FSGS with non-nephrotic proteinuria. This is an important new finding that needs widespread recognition. It is anticipated that the molecular genetic analysis with next generation sequencing will certainly offer timely correct diagnosis.

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.

Alport syndrome (AS) is a genetic disease characterized by haematuric glomerulopathy variably associated with hearing loss and anterior lenticonus. It is caused by mutations in the COL4A3, COL4A4 or COL4A5 genes encoding the α3α4α5(IV) collagen heterotrimer. AS is rare, but it accounts for >1% of patients receiving renal replacement therapy. Angiotensin-converting enzyme inhibition slows, but does not stop, the progression to renal failure; therefore, there is an urgent requirement to expand and intensify research towards discovering new therapeutic targets and new therapies. The 2015 International Workshop on Alport Syndrome targeted unmet needs in basic science, genetics and diagnosis, clinical research and current clinical care. In three intensive days, more than 100 international experts including physicians, geneticists, researchers from academia and industry, and patient representatives from all over the world participated in panel discussions and breakout groups. This report summarizes the most important priority areas including (i) understanding the crucial role of podocyte protection and regeneration, (ii) targeting mutations by new molecular techniques for new animal models and potential gene therapy, (iii) creating optimal interaction between nephrologists and geneticists for early diagnosis, (iv) establishing standards for mutation screening and databases, (v) improving widespread accessibility to current standards of clinical care, (vi) improving collaboration with the pharmaceutical/biotech industry to investigate new therapies, (vii) research in hearing loss as a huge unmet need in Alport patients and (viii) the need to evaluate the risk and benefit of novel (including ‘repurposing’) therapies on an international basis.

BackgroundAbout 40–50% of patients with familial microscopic hematuria (FMH) caused by thin basement membrane nephropathy (TBMN) inherit heterozygous mutations in collagen IV genes (COL4A3, COL4A4). On long follow-up, the full phenotypic spectrum of these patients varies a lot, ranging from isolated MH or MH plus low-grade proteinuria to chronic renal failure of variable degree, including end-stage renal disease (ESRD).MethodsHere, we performed Whole Exome Sequencing (WES) in patients of six families, presenting with autosomal dominant FMH, with or without progression to proteinuria and loss of renal function, all previously found negative for severe collagen IV mutations. Hierarchical filtering of the WES data was performed, followed by mutation prediction analysis, Sanger sequencing and genetic segregation analysis.ResultsIn one family with four patients, we found evidence for the contribution of two co-inherited variants in two crucial genes expressed in the glomerular basement membrane (GBM); LAMA5-p.Pro1243Leu and COL4A5-p.Asp654Tyr. Mutations in COL4A5 cause classical X-linked Alport Syndrome, while rare mutations in the LAMA5 have been reported in patients with focal segmental glomerulosclerosis. The phenotypic spectrum of the patients includes hematuria, proteinuria, focal segmental glomerulosclerosis, loss of kidney function and renal cortical cysts.ConclusionsA modifier role of LAMA5 on the background of a hypomorphic Alport syndrome causing mutation is a possible explanation of our findings. Digenic inheritance is another scenario, following the concept that mutations at both loci more accurately explain the spectrum of symptoms, but further investigation is needed under this concept. This is the third report linking a LAMA5 variant with human renal disease and expanding the spectrum of genes involved in glomerular pathologies accompanied by familial hematurias. The cystic phenotype overlaps with that of a mouse model, which carried a Lama5 hypomorphic mutation that caused severely reduced Lama5 protein levels and produced kidney cysts.Electronic supplementary materialThe online version of this article (10.1186/s12882-018-0906-5) contains supplementary material, which is available to authorized users.