Trimerization and genotype-phenotype correlation of COL4A5 mutants in Alport syndrome

INTRODUCTION: Alport syndrome is a hereditary glomerulonephritis that results from the disruption of collagen α345(IV) heterotrimerization caused by mutation in METHODS: We selected 9 α5(IV) missense mutants with typical glycine substitutions that clinically differed in disease progression. To quantify the trimerization of each mutant, split nanoluciferase-fused α3/α5 mutants and α4 were transfected into the cells, and intracellular and secreted heterotrimer were detected by luminescence using an assay that we developed previously. RESULTS: Trimer formation and secretion patterns tended to be similar to the wild type in most of the mutations that did not show proteinuria at a young age. On the other hand, trimer secretion was significantly reduced in all the mutations that showed proteinuria and early onset of renal failure. One of these mutants has low ability of intracellular trimer formation, and the others had the defect of low-level secretion. In addition, the mutant that is assumed to be nonpathogenic has similar trimer formation and secretion pattern as wild-type α5(IV). CONCLUSION: The result of cell-based α345(IV) heterotrimer formation assay was largely correlated with clinical genotype-phenotype. These trimerization assessments provide additional phenotypic considerations and may help to distinguish between pathogenic and nonpathogenic mutations

iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome

ヒトiPS細胞から作製した腎オルガノイドを用いたアルポート症候群病態モデルの開発. 京都大学プレスリリース. 2023-09-28.iPSC-derived kidney organoids to model a lifelong renal disease. 京都大学プレスリリース. 2023-10/17.Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS

Three Severe Cases of Viral Infections with Post-Kidney Transplantation Successfully Confirmed by Polymerase Chain Reaction and Flow Cytometry

Viral infections in patients with post-kidney transplantation are often difficult to diagnose as well as treat. We herein report three cases with severe viral infections after kidney transplantation. All their causative pathogens could be detected promptly by polymerase chain reaction and flow cytometry during the early stages of infection. These examinations would also be of great use to monitor therapeutic responses and disease activity. It is indeed true that no specific treatment is available for most of the viral infections, but we should be aware that some infections, such as Epstein-Barr virus infection, can be treatable with prompt and specific treatment, such as rituximab

A review of clinical characteristics and genetic backgrounds in Alport syndrome

Alport syndrome (AS) is a progressive hereditary renal disease that is characterized by sensorineural hearing loss and ocular abnormalities. It is divided into three modes of inheritance, namely, X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, while ADAS and ARAS are caused by those in COL4A3/COL4A4. Diagnosis is conventionally made pathologically, but recent advances in comprehensive genetic analysis have enabled genetic testing to be performed for the diagnosis of AS as first-line diagnosis. Because of these advances, substantial information about the genetics of AS has been obtained and the genetic background of this disease has been revealed, including genotype–phenotype correlations and mechanisms of onset in some male XLAS cases that lead to milder phenotypes of late-onset end-stage renal disease (ESRD). There is currently no radical therapy for AS and treatment is only performed to delay progression to ESRD using nephron-protective drugs. Angiotensin-converting enzyme inhibitors can remarkably delay the development of ESRD. Recently, some new drugs for this disease have entered clinical trials or been developed in laboratories. In this article, we review the diagnostic strategy, genotype–phenotype correlation, mechanisms of onset of milder phenotypes, and treatment of AS, among others

Matrikines in kidney ageing and age-related disease

Purpose of review Matrikines are cell-signalling extracellular matrix fragments and they have attracted recent attention from basic and translational scientists, due to their diverse roles in age-related disease and their potential as therapeutic agents. In kidney, the matrix undergoes remodelling by proteolytic fragmentation, so matrikines are likely to play a substantial, yet understudied, role in ageing and pathogenesis of age-related diseases.Recent findings This review presents an up-to-date description of known matrikines with either a confirmed or highly anticipated role in kidney ageing and disease, including their point of origin, mechanism of cleavage, a summary of known biological actions and the current knowledge which links them to kidney health. We also highlight areas of interest, such as the prospect of matrikine cross-tissue communication, and gaps in knowledge, such as the unexplored signalling potential of many kidney disease-specific matrix fragments.Summary We anticipate that knowledge of specific matrikines, and their roles in controlling processes of kidney pathology, could be leveraged for the development of exciting new future therapies through inhibition or even with their supplementation

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

Background In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. Methods We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106-17T>G, c.393+4A>G, c.517-8A>G, c.517-3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. Results We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106-17T>G). Conclusion We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants