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

Drug efficacy against aortic dissection

Objective: Aortic dissection is a life-threatening disease. At present, the only therapeutic strategies available are surgery and antihypertensive drugs. Moreover, the molecular mechanisms underlying the onset of aortic dissection are still unclear. We established a novel aortic dissection model in mice using pharmacologically induced endothelial dysfunction. We then used the Japanese Adverse Drug Event Report database to investigate the role of pitavastatin in preventing the onset of aortic dissection. Methods and results: To induce endothelial dysfunction, Nω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, was administered to C57BL/6 mice. Three weeks later, angiotensin II (Ang II) and β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, were administered with osmotic mini-pumps. False lumen formation was used as the pathological determinant of aortic dissection. The incidences of aortic dissection and death from aneurysmal rupture were significantly higher in the Nω-nitro-L-arginine methyl ester, Ang II, and BAPN (LAB) group than they were in the Ang II and BAPN (AB) group. Pitavastatin was administered orally to LAB mice. It significantly lowered the incidences of dissection and rupture. It also decreased inflammation and medial degradation, both of which were exacerbated in the LAB group. The Japanese Adverse Drug Event Report database analysis indicated that there were 113 cases of aortic dissection out of 95 090 patients (0.12%) not receiving statins but only six cases out of 16 668 patients receiving statins (0.04%) (odds ratio: 0.30; P=0.0043). Conclusion: Our results suggest that endothelial dysfunction is associated with the onset of aortic dissection and pitavastatin can help prevent this condition

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

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

Study protocol: mycophenolate mofetil as maintenance therapy after rituximab treatment for childhood-onset, complicated, frequently-relapsing nephrotic syndrome or steroid-dependent nephrotic syndrome: a multicenter double-blind, randomized, placebo-controlled trial (JSKDC07)

Abstract Background Idiopathic nephrotic syndrome (INS) is the most common chronic glomerular disease in children. Approximately 80–90% of patients with childhood INS have steroid-sensitive nephrotic syndrome (SSNS), and can obtain remission with steroid therapy, while the remainder have steroid-resistant nephrotic syndrome (SRNS). Furthermore, approximately 50% of children with SSNS develop frequently-relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS). Children with FRNS/SDNS are usually treated with immunosuppressive agents such as cyclosporine, cyclophosphamide, or mizoribine in Japan. However, 10–20% of children receiving immunosuppressive agents still show frequent relapse and/or steroid dependence during or after treatment, which is defined as complicated FRNS/SDNS. Furthermore, 30% of SRNS patients who obtain remission after additional treatments such as cyclosporine also turn out to be complicated FRNS/SDNS. For such complicated FRNS/SDNS patients, rituximab (RTX) is currently used; however, recurrence after RTX treatment also remains an open issue. Because long-term use of existing immunosuppressive drugs has limitations, development of a novel treatment for maintenance therapy after RTX is desirable. Mycophenolate mofetil (MMF) is an immunosuppressive drug with fewer side effects than cyclosporine or cyclophosphamide. Importantly, recent studies have reported the efficacy of MMF in children with nephrotic syndrome. Methods We conduct a multicenter, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of MMF after RTX therapy in children with complicated FRNS/SDNS. Patients are allocated to either RTX plus MMF treatment group, or RTX plus placebo treatment group. For the former group, MMF is administered at a dose of 1000–1200 mg/m2/day (maximum 2 g/day) twice daily for 17 months after RTX treatment. The primary endpoint is time-to-treatment failure (development of frequent relapses, steroid dependence or steroid resistance). Discussion The results will provide important data on the use of MMF as maintenance therapy after RTX to prevent complicated FRNS/SDNS patients from declining into treatment failure. In future, MMF in conjunction with RTX treatment may permit increased duration of remission in ‘complicated’ FRNS/SDNS cases. Trial registration This trial was prospectively registered to UMIN Clinical Trials Registry on June 23, 2014 (UMIN Trial ID: UMIN000014347)

Comprehensive genetic diagnosis of Japanese patients with severe proteinuria

Numerous disease-causing gene mutations have been identified in proteinuric diseases, such as nephrotic syndrome and glomerulosclerosis. This report describes the results of comprehensive genetic diagnosis of Japanese patients with severe proteinuria. In addition, the report describes the clinical characteristics of patients with monogenic disease-causing mutations. We conducted comprehensive gene screening of patients who had either congenital nephrotic syndrome, infantile nephrotic syndrome, steroid-resistant nephrotic syndrome, or focal segmental glomerular sclerosis. Using targeted next-generation sequencing, 60 podocyte-related genes were screened in 230 unrelated patients with proteinuria. A retrospective review of clinical data was conducted for these patients. We detected monogenic disease-causing mutations in 30% (69 of 230) of patients among 19 of the screened genes. Common genes with disease-causing mutations were WT1 (25%), NPHS1 (12%), INF2 (12%), TRPC6 (10%), and LAMB2 (9%). With various immunosuppressive or renoprotective therapies, remission of proteinuria in patients with unknown causative mutations was observed in 26% of patients, whereas only 5% of patients with monogenic disease-causing mutations exhibited complete remission. We assessed the genetic backgrounds of Japanese patients with severe proteinuria. The proportion of patients with gene defects was similar to that of other reports, but the disease-causing gene mutation frequency was considerably different