Evaluation of pathogenicity of WT1 intron variants by in vitro splicing analysis.

BACKGROUND: Wilms tumor 1 (WT1; NM_024426) causes Denys-Drash syndrome, Frasier syndrome, or isolated focal segmental glomerulosclerosis. Several WT1 intron variants are pathogenic; however, the pathogenicity of some variants remains undefined. Whether a candidate variant detected in a patient is pathogenic is very important for determining the therapeutic options for the patient. METHODS: In this study, we evaluated the pathogenicity of WT1 gene intron variants with undetermined pathogenicity by comparing their splicing patterns with those of the wild-type using an in vitro splicing assay using minigenes. The three variants registered as likely disease-causing genes: Mut1 (c.1017-9 T > C(IVS5)), Mut2 (c.1355-28C > T(IVS8)), Mut3 (c.1447 + 1G > C(IVS9)), were included as subjects along the 34 splicing variants registered in the Human Gene Mutation Database (HGMD)®. RESULTS: The results showed no significant differences in splicing patterns between Mut1 or Mut2 and the wild-type; however, significant differences were observed in Mut3. CONCLUSION: We concluded that Mut1 and Mut2 do not possess pathogenicity although they were registered as likely pathogenic, whereas Mut3 exhibits pathogenicity. Our results suggest that the pathogenicity of intronic variants detected in patients should be carefully evaluated.

Systematic Review of Clinical Characteristics and Genotype-Phenotype Correlation in LAMB2-Associated Disease.

Introduction: Laminin subunit beta-2 (LAMB2)-associated disease, termed Pierson syndrome, presents with congenital nephrotic syndrome, ocular symptoms, and neuromuscular symptoms. In recent years, however, the widespread use of next-generation sequencing (NGS) has helped to discover a variety of phenotypes associated with this disease. Therefore, we conducted this systematic review. Methods: A literature search of patients with LAMB2 variants was conducted, and 110 patients were investigated, including 12 of our patients. For genotype-phenotype correlation analyses, the extracted data were investigated for pathogenic variant types, the severity of nephropathy, and extrarenal symptoms. Survival analyses were also performed for the onset age of end-stage kidney disease (ESKD). Results: Among all patients, 81 (78%) presented with congenital nephrotic syndrome, and 52 (55%) developed ESKD within 12 months. The median age at ESKD onset was 6.0 months. Kidney survival analysis showed that patients with biallelic truncating variants had a significantly earlier progression to ESKD than those with other variants (median age 1.2 months vs. 60.0 months, P < 0.05). Although the laminin N-terminal domain is functionally important in laminin proteins, and variants in the laminin N-terminal domain are said to result in a severe kidney phenotype such as earlier onset age and worse prognosis, there were no significant differences in onset age of nephropathy and progression to ESKD between patients with nontruncating variants located in the laminin N-terminal domain and those with variants located outside this domain. Conclusion: This study revealed a diversity of LAMB2-associated diseases, characteristics of LAMB2 nephropathy, and genotype-phenotype correlations.

Corrigendum to "Detecting MUC1 Variants in Patients Clinicopathologically Diagnosed With Having Autosomal Dominant Tubulointerstitial Kidney Disease"Kidney International Reports, Volume 7, Issue 4, April 2022, Pages 857-866.

[This corrects the article DOI: 10.1016/j.ekir.2021.12.037.].

All reported non-canonical splice site variants in GLA cause aberrant splicing.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene. METHODS: 13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants. RESULTS: All candidate non-canonical splice site variants in GLA caused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner. CONCLUSIONS: Validation of splicing effects using an in vitro splicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.

Aberrant splicing caused by exonic single nucleotide variants positioned 2nd or 3rd to the last nucleotide in the COL4A5 gene.

BACKGROUND AND OBJECTIVES: The evident genotype-phenotype correlation shown by the X-linked Alport syndrome warrants the assessment of the impact of identified gene variants on aberrant splicing. We previously reported that single nucleotide variants (SNVs) in the last nucleotide of exons in COL4A5 cause aberrant splicing. It is known that the nucleotides located 2nd and 3rd to the last nucleotides of exons can also play an essential role in the first step of the splicing process. In this study, we aimed to investigate whether SNVs positioned 2nd or 3rd to the last nucleotide of exons in COL4A5 resulted in aberrant splicing. METHODS: We selected eight candidate variants: six from the Human Gene Variant Database Professional and two from our cohort. We performed an in-vitro splicing assay and reverse transcription-polymerase chain reaction (RT-PCR) for messenger RNA obtained from patients, if available. RESULTS: The candidate variants were initially classified into the following groups: three nonsense, two missense, and three synonymous variants. Splicing assays and RT-PCR for messenger RNA revealed that six of the eight variants caused aberrant splicing. Four variants, initially classified as non-truncating variants, were found to be truncating ones, which usually show relatively more severe phenotypes. CONCLUSION: We revealed that exonic SNVs positioned 2nd or 3rd to the last nucleotide of exons in the COL4A5 were responsible for aberrant splicing. The results of our study suggest that attention should be paid when interpreting the pathogenicity of exonic SNVs near the 5' splice site.

Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis.

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

Detecting MUC1 Variants in Patients Clinicopathologically Diagnosed With Having Autosomal Dominant Tubulointerstitial Kidney Disease.

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD)-MUC1 is predominantly caused by frameshift mutations owing to a single-base insertion into the variable number tandem repeat (VNTR) region in MUC1. Because of the complexity of the variant hotspot, identification using short-read sequencers (SRSs) is challenging. Although recent studies have revealed the usefulness of long-read sequencers (LRSs), the prevalence of MUC1 variants in patients with clinically suspected ADTKD remains unknown. We aimed to clarify this prevalence and the genetic characteristics and clinical manifestations of ADTKD-MUC1 in a Japanese population using an SRS and an LRS. Methods: From January 2015 to December 2019, genetic analysis was performed using an SRS in 48 patients with clinically suspected ADTKD. Additional analyses were conducted using an LRS in patients with negative SRS results. Results: Short-read sequencing results revealed MUC1 variants in 1 patient harboring a cytosine insertion in the second repeat unit of the VNTR region; however, deeper VNTR regions could not be read by the SRS. Therefore, we conducted long-read sequencing analysis of 39 cases and detected MUC1 VNTR variants in 8 patients (in total, 9 patients from unrelated families). With the inclusion of family-affected patients (n = 31), the median age at the development of end-stage kidney disease (ESKD) was 45 years (95% CI: 40-40 years). Conclusion: In Japan, the detection rate of MUC1 variants in patients with clinically suspected ADTKD was 18.8%. More than 20% of patients with negative SRS results had MUC1 variants detected by an LRS.

Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing.

INTRODUCTION: COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing. METHODS: In total, 20 variants were found in the Human Gene Mutation Database (n = 14) and our cohort (n = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients' samples when available. Then, we investigated genotype-phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies. RESULTS: Among the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 variants were identical to those from the minigene assay. CONCLUSION: Our study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.

Clinical features of autosomal recessive polycystic kidney disease in the Japanese population and analysis of splicing in PKHD1 gene for determination of phenotypes.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 gene. The clinical spectrum is often more variable than previously considered. We aimed to analyze the clinical features of genetically diagnosed ARPKD in the Japanese population. METHODS: We conducted a genetic analysis of patients with clinically diagnosed or suspected ARPKD in Japan. Moreover, we performed a minigene assay to elucidate the mechanisms that could affect phenotypes. RESULTS: PKHD1 pathogenic variants were identified in 32 patients (0-46 years). Approximately one-third of the patients showed prenatal anomalies, and five patients died within one year after birth. Other manifestations were detected as follows: chronic kidney disease stages 1-2 in 15/26 (57.7%), Caroli disease in 9/32 (28.1%), hepatic fibrosis in 7/32 (21.9%), systemic hypertension in 13/27 (48.1%), and congenital hypothyroidism in 3 patients. There have been reported that truncating mutations in both alleles led to severe phenotypes with perinatal demise. However, one patient without a missense mutation survived the neonatal period. In the minigene assay, c.2713C > T (p.Gln905Ter) and c.6808 + 1G > A expressed a transcript that skipped exon 25 (123 bp) and exon 41 (126 bp), resulting in an in-frame mutation, which might have contributed to the milder phenotype. Missense mutations in cases of neonatal demise did not show splicing abnormalities. CONCLUSION: Clinical manifestations ranged from cases of neonatal demise to those diagnosed in adulthood. The minigene assay results indicate the importance of functional analysis, and call into question the fundamental belief that at least one non-truncating mutation is necessary for perinatal survival.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Systematic Review of Genotype-Phenotype Correlations in Frasier Syndrome.

INTRODUCTION: Frasier syndrome (FS) is a rare inherited kidney disease caused by intron 9 splicing variants of WT1. For wild-type WT1, 2 active splice donor sites in intron 9 cause a mixture of 2 essential transcripts (with or without lysine-threonine-serine [+/KTS or -KTS]), and imbalance of the +KTS/-KTS ratio results in the development of FS. To date, 6 causative intron 9 variants have been identified; however, detailed transcript analysis has not yet been conducted and the genotype-phenotype correlation also remains to be elucidated. METHODS: We conducted an in vitro minigene splicing assay for 6 reported causative variants and in vivo RNA sequencing to determine the +KTS/-KTS ratio using patients' samples. We also performed a systematic review of reported FS cases with a description of the renal phenotype. RESULTS: The in vitro assay revealed that although all mutant alleles produced -KTS transcripts only, the wild-type allele produced both +KTS and -KTS transcripts at a 1:1 ratio. In vivo RNA sequencing showed that patients' samples with all heterozygous variants produced similar ratios of +KTS to -KTS (1:3.2-1:3.5) and wild-type kidney showed almost a 1:1 ratio (1:0.85). A systematic review of 126 cases clarified that the median age of developing ESKD was 16 years in all FS patients, and there were no statistically significant differences between the genotypes or sex chromosome karyotypes in terms of the renal survival period. CONCLUSION: Our study suggested no differences in splicing pattern or renal survival period among reported intron 9 variants causative of FS.

Examination of the predicted prevalence of Gitelman syndrome by ethnicity based on genome databases.

Gitelman syndrome is an autosomal recessive inherited salt-losing tubulopathy. It has a prevalence of around 1 in 40,000 people, and heterozygous carriers are estimated at approximately 1%, although the exact prevalence is unknown. We estimated the predicted prevalence of Gitelman syndrome based on multiple genome databases, HGVD and jMorp for the Japanese population and gnomAD for other ethnicities, and included all 274 pathogenic missense or nonsense variants registered in HGMD Professional. The frequencies of all these alleles were summed to calculate the total variant allele frequency in SLC12A3. The carrier frequency and the disease prevalence were assumed to be twice and the square of the total allele frequency, respectively, according to the Hardy-Weinberg principle. In the Japanese population, the total carrier frequencies were 0.0948 (9.5%) and 0.0868 (8.7%) and the calculated prevalence was 0.00225 (2.3 in 1000 people) and 0.00188 (1.9 in 1000 people) in HGVD and jMorp, respectively. Other ethnicities showed a prevalence varying from 0.000012 to 0.00083. These findings indicate that the prevalence of Gitelman syndrome in the Japanese population is higher than expected and that some other ethnicities also have a higher prevalence than has previously been considered.

Genotype-Phenotype Correlation in WT1 Exon 8 to 9 Missense Variants.

INTRODUCTION: WT1 missense mutation in exon 8 or 9 causes infantile nephrotic syndrome with early progression to end-stage kidney disease (ESKD), Wilms tumor, and 46,XY female. However, some patients with missense mutations in exon 8 or 9 progress to ESKD in their teens or later. Therefore, we conducted a systematic review and functional analysis of WT1 transcriptional activity. METHODS: We conducted a systematic review of 174 cases with WT1 exon 8 or 9 missense variants from our cohort (n=13) and previous reports (n=161). Of these cases, mild and severe genotypes were selected for further in vitro functional analysis using luciferase assay. RESULTS: The median age of developing ESKD was 1.17 years. A comparative study was conducted among three WT1 genotype classes: mutations of the DNA-binding site (DBS group), mutations outside the DNA-binding site but at sites important for zinc finger structure formation by 2 cysteines and 2 histidines (C2H2 group), and mutations leading to other amino acid changes (Others group). The DBS group showed the severest phenotype and the C2H2 group was intermediate, whereas the Others group showed the mildest phenotype (developing ESKD at 0.90, 2.00, and 3.92 years, respectively, with significant differences). In vitro functional analysis showed dominant-negative effects for all variants; in addition, the DBS and C2H2 mutations were associated with significantly lower WT1 transcriptional activity than the other mutations. CONCLUSION: Not only the DNA-binding site but also C2H2 zinc finger structure sites are important for maintaining WT1 transcriptional activity, and their mutation causes severe clinical symptoms.

X-chromosome inactivation patterns in females with Fabry disease examined by both ultra-deep RNA sequencing and methylation-dependent assay.

BACKGROUND: Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A. Males are usually severely affected, while females have a wide range of disease severity. This variability has been assumed to be derived from organ-dependent skewed X-chromosome inactivation (XCI) patterns in each female patient. Previous studies examined this correlation using the classical methylation-dependent method; however, conflicting results were obtained. This study was established to ascertain the existence of skewed XCI in nine females with heterozygous pathogenic variants in the GLA gene and its relationship to the phenotypes. METHODS: We present five female patients from one family and four individual female patients with Fabry disease. In all cases, heterozygous pathogenic variants in the GLA gene were detected. The X-chromosome inactivation patterns in peripheral blood leukocytes and cells of urine sediment were determined by both classical methylation-dependent HUMARA assay and ultra-deep RNA sequencing. Fabry Stabilization Index was used to determine the clinical severity. RESULTS: Skewed XCI resulting in predominant inactivation of the normal allele was observed only in one individual case with low ⍺-galactosidase A activity. In the remaining cases, no skewing was observed, even in the case with the highest total severity score (99.2%). CONCLUSION: We conclude that skewed XCI could not explain the severity of female Fabry disease and is not the main factor in the onset of various clinical symptoms in females with Fabry disease.

Spontaneous remission in adult patients with IgA nephropathy treated with conservative therapy.

BACKGROUND: There are few studies describing the clinical course and spontaneous remission of IgA nephropathy (IgAN) in adult patients receiving conservative treatment. METHOD: Data from 62 adult patients with biopsy-diagnosed IgAN, who received conservative treatment at least 5 years prior, were retrospectively investigated. No patients received corticosteroids, other immunosuppressants, or tonsillectomy. Remission of proteinuria and hematuria were defined as proteinuria <0.3 g/gCr and urine red blood cells (RBC) <5 / high power field (HPF) on three consecutive urinalyses obtained during an observation period of ≥6 months. RESULT: Thirty-eight (61.3%) patients had remission of hematuria, 24 (38.7%) had remission of proteinuria, and 19 (30.6%) had remission of both. Remission rates increased in patients with proteinuria <0.5 g/g Cr at diagnosis. The median time to remission of hematuria was 2.8 years and that of proteinuria was 2.6 years. Patients who showed renal function decline (defined as 30% decline of estimated glomerular filtration rate [eGFR] from baseline) were older, had significantly lower eGFR, and higher proteinuria at diagnosis. Two patients with preserved renal function and normal proteinuria at diagnosis experienced renal function decline. Renal function did not decline within 3 years of diagnosis in patients with proteinuria <1 g/gCr at diagnosis. CONCLUSIONS: Relatively high rates of spontaneous remission were observed. Remission of both hematuria and proteinuria were frequent within 3 years after diagnosis, and renal function was well preserved during this period. These data indicate that it is rational to use conservative treatment for 3 years after the diagnosis instead of aggressive treatments.

Development of Injectable Polydactyly-Derived Chondrocyte Sheets.

We are conducting a clinical study of the use of allogeneic polydactyly-derived chondrocyte sheets (PD sheets) for the repair of articular cartilage damage caused by osteoarthritis. However, the transplantation of PD sheets requires highly invasive surgery. To establish a less invasive treatment, we are currently developing injectable fragments of PD sheets (PD sheets-mini). Polydactyly-derived chondrocytes were seeded in RepCell™ or conventional temperature-responsive inserts and cultured. Cell counts and viability, histology, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), and flow cytometry were used to characterize PD sheets-mini and PD sheets collected from each culture. To examine the effects of injection on cell viability, PD sheets-mini were tested in four experimental conditions: non-injection control, 18 gauge (G) needle, 23G needle, and syringe only. PD sheets-mini produced similar amounts of humoral factors as PD sheets. No histological differences were observed between PD sheets and PD sheets-mini. Except for COL2A1, expression of cartilage-related genes did not differ between the two types of PD sheet. No significant differences were observed between injection conditions. PD sheets-mini have characteristics that resemble PD sheets. The cell viability of PD sheets-mini was not significantly affected by needle gauge size. Intra-articular injection may be a feasible, less invasive method to transplant PD sheets-mini.

Association of kidney transplantation with mortality on hemodialysis after graft failure.

BACKGROUND: Although a substantial number of patients return to dialysis after kidney transplant failure, it remains controversial whether transplant-failure patients have a higher mortality risk than transplant-naïve patients on dialysis who have never undergone kidney transplantation. We compared outcomes of transplant-failure and transplant-naïve patients on hemodialysis. METHODS: Data from the Japanese National Dialysis Registry (2012-2013) were analyzed, including 220,438 prevalent hemodialysis patients. Multivariable Cox models were used to compare all-cause, cardiovascular, and infection-related mortality during 1-year follow-up between transplant-failure and transplant-naïve patients. Multiple imputation and propensity score matching were utilized as sensitivity analyses. RESULTS: During 209,377 patient-years of follow-up, 18,648 all-cause deaths (8.5% of all patients), 7700 cardiovascular deaths (41% of all-cause deaths), and 3806 infection-related deaths (20% of all-cause deaths) were observed. Adjusted hazard ratios [95% confidence intervals] for all-cause, cardiovascular, and infection-related deaths among transplant-failure patients were 0.81 [0.59-1.11], 0.54 [0.30-0.98], and 1.54 [0.92-2.59], respectively. Sensitivity analyses using multiple imputation and propensity score matching yielded similar results. CONCLUSIONS: This Japanese cohort study suggested that a cardiovascular mortality risk of transplant-failure patients could be significantly lower than that of transplant-naïve patients, while there might be a trend toward a higher infection-related mortality risk in transplant-failure patients. However, this retrospective, single-country study can introduce an immortal time bias in transplant-failure patients, and limit the external validity. Further prospective studies are warranted to improve the comparability of outcomes between transplant-failure and transplant-naïve patients, and to examine worldwide the generalizability of the potential cardiovascular benefit of kidney transplantation even after returning to dialysis.

Regenerative effects of human chondrocyte sheets in a xenogeneic transplantation model using immune-deficient rats.

Although cell transplantation has attracted much attention in regenerative medicine, animal models continue to be used in translational research to evaluate safety and efficacy because cell sources and transplantation modalities are so diverse. In the present study, we investigated the regenerative effects of human chondrocyte sheets on articular cartilage in a xenogeneic transplantation model using immune-deficient rats. Osteochondral defects were created in the knee joints of immune-deficient rats that were treated as Group A, untreated (without transplantation); Group B, transplantation of a layered chondrocyte sheet containing 5.0 × 105 cells (layered chondrocyte sheet transplantation); Group C, transplantation of a synoviocyte sheet containing 5.0 × 105 cells (synoviocyte sheet transplantation); or Group D, transplantation of both a synoviocyte sheet plus a layered chondrocyte sheet, each containing 5.0 × 105 cells (synoviocyte sheet plus layered chondrocyte sheet transplantation). Histological evaluation demonstrated that Group B showed cartilage regeneration with hyaline cartilage and fibrocartilage. In Groups C and D, the defect was filled with fibrous tissue but no hyaline cartilage. Transplanted cells were detected at 4 and 12 weeks after transplantation, but the number of cells had decreased at 12 weeks. Our results indicate that layered chondrocyte sheet transplantation contributes to articular cartilage regeneration; this model proved useful for evaluating these regenerative effects.