Eriko Tanaka

Dendrin location in podocytes is associated with disease progression in animal and human glomerulopathy.

Background: Adriamycin (ADR) nephrosis in mice has been extensively studied and has enabled a greater understanding of the processes underlying the progression of renal injury. Dendrin is a novel component of the slit diaphragm with proapoptotic signaling properties, and it accumulates in the podocyte nucleus in response to glomerular injury in mice. The present study re-evaluated chronic progressive nephropathy in ADR mice and the localization of dendrin in mice and in human glomerulopathy. Methods: To investigate the localization of dendrin, a mouse model of nephrosis and glomerulosclerosis was used, in which ADR was injected once. WT-1-positive cells and apoptotic cells were counted in vivo and in vitro. To check the expression of dendrin in ADR mice, immunostaining and Western blot were performed. A survey of dendrin staining was performed on human kidney biopsy specimens. Results: The injection of ADR induced proteinuria, podocyte loss and glomerulosclerosis. It also caused the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We demonstrated the location of dendrin to podocyte nuclei in several cases of human glomerulopathy. The mean occurrence of dendrin-positive nucleus per glomerulus increased in several cases of human glomerulopathy. Conclusions: These findings suggest that the relocation of dendrin to the podocyte nuclei is useful as a novel marker of podocyte injury in human glomerulopathy.

Asymptomatic high Epstein–Barr viral load carriage in pediatric renal transplant recipients

Tanaka E, Sato T, Ishihara M, Tsutsumi Y, Hisano M, Chikamoto H, Akioka Y, Dohno S, Maeda A, Hattori M, Wakiguchi H, Fujieda M. Asymptomatic high Epstein‐Barr viral load carriage in pediatric renal transplant recipients.
Pediatr Transplantation 2011: 15: 306–313.

Notch2 activation ameliorates nephrosis

Activation of Notch1 and Notch2 has been recently implicated in human glomerular diseases. Here we show that Notch2 prevents podocyte loss and nephrosis. Administration of a Notch2 agonistic monoclonal antibody ameliorates proteinuria and glomerulosclerosis in a mouse model of nephrosis and focal segmental glomerulosclerosis. In vitro, the specific knockdown of Notch2 increases apoptosis in damaged podocytes, while Notch2 agonistic antibodies enhance activation of Akt and protect damaged podocytes from apoptosis. Treatment with triciribine, an inhibitor of Akt pathway, abolishes the protective effect of the Notch2 agonistic antibody. We find a positive linear correlation between the number of podocytes expressing activated Notch2 and the number of residual podocytes in human nephrotic specimens. Hence, specific activation of Notch2 rescues damaged podocytes and activating Notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis.

Epstein-Barr virus load for early detection of lymphoproliferative disorder in pediatric renal transplant recipients.

AIM The aims of this study were to establish a protocol for monitoring Epstein-Barr virus (EBV) infection for identification of pediatric renal transplant recipients with a high risk of developing posttransplant lymphoproliferative disorder (PTLD) and to predict the development of PTLD. SUBJECTS AND METHODS Peripheral blood mononuclear cells (PBMCs) and plasma EBV loads were measured by nested PCR (n-PCR) and real-time PCR (r-PCR) every 1 - 3 months after grafting in 17 pediatric recipients who were seronegative for EBV before grafting (4 with EBV-associated symptoms, including 2 with PTLD (Group A); 6 with asymptomatic persistent high EBV loads in PBMCs of > 1,000 copies/µgDNA for over 6 months (Group B); and 7 with neither EBV-associated symptoms nor persistent high EBV loads in PBMCs (Group C)). RESULTS n-PCR revealed EBV-DNA in PBMCs from all patients. The EBV genome was present in plasma in 3 (75%), 1 (17%), and 0 (0%) in Groups A, B and C (p < 0.01 for A vs. B and A vs. C). EBV loads detected by r-PCR in PBMCs were significantly higher in Groups A (p < 0.05) and B (p < 0.01) compared to Group C. EBV genomes in plasma were detected by n- and r-PCR in only the 2 cases with PTLD. One patient with lymphadenitis in Group A and 1 patient in Group B had EBV-DNA in plasma based on n-PCR, but the viral loads using r-PCR were < 250 copies/ml. CONCLUSION EBV loads in PBMCs alone are insufficient for predicting EBV-associated symptoms including PTLD. Plasma EBV loads (over 250 copies/ml) estimated by r-PCR may be useful to distinguish PTLD from other EBV-associated diseases or asymptomatic viremia.

Monitoring of Epstein-Barr virus load and killer T cells in pediatric renal transplant recipients.

AIM The aim of this study is to establish a monitoring method to prevent Epstein-Barr virus (EBV)-associated symptoms including post-transplant lymphoproliferative disorder (PTLD) that occur after pediatric renal transplantation. SUBJECTS AND METHODS Circulating EBV loads were quantified by real-time PCR every 1 - 3 months after grafting in 22 pediatric recipients (13 EBV-seronegative [R(-)] and 9 EBV-seropositive [R(+)] recipients before grafting). The peripheral blood cell populations of non-specific activated killer cells (CD8+HLA-DR+ phenotype) in 13 R(-) recipients and EBV-specific cytotoxic T cells (CTLs) reactive with a tetramer expressing HLA-A24-restricted EBV-specific antigens in 8 of 13 R(-) recipients were determined by flow cytometry. RESULTS EBV-associated symptoms including PTLD (2 cases) were found in 4 R(-) and none of the R(+) recipients. The maximum of EBV load in the R(-) group was significantly higher that in the R(+) group. In R(-) recipients, 4 symptomatic cases had significantly more EBV genome than asymptomatic cases. EBV-specific CTLs were detected in 6 of the 8 R(-) recipients, but these CTLs could not be detected in 1 of the 2 cases at onset of PTLD. The percentage of CD8+HLA-DR+ cells was significantly higher in asymptomatic recipients than in recipients with EBV-associated symptoms whose EBV loads were over 400 copies/microg DNA. CONCLUSION Monitoring of killer T cells and EBV loads may allow assessment of the risk of EBV-associated symptoms, and high EBV loads and low EBV-specific and/or non-specific CTL responses may be predictive for development of EBV-associated symptoms such as PTLD.

The role of Notch signaling in kidney podocytes

The Notch signaling pathway is a basic cell-to-cell communication mechanism. This pathway is activated by the interaction between Notch receptors and the ligands of adjacent cells. Once activated, Notch receptors are cleaved and the intracellular domains translocate into the nucleus, where the transcription of target genes starts. In the mammalian kidney, Notch receptors are activated during nephrogenesis. Afterwards, in the mature glomeruli, the Notch pathway becomes silent. However, many researchers have reported the activation of Notch receptors in mature podocytes under pathological conditions. In this review, we discuss the role of Notch signaling in podocytes.

Sorting Nexin 9 facilitates podocin endocytosis in the injured podocyte

The irreversibility of glomerulosclerotic changes depends on the degree of podocyte injury. We have previously demonstrated the endocytic translocation of podocin to the subcellular area in severely injured podocytes and found that this process is the primary disease trigger. Here we identified the protein sorting nexin 9 (SNX9) as a novel facilitator of podocin endocytosis in a yeast two-hybrid analysis. SNX9 is involved in clathrin-mediated endocytosis, actin rearrangement and vesicle transport regulation. Our results revealed and confirmed that SNX9 interacts with podocin exclusively through the Bin–Amphiphysin–Rvs (BAR) domain of SNX9. Immunofluorescence staining revealed the expression of SNX9 in response to podocyte adriamycin-induced injury both in vitro and in vivo. Finally, an analysis of human glomerular disease biopsy samples demonstrated strong SNX9 expression and co-localization with podocin in samples representative of severe podocyte injury, such as IgA nephropathy with poor prognosis, membranous nephropathy and focal segmental glomerulosclerosis. In conclusion, we identified SNX9 as a facilitator of podocin endocytosis in severe podocyte injury and demonstrated the expression of SNX9 in the podocytes of both nephropathy model mice and human patients with irreversible glomerular disease.

Rac1 in podocytes promotes glomerular repair and limits the formation of sclerosis

Rac1, a Rho family member, is ubiquitously expressed and participates in various biological processes. Rac1 expression is induced early in podocyte injury, but its role in repair is unclear. To investigate the role of Rac1 expression in podocytes under pathological conditions, we used podocyte-specific Rac1 conditional knock-out (cKO) mice administered adriamycin (ADR), which causes nephrosis and glomerulosclerosis. Larger areas of detached podocytes, more adhesion of the GBM to Bowman’s capsule, and a higher ratio of sclerotic glomeruli were observed in Rac1 cKO mice than in control mice, whereas no differences were observed in glomerular podocyte numbers in both groups after ADR treatment. The mammalian target of rapamycin (mTOR) pathway, which regulates the cell size, was more strongly suppressed in the podocytes of Rac1 cKO mice than in those of control mice under pathological conditions. In accordance with this result, the volumes of podocytes in Rac1 cKO mice were significantly reduced compared with those of control mice. Experiments using in vitro ADR-administered Rac1 knockdown podocytes also supported that a reduction in Rac1 suppressed mTOR activity in injured podocytes. Taken together, these data indicate that Rac1-associated mTOR activation in podocytes plays an important role in preventing the kidneys from developing glomerulosclerosis.

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.