Yu Sasaki

Role of the TNF pathway in the progression of diabetic nephropathy in KK-Ay mice

Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNF-α remains unclear. The objectives of the present study were to examine whether TNF-α inhibition with a soluble TNF receptor (TNFR)2 fusion protein, i.e., etanercept (ETN), improves the early stage of DN in the type 2 diabetic model of the KK-A(y) mouse and to also investigate which TNF pathway, TNFR1 or TNFR2, is predominantly involved in the progression of this disease. ETN was injected intraperitoneally into mice for 8 wk. Renal damage was evaluated by immunohistochemistry, Western blot analysis, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNF-α and/or high glucose (HG) and treated with ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNF-α and TNFR1, in ETN-treated KK-A(y) mice were significantly decreased compared with untreated KK-A(y) mice. mRNA levels of ICAM-1, VCAM-1, and monocyte chemoattractant protein-1 and the number of F4/80-positive cells were all decreased after treatment. Numbers of cleaved caspase-3- and TUNEL-positive cells in untreated mice were very few and were not different from ETN-treated mice. In vitro, stimulation with TNF-α or HG markedly increased both mRNA levels of TNFRs, unlike in the in vivo case. Furthermore, ETN partly recovered TNF-α-induced but not HG-induced TNFR mRNA levels. In conclusion, it appears that ETN may improve the progression of the early stage of DN predominantly through inhibition of the anti-inflammatory action of the TNF-α-TNFR2 pathway.

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.

Podocin is translocated to cytoplasm in puromycin aminonucleoside nephrosis rats and in poor-prognosis patients with IgA nephropathy

Podocytes serve as the final barrier to urinary protein loss through a highly specialized structure called a slit membrane and maintain foot process and glomerular basement membranes. Podocyte injury results in progressive glomerular damage and accelerates sclerotic changes, although the exact mechanism of podocyte injury is still obscure. We focus on the staining gap (podocin gap) defined as the staining difference between podocin and synaptopodin, which are normally located in the foot process. In puromycin aminonucleoside nephrosis rats, the podocin gap is significantly increased (p < 0.05) and podocin is translocated to the cytoplasm on days 7 and 14 but not on day 28. Surprisingly, the gap is also significantly increased (p < 0.05) in human kidney biopsy specimens of poor-prognosis IgA nephropathy patients. This suggests that the podocin gap could be a useful marker for classifying the prognosis of IgA nephropathy and indicating the translocation of podocin to the cytoplasm. Next, we find more evidence of podocin trafficking in podocytes where podocin merges with Rab5 in puromycin aminonucleoside nephrosis rats at day 14. In immunoelectron microscopy, the podocin positive area was significantly translocated from the foot process areas to the cytoplasm (p< 0.05) on days 7 and 14 in puromycin aminonucleoside nephrosis rats. Interestingly, podocin is also translocated to the cytoplasm in poor-prognosis human IgA nephropathy. In this paper, we demonstrate that the translocation of podocin by endocytosis could be a key traffic event of critical podocyte injury and that the podocin gap could indicate the prognosis of IgA nephropathy.

Significance of urinary full-length megalin in patients with IgA nephropathy.

Background and Objectives Megalin is highly expressed at the apical membranes of proximal tubular epithelial cells. A urinary full-length megalin (C-megalin) assay is linked to the severity of diabetic nephropathy in type 2 diabetes. This study examined the relationship between levels of urinary C-megalin and histological findings in adult patients with IgA nephropathy (IgAN). Design, Setting, Participants, & Measurements Urine samples voided in the morning on the day of renal biopsy were obtained from 73 patients with IgAN (29 men and 44 women; mean age, 33 years) and 5 patients with membranous nephropathy (MN). Renal pathologic variables were analyzed using the Oxford classification of IgAN, the Shigematsu classification and the Clinical Guidelines of IgAN in Japan. The levels of urinary C-megalin were measured by sandwich ELISA. Results Histological analysis based on the Oxford classification revealed that the levels of urinary C-megalin were correlated with mesangial hypercellularity in IgAN patients (OR = 1.76, 95% CI: 1.04–3.27, P<0.05). There was a significant correlation between the levels of urinary C-megalin and the severity of chronic extracapillary abnormalities according to the Shigematsu classification in IgAN patients (β = 0.33, P = 0.008). The levels of urinary C-megalin were significantly higher in all risk levels of IgAN patients requiring dialysis using the Clinical Guidelines of IgAN in Japan than in the control group. The levels of urinary C-megalin were significantly higher in the high risk and very high risk grades than in the low risk grade (P<0.05). The levels of urinary C-megalin were significantly higher in MN patients compared to the control group. Conclusions The levels of urinary C-megalin are associated with histological abnormalities in adult IgAN patients. There is a possibility that urinary C-megalin is an independent predictor of disease progression of IgAN. In addition, our results suggest that urinary C-megalin is a marker of glomerular abnormalities in various glomerular diseases as well as IgAN.

Risk of overestimation of kidney function using GFR-estimating equations in patients with low inulin clearance.

Accurate estimation of the glomerular filtration rate (GFR) is very important in clinical practice. Although renal inulin clearance (Cin) is the gold standard for measuring GFR, the procedure for Cin measurement is complicated. Use of GFR‐estimating equations has been increasing recently due to their simplicity. The objectives of the present study are to analyze the correlation between Cin and other GFR‐estimating parameters and to investigate their clinical usefulness and limitation.

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.

PD-L1/PD-L2-expressing B-1 cells inhibit alloreactive T cells in mice

B cells constitute a complex system of antigen-presenting cells (APCs) and exist as distinct subsets that differ in their lineage affiliation, surface molecule expression, and biological function, thus potentially regulating the immune response. In this study, we investigated the immune-regulatory roles of murine B cell subsets as regulatory APCs targeting alloreactive T cells. Either splenic B cells, peritoneal cavity (PerC) B cells, or non-B cells from Balb/c mice were intravenously injected into B6 mice. Serum levels of anti-Balb/c antibodies in the recipients of PerC B cells were significantly lower than those in the recipients of splenic B cells and PerC non-B cells, as determined over a 4-week period after the injection. Mixed-lymphocyte reaction (MLR) assays using splenocytes from the B6 mice at 2 weeks after the injection revealed the significantly reduced anti-Balb/c T cell-responses in the recipients of PerC B cells, as compared to those in the recipients of splenic B cells or untreated control mice. Since PerC B cells contained MHC class II+ CD80+ CD86+ PD-L1+ PD-L2+ cells among the CD5+ B-1a cell subset, PerC B cells from Balb/c mice were pre-incubated with anti-PD-L1/PD-L2 mAbs prior to injection. This treatment abrogated their immune-regulatory effects on anti-Balb/c T cells in the MLR assays. In addition, the inoculation with Balb/c PerC B cells significantly prolonged the survival of subsequently grafted Balb/c hearts in B6 mouse recipients, whereas that with SPL B cells did not. These findings indicate that the PerC B cells, including PD-L1/PD-L2 B-1a cells, may suppress T cells responding to allostimulation, and thus may be optimal for donor lymphocyte injection.

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.

Expression of Cathepsin L and Its Intrinsic Inhibitors in Glomeruli of Rats With Puromycin Aminonucleoside Nephrosis

Cathepsin L, a lysosomal cysteine proteinase, may have a key role in various biological and disease processes by intracellular and extracellular degradation of proteins. We examined the levels of cathepsin L and its intrinsic inhibitors in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. In contrast to the weak levels of cathepsin L in normal glomeruli, on days 4 and 8, strong immunostaining was detected in almost all podocytes when proteinuria and pathological changes of the podocytes developed. Cathepsin L was reduced after day 28, but remained in a focal and segmental manner. Cystatin β, an intracellular inhibitor, was not detected in podocytes. However, cystatin C, an extracellular inhibitor, was detected in podocytes after day 4, coincident with cathepsin L. Cystatin C levels were gradually reduced but sustained in many podocytes on day 28, while cystatin C was not detected in podocytes sustained cathepsin L. These results demonstrated that cathepsin L levels are not always accompanied by the levels of its inhibitors in podocytes of PAN nephrosis, suggesting a potential role of cathepsin L in podocyte injury, which is a critical process for the development and progression of tuft adhesion and sclerosis.

Evaluation of Long-Term Combination Therapy With Peritoneal Dialysis and Hemodialysis

It is well known that a combination therapy with peritoneal dialysis (PD) and hemodialysis (HD) is feasible and may improve clinical status in patients for whom adequate solute and fluid removal is difficult to achieve with PD alone. The objective of the present study was to evaluate whether the therapy is useful in the likelihood of long‐term peritoneal membrane and cardiac function. The therapy was 6 days of PD and one session of HD per week. Physical, biochemical, dialysate‐to‐plasma ratio of creatinine (D/P Cr), arteriovenous fistula (AVF) blood flow, and left ventricular mass index (LVMI) data were prospectively analyzed in 30 patients with measurements performed at 0 and 6 months, and for 21 patients, 12 or 18 months after initiation of the therapy. The levels of hemoglobin (Hb) after therapy were significantly higher than those at the initiation of therapy. The levels of LVMI and human atrial natriuretic peptide (hANP) after therapy were significantly lower than those at the initiation of therapy, whereas AVF blood flow did not change significantly. D/P Cr levels at 6 months after the therapy were significantly lower than those at the initiation of therapy. D/P Cr levels at 12 or 18 months after the therapy were not aggravated. It appears that the therapy improves Hb levels and cardiac function because of adjusting body fluid status. It was indicated that peritoneal function after therapy may be improved. Therefore, combination therapy is useful from the lifestyle viewpoint of patients in the transition period of PD to HD with end‐stage kidney disease.