Yasunori Utsunomiya

Xenobiotic Kidney Organogenesis from Human Mesenchymal Stem Cells Using a Growing Rodent Embryo

Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish a self-organ from autologous stem cells and transplant them as syngrafts back into donor patients. It was reported previously that human mesenchymal stem cells (hMSC) that are cultivated in growing rodent embryos can differentiate within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. As another step toward clinical application, the system was modified for progression to complete functional organogenesis. Rat embryos (E11.5) were isolated from uteri, and bone marrow-derived hMSC, which were transfected adenovirally with glial cell line-derived neurotrophic factor and retrovirally with LacZ, were implanted into the nephrogenic site. Forty-eight hours later, ureteric buds were elongated and initial branching was completed. The metanephroi were dissected out, developed further using in vitro organ culture for 24 h, transplanted into the omentum of a uninephrectomized rat, and grown for 2 wk. They enlarged and exhibited normal kidney structure and ultrastructure. hMSC-derived LacZ-positive cells were identified throughout the regenerated kidney and were morphologically identical to resident renal cells. Transplantation of developing metanephroi into the LacZ transgenic rat revealed that neo-kidney vasculature originated from the host circulation. Finally, fluid was collected from expanded ureters, and urea nitrogen and creatinine were measured. Levels were much higher in these fluids compared with transplanted rat sera (840.3 +/- 184.6 versus 30.4 +/- 10.8 and 10.1 +/- 3.1 versus 0.3 +/- 0.2 mg, respectively), suggesting that the neo-kidney may produce urine. Taken together, these findings suggest that hMSC can differentiate into a mature renal structure with the potential to replace lost kidney function.

Glucocorticoid protects rodent hearts from ischemia/reperfusion injury by activating lipocalin-type prostaglandin D synthase–derived PGD2 biosynthesis

Lipocalin-type prostaglandin D synthase (L-PGDS), which was originally identified as an enzyme responsible for PGD2 biosynthesis in the brain, is highly expressed in the myocardium, including in cardiomyocytes. However, the factors that control expression of the gene encoding L-PGDS and the pathophysiologic role of L-PGDS in cardiomyocytes are poorly understood. In the present study, we demonstrate that glucocorticoids, which act as repressors of prostaglandin biosynthesis in most cell types, upregulated the expression of L-PGDS together with cytosolic calcium-dependent phospholipase A2 and COX2 via the glucocorticoid receptor (GR) in rat cardiomyocytes. Accordingly, PGD2 was the most prominently induced prostaglandin in vivo in mouse hearts and in vitro in cultured rat cardiomyocytes after exposure to GR-selective agonists. In isolated Langendorff-perfused mouse hearts, dexamethasone alleviated ischemia/reperfusion injury. This cardioprotective effect was completely abrogated by either pharmacologic inhibition of COX2 or disruption of the gene encoding L-PGDS. In in vivo ischemia/reperfusion experiments, dexamethasone reduced infarct size in wild-type mice. This cardioprotective effect of dexamethasone was markedly reduced in L-PGDS-deficient mice. In cultured rat cardiomyocytes, PGD2 protected against cell death induced by anoxia/reoxygenation via the D-type prostanoid receptor and the ERK1/2-mediated pathway. Taken together, these results suggest what we believe to be a novel interaction between glucocorticoid-GR signaling and the cardiomyocyte survival pathway mediated by the arachidonic acid cascade.

A multicenter randomized controlled trial of tonsillectomy combined with steroid pulse therapy in patients with immunoglobulin A nephropathy

Background The study aim was, for the first time, to conduct a multicenter randomized controlled trial to evaluate the effect of tonsillectomy in patients with IgA nephropathy (IgAN). Methods Patients with biopsy-proven IgAN, proteinuria and low serum creatinine were randomly allocated to receive tonsillectomy combined with steroid pulses (Group A; n = 33) or steroid pulses alone (Group B; n = 39). The primary end points were urinary protein excretion and the disappearance of proteinuria and/or hematuria. Results During 12 months from baseline, the percentage decrease in urinary protein excretion was significantly larger in Group A than that in Group B (P < 0.05). However, the frequency of the disappearance of proteinuria, hematuria, or both (clinical remission) at 12 months was not statistically different between the groups. Logistic regression analyses revealed the assigned treatment was a significant, independent factor contributing to the disappearance of proteinuria (odds ratio 2.98, 95% CI 1.01–8.83, P = 0.049), but did not identify an independent factor in achieving the disappearance of hematuria or clinical remission. Conclusions The results indicate tonsillectomy combined with steroid pulse therapy has no beneficial effect over steroid pulses alone to attenuate hematuria and to increase the incidence of clinical remission. Although the antiproteinuric effect was significantly greater in combined therapy, the difference was marginal, and its impact on the renal functional outcome remains to be clarified.

Genetically Modified Bone Marrow-Derived Vehicle Cells Site Specifically Deliver an Anti-Inflammatory Cytokine to Inflamed Interstitium of Obstructive Nephropathy

In this study, we used genetically modified bone marrow-derived CD11b+CD18+ vehicle cells to deliver IL-1 receptor antagonist (IL-1ra) for treatment of inflamed renal interstitium in an animal model of unilateral ureteral obstruction (UUO). Vehicle cells that expressed the ICAM-1 ligands, CD11b and CD18, were obtained from bone marrow cells of DBA/2j mice and adenovirally transduced with the IL-1ra gene or glucocerebrosidase (GC) gene ex vivo. In kidneys treated to develop UUO, levels of ICAM-1, IL-1β, and IL-1R expression increased within 3 days compared with contralateral untreated kidneys in the same mice. Similarly, the macrophage infiltration in the cortical interstitium increased after 3 days in UUO kidneys, but not untreated kidneys. After UUO developed, DBA/2j mice were injected i.v. with either IL-1ra+ vehicle cells (IL-1ra-treated mice) or GC+ vehicle cells (GC-treated mice) at 24 h after UUO. Six days after the injection of these vehicle cells, marked increase of CD11b+ IL-1ra+ vehicle cells was observed in the ICAM-1-positive interstitium of UUO kidneys from IL-1ra-treated mice. In contrast, no CD11b+ IL-1ra+ cells appeared in ICAM-1-negative contralateral kidneys from these mice. Furthermore, the infiltration of macrophages (p < 0.001), expression of ICAM-1 (p < 0.005), and presence of α-smooth muscle actin (p = 0.005) in the interstitium of UUO kidneys were significantly decreased in IL-1ra-treated mice compared with GC-treated mice. These findings suggest that IL-1 may contribute to the development of renal interstitial injury and that our method can deliver a functioning gene encoding an antiinflammatory cytokine gene specifically at that site by interacting with local adhesion molecules.

A histologic classification of IgA nephropathy for predicting long-term prognosis: emphasis on end-stage renal disease

BACKGROUND A multicenter case-control study on IgA nephropathy (IgAN) was conducted to develop an evidence-based clinicopathologic classification of IgAN for predicting long-term renal outcome. METHODS Two hundred and eighty-seven patients including those with isolated hematuria or very mild proteinuria were enrolled. During a median follow-up of 9.3 years after biopsy, 49 patients (17%) progressed to end stage renal disease (ESRD). The associations between pathological variables and the need for chronic dialysis was examined by multivariate logistic regression analysis separately in patients who required dialysis earlier than 5 years (Early Progressors) and those who required dialysis within 5 to 10 years (Late Progressors) after biopsy. RESULTS Independent pathological variables predicting progression to ESRD were global sclerosis, segmental sclerosis and fibrous crescents for Early Progressors, and global sclerosis and cellular/fibrocellular crescents for Late Progressors. Four histological grades, HG 1, HG 2, HG 3 and HG 4, were established corresponding to <25%, 25-49%, 50-74% and =75% of glomeruli exhibiting cellular or fibrocellular crescents, global sclerosis, segmental sclerosis or fibrous crescents. Eleven (7%) patients in HG 1, 12 (16%) in HG 2, 13 (31%) in HG 3 and 13 (68%) in HG 4 progressed to ESRD. Multivariate logistic analysis revealed that the risk of progression to ESRD was significantly higher in HG 2, 3 and 4 than in HG 1 (odds ratio, 2.4, 5.7 and 27.6 vs. 1.0). CONCLUSIONS Our evidence-based histologic classification can identify the magnitude of the risk of progression to ESRD and is useful for predicting long-term renal outcome in IgAN.

Prophylaxis of antibody-induced acute glomerulonephritis with genetically modified bone marrow-derived vehicle cells

Glomerulonephritis is an inflammatory disease of the renal glomerulus, which often progresses either slowly or rapidly, ending in renal death despite the availability of various antiinflammatory drugs. Gene therapy may be a promising method of suppressing the progression of glomerulonephritis through the blockage of key inflammatory molecule(s). However, the difficulty of local gene delivery into the glomerulus has made the clinical use of gene therapy difficult. As a solution to this issue, we applied a novel ex vivo technique that may allow site-specific gene delivery into the inflamed site and thus suppress local inflammation in the glomerulus, and examined the feasibility of this system as a prophylaxis of glomerulonephritis. The gene encoding the antiinflammatory cytokine interleukin 1 receptor antagonist (IL-1ra) was delivered into animal models of inflamed glomeruli evoked by anti-glomerular basement membrane antibody; this animal model is an analog of the human Goodpasture syndrome. Vehicle cells did indeed accumulate in the glomeruli on the induction of nephritis and were confirmed to secrete recombinant IL-1ra. Renal functions as well as morphology were preserved by this intervention for up to 14 days after IL-1ra introduction. These data demonstrate the possible application of gene therapy for acute glomerulonephritis. A gene encoding an antiinflammatory molecule, IL-1 receptor antagonist, was delivered into inflamed glomeruli, using a technique that may allow site-specific gene delivery into inflamed tissues. The progression of experimental acute glomerulonephritis was effectively suppressed by this intervention for at least 14 days after gene introduction. This success may strengthen the rationale for gene therapy in the treatment of inflammatory diseases such as glomerulonephritis.

Glomerular Density in Renal Biopsy Specimens Predicts the Long-Term Prognosis of IgA Nephropathy

BACKGROUND AND OBJECTIVES An early histopathologic predictor of the renal prognosis, before the occurrence of advanced glomerular sclerosis/interstitial fibrosis and/or apparent renal dysfunction, remains to be established in IgA nephropathy (IgAN). This study aimed to determine whether the glomerular density (GD; nonsclerotic glomerular number per renal cortical area) of biopsy specimens obtained at an early stage of IgAN could predict the long-term renal outcome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The predictive value of the factors at biopsy, including the GD, on the renal outcome was retrospectively analyzed for 98 patients who had IgAN with an estimated GFR of > or =60 ml/min per 1.73 m(2) at biopsy (87 ml/min per 1.73 m(2) on average). RESULTS The individual value of GD in biopsy ranged from 1.2 to 8.1/mm(2) (i.e., approximately a seven-fold variation), and the GD showed a close inverse correlation with mean glomerular volume. Among the various clinicopathologic factors involved, both a cellular/fibrocellular crescent and the GD were found to be significant predictors of progression in multivariate analyses. A low GD in the biopsy specimens was frequently associated with a steeper slope of the renal function and a synergistically enhanced risk for progression with the presence of cellular/fibrocellular crescent. The renal function, proteinuria, degrees of glomerulosclerosis, and interstitial fibrosis at biopsy were not independent predictors of the prognosis in these patients. CONCLUSIONS A strong predictive relationship of low GD with progression observed in this study suggests that GD may serve as an early histopathologic marker of long-term renal prognosis in IgAN.

Xenotransplanted Embryonic Kidney Provides a Niche for Endogenous Mesenchymal Stem Cell Differentiation Into Erythropoietin-Producing Tissue

Recent findings have demonstrated that stem cells can differentiate into mature tissue when supplied with a niche containing factors identical to those in the normal developmental program. A niche for the development of an organ can be provided by xenotransplantation of a similar developing organ. However, this process has many technical, safety, and ethical concerns. Here, we established xenotransplantation models that control endogenous mesenchymal stem cell (MSC) differentiation into mature erythropoietin (EPO)‐producing tissue in a niche provided by a developing xenometanephros. Transplantation of rat metanephroi into mouse omentum, and similarly pig metanephroi into cat omentum, led to the recruitment of host cells and EPO production. EPO‐expressing cells were not differentiated from integrating vessels because they did not coexpress endothelial markers (Tie‐2 and VE‐cadherin). Instead, EPO‐expressing cells were shown to be derived from circulating host cells, as shown by enhanced green fluorescent protein (EGFP) expression in the grown transplants of chimeric mice bearing bone marrow from a transgenic mouse expressing EGFP under the control of the EPO promoter. These results suggest that donor cell recruitment and differentiation in a xenotransplanted developing organ may be consistent between species. The cells responsible for EPO expression were identified as MSCs by injecting human bone marrow‐derived MSCs and endothelial progenitor cells into NOD/SCID mice. Furthermore, using metanephroi from transgenic ER‐E2F1 suicide‐inducible mice, the xenotissue component could be eliminated, leaving autologous EPO‐producing tissue. Our findings may alleviate adverse effects due to long‐lasting immunosuppression and help mitigate ethical concerns. STEM CELLS2012;30:1228–1235

Significance of mesangial expression of α-smooth muscle actin in the progression of IgA nephropathy

To determine whether phenotypic modulation of mesangial and interstitial cells correlated with the long-term prognosis of IgA nephropathy (IgAN), we analyzed retrospectively 27 patients with IgAN whose creatinine clearance at the time of renal biopsy was normal. The patients were subdivided into two groups according to the course of renal function during follow-up. Thirteen patients maintained normal renal function for more than 15 years (stable group), and 14 progressed to end-stage renal disease (ESRD group). The score of mesangial cell cellularity in the ESRD group was significantly higher than in the stable group. Immunohistochemistry localized alpha-smooth muscle actin (alpha-SMA) in renal mesangial cells of approximately half these patients. Macrophages localized predominantly in the mesangial area in patients with mesangial expression of alpha-SMA, which was associated with the expression of macrophage-colony-stimulating factor. Noteworthily, the score of mesangial alpha-SMA expression and the incidence of patients with mesangial expression of alpha-SMA at the time of renal biopsy were markedly higher in the ESRD group than in the stable group. However, there was no significant difference in both the score of interstitial alpha-SMA expression and the incidence of patients with interstitial expression of alpha-SMA between these two groups. These results suggest that macrophages recruited into the mesangium may induce phenotypic modulation of mesangial cells and that mesangial alpha-SMA expression predicts a progressive decline in renal function in patients with IgAN.

Bmp in Podocytes Is Essential for Normal Glomerular Capillary Formation

Bone morphogenetic protein (BMP) 4 exerts multiple biological effects on kidney and ureter development. To examine the role of BMP4 in glomerular morphogenesis, we generated transgenic mice with altered BMP4 function in podocytes by conferring tissue-specificity with the nephrin (Nphs1) promoter. At birth, Tg(Nphs1-Nog) mice, which had loss of BMP4 function in podocytes, were found to have glomerular microaneurysms, collapsed glomerular capillary tufts, enlarged Bowmans capsules, and fewer normal proximal tubules. Conversely, Tg(Nphs1-Bmp4) mice, which had increased BMP4 function in podocytes, demonstrated defects in glomerular capillary formation, but podocytes were not appreciably affected. The Tg(Nphs1-Nog) and Tg(Nphs1-Bmp4) mice shared morphological characteristics with the previously reported podocyte-specific Vegf-A over-expressing and knockout mice, respectively. Consistent with the morphological similarity, in situ hybridization revealed an intense signal for podocyte expression of Vegf in Tg(Nphs1-Nog) mice, whereas the signal was markedly suppressed in Tg(Nphs1-Bmp4) mice. However, in vitro studies with metanephroi failed to demonstrate a direct interaction between BMP4 or Noggin and VEGF in podocytes. Instead, immunostaining showed that phosphorylated Smads, the mediators of BMP signaling, are present in endothelial and/or mesangial cells, but not in podocytes, within the developing glomeruli. Therefore, this study suggests that podocyte-derived BMP plays an important role in glomerular capillary formation, perhaps by acting on non-podocyte glomerular cells in a paracrine fashion.