Toshiyuki Imasawa

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.

Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion.

Mesangial cells maintain normal glomerular function by mediating ECM remodeling and immune complex disposal. We have recently identified megsin, a novel member of the serine protease inhibitor (serpin) superfamily predominantly expressed in the mesangium. While our previous studies suggested a role for megsin in the pathogenesis of human glomerular diseases, its exact biological significance remained unknown. Here we produced two lines of megsin transgenic mice. Overexpression of megsin led to progressive mesangial matrix expansion and an increase in the number of mesangial cells. These glomerular lesions were accompanied by an augmented immune complex deposition, together with Igs and complement. Binding and functional assays in vitro identified plasmin as one biological substrate of megsin and confirmed its activity as a proteinase inhibitor. Transgenic animals exhibiting nephritis as a result of treatment with anti--glomerular basement membrane antiserum showed significantly more persistent expansion of the mesangial ECM than was seen in parental mice. Megsin therefore exerts a biologically relevant influence on mesangial function, and on the mesangial microenvironment, such that simple overexpression of this endogenous serpin engenders elementary mesangial lesions.

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.

Blockade of sphingosine 1-phosphate receptor 2 signaling attenuates streptozotocin-induced apoptosis of pancreatic β-cells

Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that acts through five cognate G protein-coupled receptors (S1P(1)-S1P(5)) and regulates many critical biological processes. Recent studies indicated that S1P at nanomolar concentrations significantly reduces cytokine-induced apoptosis of pancreatic beta-cells in which genes for S1P(1)-S1P(4) are co-expressed. However, the S1P receptor subtype(s) involved in this effect remains to be clarified. In this study, we investigated the potential role of S1P(2) in streptozotocin (STZ)-induced apoptosis of pancreatic beta-cells and progression of diabetes. S1P(2)-deficient (S1P(2)(-/-)) mice displayed a greater survive ability, lower blood glucose levels, and smaller numbers of TUNEL-positive apoptotic beta-cells to administration of a high dose of STZ than wild-type (WT) mice. S1P(2)(-/-) mice showed higher insulin/glucose ratios (an index of relative insulin deficiency) and larger insulin-positive islet areas to administration of a low dose of STZ than WT mice. Moreover, administration of JTE-013, a S1P(2)-specific antagonist, to WT mice ameliorated STZ-induced blood glucose elevation and reduced the incidence of diabetes. Our findings indicate that blockade of S1P(2) signaling attenuates STZ-induced apoptosis of pancreatic beta-cells and decreases the incidence of diabetes.

Macrophage‐colony stimulating factor (M‐CSF) enhances proteinuria and recruitment of macrophages into the glomerulus in experimental murine nephritis

In this study, we examined the effects of macrophage‐colony stimulating factor (M‐CSF) on glomerular macrophages in lipopolysaccharide (LPS)‐induced murine nephritis. Mice injected intraperitoneally with either M‐CSF plus LPS, LPS alone, M‐CSF alone or saline every day for 8 days were examined for the degree of urine albumin excretion and lymphocyte‐function associated antigen‐1‐positive (LFA‐1+) cells in peripheral blood as well as renal pathology. From our results, LPS or M‐CSF combined with LPS emphasized the degree of proteinuria, glomerular deposition of immunoglobulins and mesangial proliferation, associated with accumulation of macrophages in the glomeruli. However, in immunohistological examination of kidneys from these nephritic mice, neither intercellular adhesion molecule‐1 (ICAM‐1), which may play an important role in the recruitment of macrophages into glomeruli, M‐CSF receptor nor the number of LFA‐1+ cells in peripheral blood was enhanced by M‐CSF. On the other hand, M‐CSF alone induced neither proteinuria nor any pathological changes and did not increase the number of glomerular Mac‐1+ cells above that in saline‐treated controls. These results indicate that M‐CSF does not directly cause glomerulonephritis but might participate in accelerating the glomerular inflammatory process by stimulating a potent chemoattractant to recruit monocytes‐macrophages into the glomeruli.

Unbalanced expression of sphingosine 1-phosphate receptors in diabetic nephropathy

Sphingosine 1-phosphate (Sph-1-P) regulates vascular homeostasis through its receptors like S1P1 and S1P2. While S1P1 works to protect vasculature, S1P2 works antagonistically against it. Therefore, the balance of S1P1 and S1P2 determines the regulation of vascular permeability. In diabetic nephropathy, one of the typical pathological changes is endothelial injury possibly as a result of changes in vascular permeability. Therefore, we hypothesized that the balance of S1P1 and S1P2 expression becomes inappropriate in glomeruli of diabetic nephropathy. To verify the hypothesis, five SD rats with diabetes induced by streptozotocin injection and six control rats injected with only the vehicle were analyzed one year after injection. The glomeruli of the diabetic rats exhibited endothelial injuries. The analysis by real-time PCR revealed that the ratio of S1P2/S1P1 mRNA in the renal cortex of the diabetic rats was significantly higher than that in the non-diabetic control group. Immunohistochemistry revealed that S1P1 was expressed by endothelial and mesangial cells, while S1P2 was mainly expressed by mesangial cells in glomeruli. Furthermore, the ratio of the staining intensity of S1P2 to that of S1P1 in the glomeruli was significantly higher in the diabetic rats. The number of cells expressing PDGF-B, which enhances S1P2 expression, was also higher in the glomeruli of the diabetic rats than in the controls. In conclusion, Sph-1-P signals are preferentially transmitted through S1P2, rather than S1P1, in the glomeruli of rats with diabetic nephropathy. Such unbalanced delivery of the Sph-1-P signals might be involved in the pathogenesis of endothelial injuries.

Stem Cells in Renal Biology: Bone Marrow Transplantation for the Treatment of IgA Nephropathy

The pathogenesis of IgA nephropathy (IgAN) is still obscure. In this study, we investigated whether the fundamental pathogenesis of IgAN lies in bone marrow stem cells (BMCs) and whether bone marrow transplantation from normal C57BL/6j (B6) mice can attenuate glomerular lesions in a murine IgAN model (high serum level IgA ddY mouse; HIGA mouse). Mesangial deposits of IgA and C3 and glomerular sclerosis in HIGA recipients of BMCs from B6 mice (B6→HIGA) were decreased as compared with those in HIGA recipients of BMCs from HIGA mice (HIGA→HIGA). Furthermore, the serum levels of IgA and macromolecular IgA were notably lower in B6→HIGA mice than in HIGA→HIGA mice. Of note, bone marrow derived H-2b-positive cells from B6 donors were observed in the glomeruli of H-2b-negative HIGA recipients. Our data suggest that qualitative and quantitative changes of serum IgA are determined at the level of stem cells and that bone marrow transplantation from normal mice may not only replace recipients’ immune cells with donors’ BMCs, but also regenerate glomerular cells in HIGA mice. This approach offers a promising strategy for the treatment of IgAN.

Predicting the outcome of chronic kidney disease by the estimated nephron number: The rationale and design of PRONEP, a prospective, multicenter, observational cohort study

BackgroundThe nephron number is thought to be associated with the outcome of chronic kidney disease (CKD). If the nephron number can be estimated in the clinical setting, it could become a strong tool to predict renal outcome. This study was designed to estimate the nephron number in CKD patients and to establish a method to predict the outcome by using the estimated nephron number.Methods/DesignThe hypothesis of this study is that the estimated nephron number can predict the outcome of a CKD patient. This will be a multicenter, prospective (minimum 3 and maximum 5 years follow-up) study. The subjects will comprise CKD patients aged over 14 years who have undergone a kidney biopsy. From January 2011 to March 2013, we will recruit 600 CKD patients from 10 hospitals belonging to the National Hospital Organization of Japan. The primary parameter for assessment is the composite of total mortality, renal death, cerebro-cardiovascular events, and a 50% reduction in the eGFR. The secondary parameter is the rate of eGFR decline per year. The nephron number will be estimated by the glomerular density in biopsy specimens and the renal cortex volume. This study includes one sub-cohort study to establish the equation to calculate the renal cortex volume. Enrollment will be performed at the time of the kidney biopsy, and the data will consist of a medical interview, ultrasound for measurement of the kidney size, blood or urine test, and the pathological findings of the kidney biopsy. Patients will continue to have medical consultations and receive examinations and/or treatment as usual. The data from the patients will be collected once a year after the kidney biopsy until March 2016. All data using this study are easily obtained in routine clinical practice.DiscussionThis study includes the first trials to estimate the renal cortex volume and nephron number in the general clinical setting. Furthermore, this is the first prospective study to examine whether the nephron number predicts the outcome of CKD patients. The results from this study should provide powerful new tools for nephrologists in routine clinical practice.Trial registrationUMIN-Clinical Trial Registration, UMIN000004784.

Galactosialidosis associated with IgA nephropathy: Morphological study of renal biopsy

Galactosialidosis is an autosomal recessive lysosomal disease associated with a deficiency of β‐galactosidase and neuraminidase. Described herein is the case of a young adult who had been diagnosed with galactosialidosis at 8 years of age. At the age of 30 years, proteinuria and hematuria appeared and the patient underwent a renal biopsy 1 year later. Light microscopy of the kidney sections indicated fine granular contents in the cytoplasm of glomerular endothelial and epithelial cells, arteriolar smooth muscles and proximal tubular epithelial cells on periodic acid silver–methenamin (PAM) stain. Electron microscopy of these cells indicated enlarged, smooth endoplasmic reticulum and lysosomes containing 150 nm‐wide rods with a fine lattice structure at 66 Å periodicity. Moreover, electron‐dense deposits were located in the paramesangial area. Immunofluorescence staining indicated diffuse and global anti‐human IgA and C3‐positive staining as a mesangial pattern. Given these findings this patient was therefore diagnosed with both galactosialidosis and IgA nephropathy. This is the first report to describe light and electron microscopy observations of storage materials in the kidneys in young/adult galactosialidosis.