Kenichiro Kojima

Podocyte Flattening and Disorder of Glomerular Basement Membrane Are Associated with Splitting of Dystroglycan-Matrix Interaction

The transmembrane component of the dystroglycan complex, a heterodimer of alpha- and beta-dystroglycan, was recently localized at the basal cell membrane domain of podocytes, and it was speculated that it serves as a device of the podocyte for maintaining the complex podocyte foot process architecture, and for regulating the exact position of its ligands, the matrix proteins laminin and agrin, in the glomerular basement membrane (GBM). The redistribution of dystroglycan in two experimental rat models of foot process flattening and proteinuria-i.e., podocyte damage induced by polycationic protamine sulfate perfusion, and reactive oxygen species (ROS)-associated puromycin aminonucleoside nephrosis-was examined. In both experimental diseases, aggregation and reduced density of alpha-dystroglycan by endocytosis by podocytes was observed. In in vitro solid-phase binding assays, protamine and ROS competed with the binding of alpha-dystroglycan with purified laminin and a recombinant C-terminal fragment of agrin that contains the dystroglycan-binding domain. These changes were associated with disorder of the fibrillar components of the lamina rara externa of the GBM, as confirmed quantitatively by fractal analysis. These results indicate that both polycation and ROS induce similar changes in the distribution of podocyte alpha-dystroglycan that involve competitive disruption of alpha-dystroglycan/matrix protein complexes, endocytosis of the liberated receptor by podocytes, and disorganization of the matrix protein arrangement in the lamina rara externa. This links functional damage of the dystroglycan complex with structural changes in the GBM.

Protection of α3 integrin-mediated podocyte shape by superoxide dismutase in the puromycin aminonucleoside nephrosis rat

Because reactive oxygen species (ROS) are involved in the development of puromycin aminonucleoside nephrosis (PAN), we examined whether superoxide dismutase (SOD) could ameliorate this condition. Phosphatidyl choline-bound SOD (PC-SOD) has higher affinity for the cell membrane than recombinant human SOD (rhSOD). In this study, PC-SOD had a longer half-life in the circulation and also higher affinity to renal fractions (glomerulus, brush border, and tubulus) than rhSOD. PAN was induced in rats with single injections of puromycin aminonucleoside. Rats were divided into four groups: group P, PAN rats without treatment; group PC-T and group rh-T, PAN rats treated with 30,000 U/kg PC-SOD and rhSOD, respectively; and group C, normal controls. The effect of PC-SOD versus rhSOD on PAN was evaluated by morphological podocyte changes (podocyte density along the GBM) and alpha(3) integrin expression at days 4 and 10. Proteinuria was measured over time until day 14. Distribution and quantitation of alpha(3) integrin were studied by confocal laser scan microscopy. On day 4, glomerular ROS was measured by chemiluminescence without stimulation. PC-SOD decreased proteinuria to the control level, but rhSOD only decreased proteinuria by 31%. PC-SOD significantly improved podocyte density (P < 0.05 versus group P). Total alpha(3) integrin expression decreased in the P and rh-T groups at day 4 and then had recovered by day 10, but the polarity of the site of expression did not recover. PC-T preserved both the amount and polarity of integrin expression on days 4 and 10. PC-SOD significantly suppressed ROS generation in PAN (P < 0.05). These findings suggest that alpha(3) integrin regulates glomerular permeability by maintaining podocyte shape and adhesion, which is disrupted by ROS overproduction.

Defective glycosylation of α-dystroglycan contributes to podocyte flattening

In addition to skeletal muscle and the nervous system, α-dystroglycan is found in the podocyte basal membrane, stabilizing these cells on the glomerular basement membrane. Fukutin, named after the gene responsible for Fukuyama-type congenital muscular dystrophy, is a putative glycosyltransferase required for the post-translational modification of α-dystroglycan. Chimeric mice targeted for both alleles of fukutin develop severe muscular dystrophy; however, these mice do not have proteinuria. Despite the lack of a functional renal defect, we evaluated glomerular structure and found minor abnormalities in the chimeric mice by light microscopy. Electron microscopy revealed flattening of podocyte foot processes, the number of which was significantly lower in the chimeric compared to wild-type mice. A monoclonal antibody against the laminin-binding carbohydrate residues of α-dystroglycan did not detect α-dystroglycan glycosylation in the glomeruli by immunoblotting or immunohistochemistry. In contrast, expression of the core α-dystroglycan protein was preserved. There was no statistical difference in dystroglycan mRNA expression or in the amount of nephrin and α3-integrin protein in the chimeric compared to the wild-type mice as judged by immunohistochemistry and real-time RT-PCR. Thus, our results indicate that appropriate glycosylation of α-dystroglycan has an important role in the maintenance of podocyte architecture.

Source of Reactive Oxygen Species in Anti-Thy1 Nephritis

In proliferative glomerulonephritis, both macrophages and mesangial cells generate reactive oxygen species (ROS), contributing to the development of glomerular injury. We have attempted to determine which cell produces ROS during anti-Thy1 nephritis (ATN) in rats. The generation of ROS was studied using luminol amplified chemiluminescence (GCL) on isolated glomeruli. Immunohistochemical studies used avidin-biotin complex (ABC) to label macrophages and mesangial cells. Immediately after ATN induction, mesangiolysis and infiltration with ED-1 positive cells (referred to as macrophage) was noted with a peak at day 1. After day 4, mesangial proliferation appeared with a decrease of the ED-1 positive cells and a prominent increase of PCNA positive cells (regarded as mesangial cells). In the early phase of ATN, GCL, reflecting ROS generation, increased along with the appearance of ED-1 positive cells. GCL subsequently decreased as mesangial cells increased. This suggested that macrophage were the principal participants in ROS generation in the early phase of ATN although mesangial cells cannot be completely disregarded in the generation of ROS and development of glomerular injury.

Glomerular handling of immune complex in the acute phase of active in situ immune complex glomerulonephritis employing cationized ferritin in rats : Ultrastructural localization of immune complex, complements and inflammatory cells

Abstract The ultrastructural localization of immune complex (IC) and inflammatory mediator systems in the glomerulus was investigated in active in situ IC glomerulonephritis employing cationized ferritin in rats. Glomerulonephritis was induced by unilateral renal perfusion of cationized ferritin as antigen (Ag) in preimmunized rats, and anti-ferritin antibody (Ab), C3 and the rat C5b–9 complex were localized by means of immunogold electron microscopy. Ag–Ab complexes were initially formed subendothelially, associated with C3, and attracted platelets, polymorphonuclear leucocytes (PMN) and monocytes. Then Ag–Ab complexes, without C3, passed across the glomerular basement membrane to re-aggregate subepithelially accompanied by C3 deposition after 1 day. Ag–Ab complexes without C3 accumulated in the inter-podocyte space within 1 day and were seen in the epithelial cells at 6 h. C5b–9 complexes were found in subepithelial immune deposits and in membrane vesicles of the epithelial cells, but only in very small amounts in subendothelial immune deposits. Accumulated platelets, PMN, and monocyte were in direct contact with endothelial cells or subendothelial IC. PMN and monocytes contained Ag, Ab and C3 in intracytoplasmic vacuoles. Ag–Ab complexes were also found in the mesangial matrix adjacent to the subendothelial region after 2 h and increased slightly in number, with expansion of the mesangial area thereafter. Most ICs formed in the subendothelial space rapidly formed lattices of a size that activated C3 and were then translocated to the subepithelial space. The potential ability of C3 to solubilize ICs in the subendothelial region may be important in this process. Endocytosis of subendothelial ICs by PMN and/or monocytes and the movement of ICs to the mesangial matrix may also contribute to the removal of IC from the subendothelial space.

Blood pressure control and satisfaction of hypertensive patients following a switch to combined drugs of an angiotensin receptor blocker and a calcium channel blocker in clinical practice of nephrology

BackgroundCombination drugs containing an angiotensin receptor blocker and a calcium channel blocker have been widely commercialized in recent years, and their advantages, such as improvements in adherence, and reductions in medication costs, have been greatly emphasized. However, the actual situations and the impact of switching to combination drugs in clinical practice of nephrology are not fully understood.MethodsThis study was conducted in outpatients of nephrology who received antihypertensive medicines, and who switched to combination drugs. Changes in the potency of the antihypertensive drugs, and blood pressure were examined retrospectively before and after changing treatments. In addition, the study also involved patients’ questionnaire, which examined changes in blood pressure at home, the presence or absence of missed doses, the impact on medication-related expenses, and the level of patients’ satisfaction with regard to combination drugs.ResultsSurvey results from 90 participants revealed that changing to combination drugs resulted in a reduction of missed doses, a decrease in blood pressure measured in an outpatient setting, and a reduction in medication-related expenses in total patients, non-chronic kidney disease (CKD) patients, and CKD patients.ConclusionOur study shows that switching to combination antihypertensive drugs resulted in an improvement in adherence and a reduction in medication-related expenses, and revealed that patient satisfaction was high. Combination drugs for hypertensive patients may be beneficial in both medical and economical viewpoints.

Do obesity and non-insulin dependent diabetes mellitus aggravate exercise-induced microproteinuria?

We evaluated the role of obesity in proteinuria by treadmill exercising of simple obese subjects and non-obese subjects with non-insulin dependent diabetes mellitus in whom the albumin excretion rate at rest was within normal range. Non-obese healthy volunteers were studied as the controls. The fractional renal clearances of four endogenous proteins, albumin, IgG, IgG4, and beta2-microglobulin were measured before, during, and after treadmill exercise in 17 simple obese and 15 non-obese diabetic subjects, and in 21 normal subjects. Exercise increased the fractional albumin clearance in all groups. In diabetic subjects, the fractional IgG4 clearance also increased: fractional beta2-microglobulin clearance increased in normal controls and in diabetics. In obese subjects, the fractional clearances of albumin, IgG, and IgG4 were similar to those in normal controls, but fractional beta2-microglobulin clearance was significantly lower. These results suggest that enhanced microalbuminuria in obese subjects is probably of glomerular origin. In normal subjects and diabetics, exercise-induced microproteinuria is probably of both glomerular and tubular origin. Defect in the charge-selective barrier of the glomerular capillary wall has been implicated in diabetics. Thus some additional factors relevant to obesity must be taken into account in the consideration of the mechanism of microalbuminuria in diabetics with obesity.

A Rare Adult Case with Diffuse Segmental Membranous Glomerulonephritis

A 71-year-old man with hypertension and diabetes mellitus presented with proteinuria. Laboratory data showed proteinuria of 3.1 g/g creatinine, serum albumin of 3.5 g/dL and serum creatinine of 1.03 mg/dL without autoantibodies. A renal biopsy revealed segmental granular IgG depositions on glomerular capillary walls. Electron microscopy showed segmentally subepithelial, intramembranous and mesangial deposits. Diffuse segmental membranous glomerulonephritis (MGN) was diagnosed with only IgG1 deposition and without M-type phospholipase A2 receptor or thrombospondin type-1 domain-containing 7A staining, suggesting secondary MGN with an unknown target antigen in immune deposits. Physicians should keep in mind the existence of segmental MGN to better understand the clinicopathological characteristics.