Takashi Oite

Epitope-specific induction of mesangial lesions with proteinuria by a MoAb against mesangial cell surface antigen.

A MoAb 1–22–3 (igG3) was produced in mice immunized with rat glomeruli. A blotting study indicated the antigen molecule recognized by this MoAb has a molecular weight of about 25 kD, which is the same as that of the Thy 1.1 molecule. This MoAb is capable of inducing the morphological changes similar to those induced by anti‐thymocyte serum or the anti‐Thy 1.1 MoAb, ER4 and massive proteinuria in rats by a single i.v. injection. Proteinuria started immediately after MoAb 1–22–3 injection and peaked on day 5. Reaction products were detected by immunoelectron microscopy in vitro on the limited mesangiai cell surface facing endothelial cells and in vivo in partially lysed mesangial cells 30 min alter injection. Unlike the proteinuria‐inducing MoAb ER4, reactivity of MoAb 1–22–3 was detected neither on endothelial cells, epithelial cells, nor along the glomerular basement membrane in vivo and in vitro. There is a difference in reactivity toward guinea‐pig and rabbit materials between MoAb 1–22–3 and the commercial anti‐Thy 1.1 MoAb(OX7). The antigenic determinant of MoAb 1–22–3 is concluded to be a new epitope and only the binding of MoAb 1–22–3 to this epitope proved to lead to an abnormal increase of glomerular capillary wall permeability.

Up-Regulation of Connexin43 in Glomerular Podocytes in Response to Injury

Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis. However, it is poorly understood how podocytes respond to injury. In this study, glomerular expression of connexin43 (Cx43) gap junction protein was examined at both protein and transcript levels in an experimental model of podocyte injury, puromycin aminonucleoside (PAN) nephrosis. A striking increase in the number of immunoreactive dots with anti-Cx43 antibody was demonstrated along the glomerular capillary wall in the early to nephrotic stage of PAN nephrosis. The conspicuous change was not detected in the other areas including the mesangium and Bowmans capsule. Immunoelectron microscopy showed that the immunogold particles for Cx43 along the capillary wall were localized predominantly at the cell-cell contact sites of podocytes. Consistently, Western blotting and ribonuclease protection assay revealed a distinct increase of Cx43 protein, phosphorylation, and transcript in glomeruli during PAN nephrosis. The changes were detected by 6 hours after PAN injection. These findings indicate that the increase of Cx43 expression is one of the earliest responses that have ever been reported in podocyte injury. To show the presence of functional gap junctional intercellular communication (GJIC) in podocytes, GJIC was assessed in podocytes in the primary culture by transfer of fluorescent dye, Lucifer yellow, after a single-cell microinjection. Diffusion of the dye into adjacent cells was observed frequently in the cultured podocytes, but scarcely in cultured parietal epithelial cells of Bowmans capsule, which was compatible with their Cx43 staining. Thus, it is concluded that Cx43-mediated GJIC is present between podocytes, suggesting that podocytes may respond to injury as an integrated epithelium on a glomerulus rather than individually as a separate cell.

ATP-Dependent Mechanism for Coordination of Intercellular Ca2+ Signaling and Renin Secretion in Rat Juxtaglomerular Cells

Abstract— A change in intracellular Ca2+ is considered to be the common final signaling pathway through which renin secretion is governed. Therefore, information relating to the generation, control, and processing of Ca2+ signaling in juxtaglomerular cells (JG) will be critical for understanding JG cell behavior. In this study, we investigated the means by which JG cells harmonize their intracellular Ca2+ signals and explored the potential role of these mechanisms in renin secretion. Mechanical stimulation of a single JG cell initiated propagation of an intercellular Ca2+ wave to up to 11.9±4.1 surrounding cells, and this was prevented in the presence of the ATP-degrading enzyme, apyrase (1.7±0.7 cells), or by desensitization of purinergic receptors via pretreatment of cells with ATP (1.8±0.9 cells), thus implicating ATP as a mediator responsible for the propagation of intercellular Ca2+ signaling. Consistent with this, JG cells were demonstrated not to express the gap junction protein connexin43, and neither did they possess functional gap junction communication. Furthermore, massive mechanical stretching of JG cells elicited a 3-fold increase in ATP release. Administration of ATP into isolated perfused rat kidneys induced a rapid, potent, and persistent inhibition of renin secretion, together with a transient elevation of renal vascular resistance. ATP (1 mmol/L) caused up to 79% reduction of the renin secretion activated by lowering the renal perfusion flow (P <0.01). Taken together, our results indicate that under mechanical stimulation, ATP functions as a paracellular mediator to regulate renin secretion, possibly through modulating intra- and intercellular Ca2+ signals.

Effect of High Glucose on Nitric Oxide Production and Endothelial Nitric Oxide Synthase Protein Expression in Human Glomerular Endothelial Cells

Background: Hyperglycemia directly contributes to the development of diabetic nephropathy. Nitric oxide (NO), a potent endothelium-derived vasodilator, has been suggested to participate in the regulation of renal blood flow, glomerular filtration rate, and mesangial matrix accumulation. Human vascular endothelial cells are known to exhibit functional heterogeneity, this prompted us to do the first study of NO bioavailability in human glomerular endothelial cells (HGECs), in response to high glucose exposure. Methods: NO release was examined by detecting nitrite generation by the Griess assay in HGECs exposed to control-level (5.5 mM) and high-level (15, 30 and 60 mM) glucose solutions at various time periods (24, 48 and 72 h) in the presence or absence of L-arginine (1 mM), or superoxide dismutase (SOD) (250 U/ml). In addition, we evaluated the effect of glucose on the expression of endothelial nitric oxide synthase (eNOS) in HGECs by Western blotting. Results: Final levels of nitrite generated in HGECs were reduced significantly, in a time- and concentration-dependent manner, after high glucose exposure. However, Western blot analysis revealed that eNOS protein expression was significantly upregulated at 12 h after exposure to high glucose concentrations (30 mM), reaching a peak at 48 h (twofold increase over baseline levels). The inhibitory effect of high glucose on NO production was restored by the addition of SOD. Addition of L-arginine (1 mM) to external media also reversed the inhibitory effect of high glucose on NO production of HGECs as well. Conclusions: The present study demonstrated that high glucose increased eNOS protein expression, but decreased NO release finally. Decreased NO bioavailability seems to be associated with overproduction of superoxide and L-arginine deficiency. These findings provide an important clue in clarifying the molecular basis of the mechanisms by which elevated glucose leads to an imbalance between NO and superoxide, resulting in impaired endothelial function. In addition, restoration of NO function by both administration of L-arginine and adequate intake of antioxidants suggests a potential supportive treatment for patients with diabetic nephropathy.

Progressive renal lesions induced by administration of monoclonal antibody 1-22-3 to unilaterally nephrectomized rats

A new animal model of progressive glomerulosclerosis was developed by administering a single i.v., injection of MoAb 1–22–3 to unilaterally nephrectomized rats. Renal morphological analysis revealed that glomerular lesions characterized by mesangial cell proliferation and mesangial matrix expansion were induced in about 95% of the glomeruli. Approximately 20% of the glomeruli of the unilaterally nephrectomized rats showed sclerosis or segmental sclerosis by week 6 after MoAb injection and crescent formation was observed in some glomeruli (ca 4%). Cellular infiltration was also noted in some parts of the interstitium. Increased expression of transforming growth factor–beta (TGF‐β) was observed in the unilaterally nephrectomized rats treated with MoAb 1‐22‐3, but we could not demonstrate pathological involvement of platelet‐derived growth factor (PDGF). even though early‐stage mesangial cell proliferation was observed. The mechanism of mesangial cell proliferation in this model remains to be elucidated. The relatively short period of time needed to induce the sclerotic changes is considered to be a great advantage of this model for clarifying the mechanisms involved in the chronic progression of mesangial proliferative glomerulonephritis.

Mesangial sclerotic change with persistent proteinuria in rats after two consecutive injections of monoclonal antibody 1-22-3.

Irreversible mesangial changes with persistenl proteinuria were induced in rats given two consecutive injections 2 weeks apart of a MoAb 1–22–3 to rat mesangial cell. The characteristics of the resulting lesions were investigated and compared with those of the reversible change induced by a single injection. At 24 h after the second injection, mesangiolytic changes similar to those after a single injection were evident. The accumulation of macrophagc‐like cells in glomeruli observed at I week after the first injection was not evident during the experimental period after the second injection. Hypercellularity with the characteristics of intrinsic mesangial cell and increased mesangial matrix were already present I week after the second injection. And mesangial sclerotic change progressed up to 6 months. Deposition of collagen type I and type III and accumulation of collagen fibril at the ullraslructural level were evident in rats 6 months after the second injection. Proteinuria started immediately and continued for more than 6 months after the second injection. The mesangial sclerotic change with persistent proteinuria described here is considered to be a better model for investigating the mechanism of chronic progression of human mesangial proliferative glomerulo‐nephritis.

Quantitative studies of monoclonal antibody 5-1-6-induced proteinuric state in rats

Murine monoclonal antibody (MoAb) 5‐1‐6 was already reported to bind to epithelial cell fool processes and to cause proteinuria in rats. In vivo kinetics of the injected MoAb 5‐1‐6, relationship between the quantity of kidney‐binding antibody and proteinuria. and changes in the amount of antigenic molecule recognized by this MoAb in the proteinuric state were studied. The amount of total kidney‐binding antibody (TK Ab) as determined I h after a 2 mg administration was 50.8 ± 10.4 μg/2 kidneys, and TKAb declined to 1.9 ± 0.4 at day 15. The minimum dose of MoAb required to induce proteinuria was 125 μg as the injected dose. This dose corresponded to 12‐8 μg of TKAb at I h and 0.34 μg of TKAb at day 5. The amount of MoAb 5‐1 ‐6 binding to isolated normal glomeruli was also shown to exceed 147‐7 μg/76000 glomeruli, indicating proteinuria to be induced provided more than 8.7% (= 12.8/147.7) of the critical epitopes is specifically occupied by MoAb. The total amount of MoAb 5‐1‐6 bound to glomeruli in vivo and in vitro was assayed with [125I]‐labelled anti‐mouse IgG. The amount of [125I] anti‐mouse IgG bound to glomeruli was 6.93 ± 0.45 μg/10 000 glomeruli from rat 5 days after this MoAb injection and 26.58 ± 0.66 μg/10000 control glomeruli, indicating the decrease in the number of MoAb 5‐1‐6‐rccognizcd antigen molecules in glomeruli isolated from the rat in proteinuric state induced by this MoAb. Thus, the MoAb 5‐1‐6‐recognized molecule itself may principally function to regulate the permeability of the glomerular capillary wall and the decrease of the molecule may lead to proteinuria.

Coordination of Mesangial Cell Contraction by Gap Junction–Mediated Intercellular Ca2+ Wave

Gap junction intercellular communication (GJIC) plays a fundamental role in mediating intercellular signals and coordinating multicellular behavior in various tissues and organs. Glomerular mesangial cells (MC) are rich in GJ, but the functional associations of these intercellular channels are still unclear. This study examines the potential role of GJ in the transmission of intercellular Ca(2+) signals and in the coordination of MC contraction. First, the presence of GJ protein Cx43 and functional GJIC was confirmed in MC by using immunochemical staining or transfer of Lucifer yellow (LY) after a single cell injection, respectively. Second, mechanical stimulation of a single MC initiated propagation of an intercellular Ca(2+) wave, which was preventable by the GJ inhibitor heptanol but was not altered by pretreatment of MC with ATP or addition of apyrase into the assay system. Third, the phospholipase C (PLC) inhibitor U73122 could largely eliminate the mechanically elicited propagation of intercellular Ca(2+) waves, suggesting a possibly mediating role of inositol trisphosphate (IP(3)) in the initiation and transmission of intercellular Ca(2+) signaling. Fourth, injection of IP(3) into a single cell caused contraction, not only in the targeted cell, but also in the adjacent cells, as indicated by the reduction of cellular planar area. Fifth, addition of two structurally unrelated GJ inhibitors, heptanol and alpha-glycyrrhetinic acid (GA), into MC embedded in collagen gels significantly attenuated the reduction of gel areas after exposure to serum. This study provides the first functional evidence supporting the critical role of GJIC in the synchronization of MC behaviors.

Glomerular angiotensinogen protein is enhanced in pediatric IgA nephropathy

Enhanced intrarenal renin-angiotensin system (RAS) is implicated in the development and progression of renal injury. To investigate whether angiotensinogen (AGT) expression is involved in glomerular RAS activity and glomerular injury, we examined glomerular AGT expression and its correlation with expression of other RAS components, and levels of glomerular injury in samples from patients with immunoglobulin A nephropathy (IgAN) (23) and minor glomerular abnormalities (MGA) (8). Immunohistochemistry showed that AGT protein was highly expressed by glomerular endothelial cells (GEC) and mesangial cells in nephritic glomeruli of IgAN compared with glomeruli of MGA. Levels of glomerular AGT protein were well correlated with levels of glomerular angiotensin II (ang II), transforming growth factor-β (TGF-β), α-smooth-muscle actin, glomerular cell number, and glomerulosclerosis score but not with those of glomerular angiotensin-converting enzyme and ang II type 1 receptor. Real-time polymerase chain reaction (RT-PCR) and Western blot analyses using cultured human GEC indicated that ang II upregulated AGT messenger ribonucleic acid (mRNA) and protein expression in a dose- and time-dependent manner. These data suggest that activated glomerular AGT expression is likely involved in elevated local ang II production and, thereby, may contribute to increased TGF-β production and development of glomerular injury in IgAN. Augmentation of GEC-AGT production with ang II stimulation might drive further glomerular injury in a positive-feedback loop.

Quantitative study of mesangial injury with proteinuria induced by monoclonal antibody 1-22-3

Murine MoAb 1‐22‐3 has already been reported to bind to the mesangial cell surface and to cause transient proteinuria and mesangial morphological changes characterized by mesangiolysis. subsequent mesangial cell proliferation and mesangial matrix increase by a single i.v. injection. In this study, MoAb‐induced glomerulopathy was quantitatively analysed. No correlation between the severity of mesangial morphological changes and the degree of proteinuria was detected (r= 0.190). The minimum dose injected to induce abnormal proteinuria was 25 μg. This dose corresponded to 1.79 μg/2 kidneys 30 min after MoAb injection. The highest average level of proteinuria was observed in rats injected with 500 μg of MoAb, and less proteinuria was observed in rats injected with 10.0, 5.0 and 2.0mg. Although the amounts of kidney‐fixing MoAb and the subsequent deposition of rat C3 in the high‐dose‐injected group were larger than in the 500 ng injected group, the numbers of infiltrating inflammatory cells were the same in both groups. No correlations between the degrees of such mediators and proteinuria were observed.