Isao Shirato

MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis

Microtubule‐associated protein 1 light chain 3 (LC3) is a unique modifier protein. LC3‐I, the cytosolic form, is modified to LC3‐II, the membrane‐bound form, by a mechanism similar to ubiquitylation by E1‐ and E2‐like enzymes, Apg7p and Apg3p, respectively. In the present study, we found that LC3‐I is processed to LC3‐II during the differentiation and recovery from puromycin aminonucleoside‐induced nephrosis of podocytes. LC3 is especially expressed in the podocytes of rat kidney as the membrane‐bound form LC3‐II. Biochemical analysis using a conditionally immortalized mouse podocyte clone (MPC) revealed that LC3‐I is processed to LC3‐II during the differentiation of cells into mature podocytes and accumulates in the membrane‐rich fraction of the cell lysate. LC3‐II‐localized vesicles, which differ from lysosomes and endosomes, in differentiated MPC cells are morphologically similar to autophagic vacuoles during starvation‐induced autophagy. During starvation‐induced autophagy, autophagosomes fuses with lysosome and LC3‐II on autophagosomes is finally degraded by lysosomal proteases. However, in differentiated MPC cells, little LC3‐II on the vesicles is degraded by lysosomal proteases, suggesting that little LC3‐II‐localized vesicles in differentiated MPC cells fuse with lysosome. Furthermore, the LC3‐II level in differentiated MPC cells increases with recovery from damage caused by experimental puromycin aminonucleoside‐induced nephrosis. These results suggest that LC3‐II‐localized vesicles play an important role in the physiological function of podocytes.

The podocyte's response to stress: the enigma of foot process effacement

Progressive loss of podocytes is the most frequent cause accounting for end-stage renal failure. Podocytes are complex, terminally differentiated cells incapable of replicating. Thus lost podocytes cannot be replaced by proliferation of neighboring undamaged cells. Moreover, podocytes occupy a unique position as epithelial cells, adhering to the glomerular basement membrane (GBM) only by their processes, whereas their cell bodies float within the filtrate in Bowmans space. This exposes podocytes to the danger of being lost by detachment as viable cells from the GBM. Indeed, podocytes are continually excreted as viable cells in the urine, and the rate of excretion dramatically increases in glomerular diseases. Given this situation, it is likely that evolution has developed particular mechanisms whereby podocytes resist cell detachment. Podocytes respond to stress and injury by undergoing tremendous changes in shape. Foot process effacement is the most prominent and, yet in some ways, the most enigmatic of those changes. This review summarizes the various structural responses of podocytes to injury, focusing on foot process effacement and detachment. We raise the hypothesis that foot process effacement represents a protective response of podocytes to escape detachment from the GBM.

ECM Gene Expression and Its Modulation by Insulin in Diabetic Rats

The steady-state levels of mRNA encoding for the α1(IV) collagen chain, laminin B1 and B2 chains, basement membrane HSPG, and α1(I) and a1(III) collagen chains were examined in rat glomeruli at 4, 12, and 24 wk after injection of STZ. The mRNA levels for the α1(IV) collagen chain, laminin B1 and B2 chains, and α1 (I) and α1(I) and α1(III) collagen chains increased significantly with age in the STZ-induced diabetic rats before morphological thickening of basement membrane occurred. In contrast, the mRNA levels for HSPG decreased markedly 4 wk after STZ injection and then increased with age compared with those for control rats. The mRNA levels for these ECM components showed a continuous decline with age in controls. Treating the diabetic rats with insulin for 4 wk ameliorated the abnormally regulated ECM gene expression in the glomeruli. These data suggest that the abnormal regulation of ECM gene expression in the glomeruli may contribute to the expansion of mesangial matrix and basement membrane thickening in diabetic rats, and that hyperglycemia may play a role in the abnormal ECM gene expression.

Hydrogen Peroxide Induces Necrosis, Apoptosis, Oncosis and Apoptotic Oncosis of Mouse Terminal Proximal Straight Tubule Cells

Hydrogen peroxide (H₂O₂) has been shown to be an important mediator of ischemic and toxic tubular damage. The purpose of this study was to identify the mode of cell death observed in H₂O₂-exposed cultures of mouse terminal proximal straight tubule (S₃) cells. H₂O₂ induced a dose- and time-dependent decrease in viability of S₃ cells. Morphologically, S₃ cells exposed to H₂O₂ (0.05–0.1 mM) showed features of necrosis, apoptosis, oncosis and apoptotic oncosis, whereas necrosis occurred most frequently in every experimental condition tested. On the other hand, agarose gel electrophoresis of DNA extracted from S₃ cells exposed to H₂O₂ revealed a typical DNA ladder pattern. These data suggest that H₂O₂-induced proximal tubule damages are associated with the induction of various modes of cell death including necrosis, apoptosis, oncosis and apoptotic oncosis, and with the activation of endonuclease.

Serum Cystatin C Is a More Sensitive Marker of Glomerular Function than Serum Creatinine

We determined the relationship between the levels of serum cystatin C or creatinine (s-Cr) and the grade of creatinine clearance (CCr) in patients with various glomerular diseases. Serum samples from 96 patients with glomerular diseases were obtained from our hospital. The levels of serum cystatin C were measured using the Dade Behring Cystatin C assay with the automated Dade Behring Nephelometer II (BNII). CCr levels were classified into six groups according to the Guidelines of the Japanese Society of Nephrology as follows: grade 1 (normal renal function); grade 2 (slight decrease of renal function); grade 3 (moderate decrease of renal function); grade 4 (severe decrease of renal function); grade 5 (renal failure), and grade 6 (uremia). The mean levels of serum cystatin C in grade 3 patients were significantly higher than those in grade 1. The mean levels of serum cystatin C in grades 4, 5 and 6 patients were also significantly higher than those in grade 1. However, the mean levels of serum Cr in grade 3 patients were not significantly higher than those in grade 1. The levels of s-Cr in grades 4, 5 or 6 patients were significantly higher than those in grade 1. In this study, an increase of serum cystatin C levels occurred earlier than that of s-Cr in various glomerular diseases. It appears that the levels of serum cystatin C may provide early prognostic marker of patients with various glomerular diseases rather than the levels of s-Cr.

The development of focal segmental glomerulosclerosis in Masugi nephritis is based on progressive podocyte damage

We analysed the sequence of structural changes leading to focal segmental glomerulosclerosis (FSGS) in chronic Masugi nephritis. The protocol resulted in an immediate onset of the disease and the development of segmental sclerosis in a considerable proportion of glomeruli within 28 days of serum injection. Throughout the study, the degree of structural damage was significantly correlated with protein excretion. Even 1 day after injection of the serum, the whole spectrum of early lesions was encountered involving all three cell types. Endothelial detachments, mesangiolysis and podocyte foot process effacement were most prominent. There was focal persistence of capillary microthrombosis but, generally, mesangial and endothelial injuries recovered. The development of podocyte lesions was different: on one hand recovery was seen leading to the re-establishment of an interdigitating foot process pattern, and on the other persistent podocyte detachments from peripheral capillaries allowed the attachment of parietal epithelial cells to “naked” portions of the glomerular basement membrane (GBM), and thus to the formation of a tuft adhesion to Bowmans capsule. Progressive podocyte degeneration at the flanks of an adhesion permitted expansion of the adhesion by encroachment of parietal cells onto the tuft along the denuded GBM. Inside an adhesion, capillaries and mesangial areas either collapse or become obstructed by hyalinosis or thrombosis. Resident cells disappear progressively from inside an adhesion; macrophages may invade. Segmental sclerosis in this model consists of collapsed tuft structures adhering broadly to the cortical interstitium. Proliferation of mesangial cells did not contribute to this development. Recovery of endothelial and mesangial lesions was associated with cell proliferation in early stages of the disease; podocyte proliferation was not encountered at any stage. We conclude that the inability to replace an outmatched podocyte crucially underlies the development of sclerosis. Severe podocyte damage cannot be repaired but leads to tuft adhesions to Bowmans capsule followed by progressive collapse of tuft structures inside an adhesion, resulting in segmental glomerulosclerosis.

Detection of Serum IL-6 in Patients with Diabetic Nephropathy

Yasuhiko Tomino. MD, Division of Nephrology, Department of Medicine, Juntentto University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113 (Japan) Dear Sir, A study on the detection of serum IL-6 in patients with non-insulin-dependent diabetes mellitus (NIDDM) with or without nephropathy is described. IL-6 is generally regarded as a multifunctional cytokine which has a variety of biological activities, including the ability to stimulate bone marrow stem cell proliferation, B cell differentiation, immuno-globulin secretion, T cell activation, and acute phase protein synthesis [1, 2], IL-6 is also produced by the renal glomerular mesan-gial cells. Cytokines are known to play an important role in autoimmunity and appear to be involved in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). However, Cavallo et al. [3] reported that detectable levels of serum IL-6 were observed in only 10% of IDDM patients. Serum samples were obtained from 9 patients with NIDDM with nephropathy (diabetic nephropathy), 9 patients with NIDDM without nephropathy and 29 patients with chronic glomerulonephritis (CGN). NIDDM was diagnosed with a 75-gram glucose tolerance test. Patients with diabetic nephropathy continuously showed more than 200 mg/24 h. Serum IL-6 levels were measured with ELISA as described previously [4]. Mouse monoclonal anti-IL-6 antibody (HH61-10) and monoclonal horse radish peroxidase-conjugated anti-IL-6 antibody (HH61-2 Fab’) were used in a double-antibody sandwich ELISA [5]. Levels of serum IL-6 of healthy controls were less than 4.0 pg/ml [5]. The mean levels of serum IL-6 in all patients with NIDDM were significantly higher than those in patients with CGN (p < 0.05). The levels of serum IL-6 in patients with diabetic nephropathy were significantly higher than those in cases of CGN or NIDDM without nephropathy (p < O.Ol and p < 0.05, respectively; table 1). It appears that the presence of IL-6 in the patients’ sera may reflect increased localized production of this cytokine at the pancreatic and/or glomerular me-sangial levels. The measurement in serum IL-6 may add

Blockade of TGF-β Signaling in T Cells Prevents the Development of Experimental Glomerulonephritis

Anti-glomerular basement membrane (GBM) Ab-induced glomerulonephritis (GN) at late stage is thought to be mediated by T cells. However, signaling pathways of T cells that are involved in the development of anti-GBM Ab-induced GN are unclear. We have recently established transgenic mice expressing Smad7, an inhibitor of TGF-β signaling, in mature T cells, where signaling by TGF-β was blocked specifically in T cells. In this study, we showed that anti-GBM Ab-induced GN was suppressed in several measures in the transgenic mice including the severity of glomerular changes, proteinuria, renal function, and CD4 T cell infiltration into the glomeruli without down-regulation of CD62 ligand (CD62L) (L-selectin) expression on CD4 T cells. Furthermore, treatment with the soluble fusion protein of CD62L and IgG enhanced anti-GBM Ab-induced GN. These findings indicated that blockade of TGF-β signaling in T cells prevented the development of anti-GBM Ab-induced GN. Because CD62L on T cells appears to be inhibitory for the development of anti-GBM Ab-induced GN, persistent expression of CD62L on CD4 T cells may explain, at least in part, the suppression of anti-GBM Ab-induced GN in the transgenic mice. Our findings suggest that the development of anti-GBM Ab-induced GN requires TGF-β/Smad signaling in T cells.

Cisplatin-induced apoptosis of immortalized mouse proximal tubule cells is mediated by interleukin-1β converting enzyme (ICE) family of proteases but inhibited by overexpression of Bcl-2

Abstract Cisplatin is known to induce serious renal damage including acute renal failure, the major site of renal injury appears to be localized to the third segment of the proximal tubule (S3). Apoptosis occurs during a variety of acute injuries to tubule cell. The purpose of this study was to determine whether cisplatin induces apoptosis of immortalized mouse S3 cells, and to define the intracellular pathways leading to cell death. S3 cells exposed to cisplatin exhibited biochemical, morphological, and flow cytometric changes characteristic of apoptosis associated with slight necrosis. Cisplatin-induced apoptosis could be inhibited by overexpression of crmA, a cowpox virus gene, of which the product is known to suppress activities of the interleukin-1β converting enzyme (ICE) family proteases. On the other hand, overexpression of bcl-2, an antiapoptotic oncogene, rendered S3 cells partially resistant to cisplatin. These results indicate that cisplatin-induced proximal tubule damage is associated with apoptosis, which is positively modulated by the ICE family of proteases and negatively by the product of bcl-2.

Diffuse Proliferative Glomerulonephritis with Hepatitis C Virus-Like Particles in Paramesangial Dense Deposits in a Patient with Chronic Hepatitis C Virus Hepatitis

A 62-year-old man with hepatitis C virus (HCV) infection developed proliferative glomerulonephritis with IgM and C3 deposits. Electron microscopy showed HCV-like particles in the paramesangial dense deposits, which are similar in shape to HCV previously described. These findings suggest HCV-related proliferative glomerulonephritis.