Michio Nagata

The Classification of Glomerulonephritis in Systemic Lupus Erythematosus Revisited

The currently used classification reflects our understanding of the pathogenesis of the various forms of lupus nephritis, but clinicopathologic studies have revealed the need for improved categorization and terminology. Based on the 1982 classification published under the auspices of the World Health Organization (WHO) and subsequent clinicopathologic data, we propose that class I and II be used for purely mesangial involvement (I, mesangial immune deposits without mesangial hypercellularity; II, mesangial immune deposits with mesangial hypercellularity); class III for focal glomerulonephritis (involving <50% of total number of glomeruli) with subdivisions for active and sclerotic lesions; class IV for diffuse glomerulonephritis (involving > or =50% of total number of glomeruli) either with segmental (class IV-S) or global (class IV-G) involvement, and also with subdivisions for active and sclerotic lesions; class V for membranous lupus nephritis; and class VI for advanced sclerosing lesions. Combinations of membranous and proliferative glomerulonephritis (i.e., class III and V or class IV and V) should be reported individually in the diagnostic line. The diagnosis should also include entries for any concomitant vascular or tubulointerstitial lesions. One of the main advantages of the current revised classification is that it provides a clear and unequivocal description of the various lesions and classes of lupus nephritis, allowing a better standardization and lending a basis for further clinicopathologic studies. We hope that this revision, which evolved under the auspices of the International Society of Nephrology and the Renal Pathology Society, will contribute to further advancement of the WHO classification.

Abnormal Extracellular Matrix Protein Transport Associated With Increased Apoptosis of Vascular Smooth Muscle Cells in Marfan Syndrome and Bicuspid Aortic Valve Thoracic Aortic Aneurysm

Background—Marfan syndrome (MS) is a genetic disorder caused by a mutation in the fibrillin gene FBN1. Bicuspid aortic valve (BAV) is a congenital heart malformation of unknown cause. Both conditions are associated with ascending aortic aneurysm and premature death. This study examined the relationship among the secretion of extracellular matrix proteins fibrillin, fibronectin, tenascin, and vascular smooth muscle cell (VSMC) apoptosis. The role of matrix metalloproteinase (MMP)-2 in VSMC apoptosis was studied in MS aneurysm. Methods and Results—Aneurysm tissue was obtained from patients undergoing surgery (MS: 4 M, 1 F, age 27–45 years; BAV: 3 M, 2 F, age 28–65 years). Normal aorta from subjects with nonaneurysm disease was also collected (4 M, 1 F, age 23–93 years). MS and BAV aneurysm histology showed areas of cystic medial necrosis (CMN) without inflammatory infiltrate. Immunohistochemical study of cultured MS and BAV VSMC showed intracellular accumulation and reduction of extracellular distribution of fibrillin, fibronectin, and tenascin. Western blot showed no increase in expression of fibrillin, fibronectin, or tenascin in MS or BAV VSMC and increased expression of MMP-2 in MS VSMCs.There was 4-fold increase in loss of cultured VSMC incubated in serum-free medium for 24 hours in both MS (27±8%) and BAV (32±14%) compared with control (7±5%). Conclusions—In MS and BAV there is alteration in both the amount and quality of secreted proteins and an increased degree of VSMC apoptosis. Up-regulation of MMP-2 might play a role in VSMC apoptosis in MS VSMC. The findings suggest the presence of a fundamental cellular abnormality in BAV thoracic aorta, possibly of genetic origin.

Podocyte Injury Promotes Progressive Nephropathy in Zucker Diabetic Fatty Rats

The zucker diabetic fatty (ZDF-fa/fa) rat is one of the attractive models for type II diabetes based on impaired glucose tolerance caused by the inherited insulin-resistance gene fa. Characterization of nephropathy in this model may provide useful insights into the mechanism of the progression of diabetic nephropathy. The present study analyzed the pathophysiology of diabetes and nephropathy, including the process of glomerulosclerosis in this model by biochemical and morphometric analyses. In addition, we conducted studies in podocytes in culture to examine the direct effects of high glucose on podocytes. ZDF-fa/fa rats showed overt diabetes despite hyperinsulinemia as early as 3 months of age. Blood glucose levels increased further with a considerable decrease of insulin levels at 5 months. Glomerular filtration rate (GFR) was significantly elevated until 3 months, but fell to the level seen in lean rats by 7 months. Proteinuria started to rise during the period of increased GFR, and increased further after GFR had fallen to within the normal range. Renal fibronectin, collagen iv, and vascular endothelial growth factor mRNA levels were increased at 7 months. Glomerulosclerosis commenced as early as 5 months of age, and was associated with glomerular hypertrophy and mild mesangial expansion with evidence of accentuated podocyte injury, as revealed by increased expression of desmin. Electron microscopy suggested that degeneration of podocytes and the development of tuft adhesions were responsible for the glomerular sclerosis in this model. In addition, glomeruli from the diabetic rats showed up-regulation of the cyclin kinase inhibitors, p21 and p27. Further studies suggested that the increase in p27 expression was predominantly caused by podocytes, because predominant immunolocalization of p27 in podocytes in diabetic rats and high glucose medium induced cell hypertrophy accompanied by p27 up-regulation in differentiated podocyte cell lines. In conclusion, progressive diabetic nephropathy in ZDF-fa/fa rats is associated with evidence of podocyte injury. High concentrations of ambient glucose induced podocyte hypertrophy and stress in vitro, suggesting that the podocyte is a likely target of the diabetic milieu.

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.

Cell cycle regulation and differentiation in the human podocyte lineage

Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin-dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (approximately 20% of the Ki-67-positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells), cyclin D1 and CKIs were markedly down-regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl-2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans.

MafB Is Essential for Renal Development and F4/80 Expression in Macrophages

ABSTRACT MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. mafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

Increased immunoreactivity of endothelin-1 and endothelin B receptor in human atherosclerotic lesions. A possible role in atherogenesis.

This study was designed to analyze the distribution and localization of endothelin-1 (ET-1) and ET receptors (ET(A) and ET(B)) at different stages of human atherosclerotic lesions by immunohistochemistry. Compared with ET(A) receptors, there was increased immunoreactivity of ET-1 and ET(B) receptor in both unfoamy and foamy macrophages and T lymphocytes in fatty streak and fibrous plaque lesions. In addition, medial SMCs located just beneath the foam cell lesions revealed a higher intensity of ET(B) receptor immunoreactivity than those located beneath the normal-looking intima without foam cells. In fibrous plaques, intimal SMCs near foam cells showed an increased density of ET receptors with predominant ET(B) immunoreactivity. In the areas where SMCs showed ET(B) receptor, ET-1 immunoreactivity was also enhanced. These results suggest that accumulation of foamy macrophages and T lymphocytes may modulate the switching of ET receptor subtypes from ET(A) to ET(B) in vascular SMCs. and that the enhanced ET system mediated by ET(B) receptors may play active roles in the progression of atherosclerosis.

Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.

Loss of mitotic activity and the expression of vimentin in glomerular epithelial cells of developing human kidneys.

Glomerular epithelial cells (GECs) have been considered as post-mitotic cells. In order to establish the stage when GECs stop dividing, the expression of proliferating cell nuclear antigen (PCNA/cyclin) in GECs during glomerulogenesis in human kidneys was studied by the streptavidin-biotin staining technique with monoclonal antibodies. Antibody specific for PCNA/cyclin reacted with almost all the cell nuclei of nephrogenic vesicles, as well as those of S-shaped bodies, the cells of which in the lower limb are progenitors of GECs. However, this reaction was markedly reduced in GECs at the capillary loop stage and entirely disappeared at the maturational stage. In contrast to the expression of PCNA/cyclin, vimentin-specific antibody did not react with nephrogenic vesicles and the lower limb of S-shaped bodies, whereas vimentin was ubiquitously expressed in the GECs at the capillary loop stage, as well as at the maturational stage. Furthermore, these two antigens were not co-expressed in the same glomerulus during glomerulogenesis, as revealed by analysis of serial sections. These results lead to the conclusion that expression of PCNA/cyclin and vimentin during glomerulogenesis is fairly stage-dependent, and GECs rapidly lose their mitotic activity at the capillary loop stage.

Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development.

The cyclin-dependent kinase (CDK) inhibitors p27 Kip1 and p57 Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27–/– mice are substantially different from those of p57–/– mice, suggesting that spatial and temporal expression patterns of p27 Kip1 and p57 Kip2 might be distinct. In this study, the roles of p27 Kip1 and p57 Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27 Kip1 (thymus, spleen,retina, testis and ovary) or only p57 Kip2 (gut, palate,pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27 Kip1 and p57 Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57 Kip2 was expressed in the cortex but not in the medulla, whereas p27 Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57 Kip2 in most tissues was restricted to embryogenesis, expression of p27 Kip1 in many tissues was maintained in adult animals. Double- label immunofluorescence staining with either anti-p27 Kip1 or anti-p57 Kip2 and anti-BrdU revealed that the expression of p27 Kip1 and p57 Kip2 was inversely correlated with cell proliferation, suggesting that p27 Kip1 and p57 Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27 Kip1 or p57 Kip2 , and they suggest that these proteins might play important roles in tissue development.