Toshio Yanagihara

Urinary Podocytes in Primary Focal Segmental Glomerulosclerosis

Background/Aim: Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome. Although the pathogenesis is not known, recent studies suggest that FSGS may be a podocyte disease. The aim of this study was to look for podocyte injury in this disease, using measurements of urinary podocytes. Methods: We examined the first morning urine of the day collected from 71 patients (45 men and 26 women, median age and range 11.2 and 3–29 years) diagnosed as having nephrotic syndrome. Freshly voided urine samples were examined by immunofluorescence labeling using monoclonal antibodies against human podocalyxin. Renal histological examinations were performed in 58 of the 71 patients: 28 had minimal-change disease, 20 had FSGS, and 10 had membranous nephropathy. Results: Median and range of urinary podocytes measured were 0.2 and 0–40.8 cells/ml for 71 patients with nephrotic syndrome and 0 and 0–0.8 cells/ml for normal healthy control subjects (n = 200). Patients with FSGS had significantly higher levels of urinary podocytes (median and range 1.3 and 0–40.8 cells/ml) than those with minimal-change disease (median and range 0 and 0–6.9 cells/m; p = 0.003) or membranous nephropathy (median and range 0 and 0–1.4 cells/ml; p = 0.02). Conclusions: The urinary excretion of podocytes is significantly higher in patients with FSGS as compared with those having membranous nephropathy or minimal-change disease. These findings suggest that podocyte injury and loss in the urine may have an important role in the pathogenesis of FSGS.

Apical cell membranes are shed into urine from injured podocytes: a novel phenomenon of podocyte injury.

Previously it was shown that urine from patients with nephritis contains podocytes and their fragments (podocalyxin [PCX]-positive granular structures [PPGS]), reflecting the degree of podocyte injury. The present study was designed to trace PPGS to their origin. Urine samples and renal biopsy specimens from 53 children with nephrotic syndrome and nephritis were examined immunohistochemically. Immunofluorescence studies of kidney sections using an anti-PCX antibody demonstrated that PPGS originated from the glomerulus and flowed into the tubular lumen. Electron microscopic examination revealed that PPGS originated from microvillous or vesicle-like structures on injured podocytes in the glomerulus. For examining the origin of the PPGS, apical, slit-diaphragmatic, and basal portions of the podocytes were specifically stained, revealing that PPGS are composed primarily of apical podocyte membranes. Several newly developed antibodies that are reactive with various segments of the PCX molecule were used to analyze more detailed membrane structures, and it was found that PPGS contained intact PCX molecules, indicating that cell membrane structures are excreted in urine. The quantification of PCX content and podocyte numbers revealed that urinary sediment PCX (u-sed-PCX) content per urinary podocyte was much higher than PCX content per podocyte from isolated glomeruli of normal controls, suggesting that u-sed-PCX are derived from sources other than just the cell debris of detached podocytes. Analysis of the correlation between u-sed-PCX and renal histology revealed that the presence of PPGS reflects acute glomerular injury. In conclusion, podocyte apical cell membranes are shed into the urine from injured podocytes, indicating a previously unrecognized manifestation of podocyte injury.

Urinary Excretion of Podocytes Reflects Disease Activity in Children with Glomerulonephritis

The significance of the presence of podocytes in the urine was studied in various renal diseases in children. The podocytes were detected by immunofluorescence using monoclonal antibodies against the podocalyxin that is present on the surface of podocytes which serves as a glycocalyx. They were scored according to the numbers per partitioned area on cytospun urine sediments. Urine podocytes were absent in normal control, nonglomerular diseases such as urinary tract infection and nonglomerular hematuria, and glomerular, noninflammatory diseases such as minimal change nephrotic syndrome and membranous nephropathy. Conversely, the excretion of podocytes in the urine were detected in various glomerular, inflammatory diseases. A significantly higher level of the podocyte score was found in the acute state of glomerular diseases which was defined as within 6 months after disease onset. Positive correlations were obtained between the presence of urinary podocytes and the histological features of active extracapillary changes and mesangial proliferation. Urinary podocytes were examined monthly for 12 months in 7 cases with IgA nephropathy and 2 cases with Henoch-Schönlein purpura nephritis, and a consistently higher urinary podocyte score was observed in the patients with histological progression. The scoring of urinary podocytes was found to be useful clinically, as a diagnostic tool for glomerular or nonglomerular diseases, inflammatory or noninflammatory diseases, a marker for the estimation of the severity of active glomerular injury and also as a predictor of disease progression.

Cumulative Excretion of Urinary Podocytes Reflects Disease Progression in IgA Nephropathy and Schönlein-Henoch Purpura Nephritis

Recent studies have revealed that podocytopenia leads to glomerular scarring and that the loss of podocytes into the urine may be a cause of podocytopenia. The purpose of this study was to examine whether serial examinations of urinary podocytes (u-podo) could be a useful predictor of disease progression in children with glomerulonephritis. Urine samples and renal biopsy specimens from 20 patients (10 males and 10 females; mean age 11.8 yr; range 4 to 24 yr) with IgA nephropathy (n = 17) and Henoch-Schönlein purpura nephritis (n = 3) were analyzed. Forty-four renal biopsies were performed on 20 patients. Proteinuria (g/d per 1.73 m2), hematuria (score), and u-podo (cells/ml) were examined twice a month in 24 intervals between two biopsies (mean 16.7 mo; range 4 to 58 mo) and average and cumulative values were determined for the intervals. Renal histologic changes were scored on the basis of acute intracapillary, acute extracapillary, acute tubulointerstitial, chronic intracapillary, chronic extracapillary, and chronic tubulointerstitial lesions, as well as glomerulosclerosis. It was found that hematuria, proteinuria, u-podo, and acute lesion scores decreased during the intervals examined, whereas chronic lesion scores increased. Changes in acute histology scores correlated well with hematuria, proteinuria, and u-podo excretion, whereas chronic histology scores and glomerulosclerosis both correlated well with cumulative u-podo excretion. Patients with severe histologic progression of disease also had persistent u-podo excretion. These findings provide additional data to support a potential causative role for prolonged urinary loss of podocytes in disease progression in children with IgA nephropathy and Henoch-Schönlein purpura nephritis.

Urinary Excretion of Podocalyxin Indicates Glomerular Epithelial Cell Injuries in Glomerulonephritis

Immunofluorescent study of urine sediments was performed to detect glomerular epithelial cell injuries using specific monoclonal antibody against podocalyxin, a glycoprotein that is prominently expressed on glomerular epithelial cells. Three kinds of structures (casts, granules and cells) were stained in urine sediments from various glomerular diseases. The presence of podocalyxin in urines was confirmed by absorption test, immunoelectron microscopy and Western blotting. Podocalyxin was detected in the urinary sediments of patients with various forms of glomerulonephritis, particularly those with acute onset. The amount of their urinary excretion apparently indicates the degree of the glomerular epithelial cell injuries in glomerular diseases.

Podocyte membrane vesicles in urine originate from tip vesiculation of podocyte microvilli

Podocyte injury is involved in both the onset and progression of glomerular diseases. Our previous studies revealed that apical cell membranes of podocyte are shed into urine sediment and that urinary podocalyxin is a useful biomarker of podocyte injury. In this study, we examined the origin of urinary podocalyxin. Urine samples and kidney specimens from healthy children (n = 126) and patients with glomerular diseases (n = 77) were analyzed by immunohistologic methods. Immunofluorescence studies demonstrated that urinary podocalyxin was shed as granular structures into both the urine sediment and supernatant. Large amounts of podocalyxin were shed into both the urine sediment (17.2 +/- 3.2 ng/mg creatinine) and the supernatant (172.6 +/- 24.6 ng/mg creatinine) of patients, compared with the small amounts of urinary podocalyxin in healthy controls (sediment, 0.5 +/- 0.1 ng/mg creatinine; supernatant, 24.3 +/- 3.5 ng/mg creatinine). Electron and immunoelectron microscopic examinations showed that podocalyxin-positive vesicles in the sediment (125.6 +/- 8.8 nm) and the supernatant (121.2 +/- 6.4 nm) were similar in size to podocyte microvilli in biopsy specimens (123.6 +/- 8.9 nm), differentiating them from the much smaller urine exosomes (30-80 nm in diameter). Urine podocalyxin-positive vesicles tested negative in immunofluorescence microscopy on both exosomal markers CD24 and CD63. Podocalyxin-positive vesicles also tested negative for cytoskeletal markers, and electron microscopic examination revealed tip vesiculation of microvilli. We conclude that human urinary apical cell membrane vesicles appear to originate not from podocyte exosomes but from tip vesiculation of glomerular podocyte microvilli.

Immunohistochemical and urinary markers of podocyte injury

Abstract. Renal podocytes play an important role in glomerular filtration. We estimated podocyte injury by light microscopic examination of kidney specimens or by urinary excretion of substances related to podocytes. Fresh kidney sections from 69 patients and urinary sediments from 84 patients with various renal diseases were examined immunohistochemically using fluorescent labelling. Four kinds of monoclonal antibodies which recognize podocytes were used: (1) anti-podocalyxin (anti-PCX) antibody, (2) anti-C3b receptor (anti-C3bR) antibody, (3) anti-podocyte protein, 44 kilodalton, (anti-pp44) antibody, and (4) anti-alpha 3 integrin (anti-Intα3) antibody. Labelling of kidney sections by anti-C3bR (k-C3bR) was reduced in cases of severe glomerular injury, although there were no changes in k-PCX, k-pp44, or k-Intα3 labelling. PCX labelling of urinary sediments (u-PCX) was detected in nearly all cases of glomerulonephritis, u-C3bR was seen in some cases of glomerular injury, u-Intα3 was seen in only a small number of cases of severe glomerular injury, and u-pp44 was not detected in any urinary samples. u-PCX, u-C3bR, u-Intα3, and k-C3bR correlated with the degree of pathological change, the degree of proteinuria and hematuria. These results suggest that immunostaining kidney sections with anti-C3bR antibody and urinary sediments with anti-PCX, anti-C3bR, and anti-Intα3 antibodies might be useful in detecting podocyte injury.

Origin of hyperplastic epithelial cells in idiopathic collapsing glomerulopathy

Glomerular epithelial cell hypertrophy and hyperplasia are listed as the primary criteria for the diagnosis of collapsing glomerulopathy (CG), a distinct variant of focal segmental glomerulosclerosis. However, the extent of podocyte phenotypic alterations that occur in CG, and the origin of the hyperplastic epithelial cells remain to be established.

Podocalyxin on the glomerular epithelial cells is preserved well in various glomerular diseases.

Podocalyxin on the Glomerular Epithelial Cells Is Preserved Well in Various Glomerular Diseases M. Masanori Hara T. Toshio Yanagihara T. Tsuneo Takada M. Michio Itoh Y. Yuichi Adachi A. Akira Yoshizumi K. Katsutoshi Kawasaki T. Tadashi Yamamoto I. Itaru Kihara Department of Pediatrics, Yoshida Hospital, andDepartment of Pathology, Institute of Nephrology, School of Medicine, Niigata University, Niigata, Japan

Influence of Prolonged Corticosteroid Therapy on the Outcome of Steroid-Responsive Nephrotic Syndrome

Eighty-six patients (59 males and 27 females) diagnosed with steroid-responsive nephrotic syndrome during childhood were identified. The patients were 20–40 years of age (mean 27.0 ± 5.0) with a mean follow-up period of 19.5 ± 5.9 years. All patients had been treated with a long-term tapering corticosteroid therapy. Thirty patients had also received a course of cyclophosphamide (2 mg/kg/day for 12 weeks). Sixty-six had achieved sustained remission off corticosteroids, while 20 were still receiving corticosteroids to maintain remission. None of the 86 patients had proteinuria or renal insufficiency at the time of the study. Mean final heights in males and females were similar (–0.51 ± 1.21 and –0.23 ± 1.16 standard deviation score). Mean final height of 20 steroid-dependent patients was significantly less than that of 66 in remission off corticosteroids (p < 0.005). Ten cyclophosphamide-treated patients got married and 9 had at least 1 healthy child. In children with steroid-responsive nephrotic syndrome, the need for corticosteroid therapy to maintain remission may be associated with decreased adult height. Patients who received a 12-week course of cyclophosphamide are likely to be normally fertile as adults.