Itaru Kihara

Molecular cloning of a new aquaporin from rat pancreas and liver.

A new water channel (aquaporin-8, gene symbol AQP8) was isolated from rat pancreas and liver by homology cloning. Ribonuclease protection assay showed intense expression of the gene in pancreas and liver, less intense in colon and salivary gland, and negligible in other organs. The full-length cDNA was obtained by ligation of ∼1.4-kilobase (kb) cDNA isolated from the rat liver cDNA library to ∼0.5 kb of the 5′-end fragment obtained by the rapid amplification of cDNA ends method. A major transcript of ∼1.45 kb was demonstrated in liver and colon by Northern blot analysis. Expression of the cRNA in Xenopusoocytes markedly enhanced osmotic water permeability in a mercury-sensitive manner, indicating a water channel function of this molecule. The open reading frame encoded a 263-amino acid protein with a predicted molecular size of 28 kDa. Hydropathy analysis represented six membrane-spanning domains and five connecting loops containing two sites of NPA motif as preserved in other aquaporins. Unlike other mammalian aquaporins, AQP8 has an unusual structure with a long N terminus and a short C terminus, which are found in plant aquaporin, γ-tonoplast intrinsic protein. By in situ hybridization,AQP8 mRNA expression was assumed in hepatocytes, acinal cells of pancreas and salivary gland, and absorptive colonic epithelial cells. The physiological role(s) of AQP8 remain to be elucidated.

Expression and localization of aquaporins in rat gastrointestinal tract.

A family of water-selective channels, aquaporins (AQP), has been demonstrated in various organs and tissues. However, the localization and expression of the AQP family members in the gastrointestinal tract have not been entirely elucidated. This study aimed to demonstrate the expression and distribution of several types of the AQP family and to speculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1-5 and AQP8 was examined in various portions through the gastrointestinal tract. AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon, and their expression was relatively intense in the small intestine and colon. In contrast, AQP4 mRNA was selectively expressed in the stomach and small intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated cellular localization of these AQP in these portions. AQP1 was localized on endothelial cells of lymphatic vessels in the submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma membranes of stratified squamous epithelial cells in the esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in the colon. However, AQP3 was not apparently detected in the small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland cells in the small intestine. AQP8 mRNA expression was demonstrated in the absorptive columnar epithelial cells of the jejunum and colon by in situ hybridization. These findings may indicate that water crosses the epithelial layer through these water channels, suggesting a possible role of the transcellular route for water intake or outlet in the gastrointestinal tract.

Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells

Aquaporin (AQP) 5 gene was recently isolated from salivary gland and identified as a member of the AQP family. The mRNA expression and localization have been examined in several organs. The present study was focused on elucidation of AQP5 expression and localization in the eye, salivary gland, and lung in rat. RNase protection assay confirmed intense expression of AQP5 mRNA in these organs but negligible expression in other organs. To examine the mRNA expression sites in the eye, several portions were microdissected for total RNA isolation. AQP5 mRNA was enriched in cornea but not in other portions (retina, lens, iris/ciliary body, conjunctiva, or sclera). AQP5 was selectively localized on the surface of corneal epithelium in the eye by immunohistochemistry and immunoelectron microscopy using an affinity-purified anti-AQP5 antibody. AQP5 was also localized on apical membranes of acinar cells in the lacrimal gland and on the microvilli protruding into intracellular secretory canaliculi of the serous salivary gland. In the lung, apical membranes of type I pulmonary epithelial cells were also immunostained with the antibody. These findings suggest a role of AQP5 in water transport to prevent dehydration or to secrete watery products in these tissues.

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.

A hitherto unreported vascular tumor of the kidney: a proposal of "juxtaglomerular cell tumor".

A well encapsulated round tumor was found in the kidney nephrectomized from a young hypertensive woman. The main body of the tumor was composed of small vessels similar in caliber to the glomerular afferent arterioles and polygonal cells derived from them inbetween. These cells contained characteristic granules demonstrable by the Bowies procedure. Histological assumption of a renin‐producing tumor was substantiated by the demonstration of plasma renin activities before and after the surgery. The patients blood pressure returned immediately to normal after nephrectomy. A term of ‘iuxtaglomerular cell tumor’ was proposed to this hitherto unreported tumor. ACTA PATH. JAP. 18: 197–206, 1968.

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.