Katsutoshi Kawasaki

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.

Selective depletion of neutrophils by a monoclonal antibody, RP-3, suppresses dextran sulphate sodium-induced colitis in rats

Administration of dextran sulphate sodium to animals induces acute colitis characterized by infiltration of large numbers of neutrophils into the colonic mucosa, which histologically resembles human active ulcerative colitis. It has been reported that neutrophils and the reactive oxygen metabolites produced by them are involved in the progress of ulcerative colitis. This study was intended to clarify their roles by using this animal model. First, possible sources and species of reactive oxygen metabolites were determined using luminol‐dependent chemiluminescence with addition of enzyme inhibitors and reactive oxygen metabolite scavengers. Next, to examine whether neutrophils and hypochlorous acid derived from them contribute to tissue injury, we administered RP‐3, a monoclonal antibody capable of selectively depleting neutrophils, and taurine, a hypochlorous acid scavenger, to rats treated with dextran sulphate sodium. Addition of azide, taurine, catalase, superoxide dismutase and dimethyl sulphoxide into colonic mucosal scrapings significantly inhibited chemiluminescence production, but allopurinol and indomethacin had no effects. These results suggest that excessive hypochlorous acid, hydrogen peroxide, superoxide anion and hydroxyl radical are generated by the inflamed colonic mucosa. Intraperitoneal injections of RP‐3 significantly suppressed bleeding, tissue myeloperoxidase activity, chemiluminescence production and erosion formation. On the other hand, administration of taurine tended to inhibit bleeding and erosion formation to some extent, although it could not significantly suppress them. These data suggest that neutrophils play an important role in the development of this colitis and that hypochlorous acid might be one of the causes of tissue injury induced by neutrophils.

Immunoelectron Microscopic Demonstration of Thy-1 Antigen on the Surfaces of Mesangial Cells in the Rat Glomerulus

Mesangial cells of F344 rats degenerated and then disappeared within 2 days after the intravenous administration of rabbit antirat thymocyte serum (ATS). Rabbit IgG and rat C3 were identified in the mesangium in the rat glomeruli. To establish the glomerular binding site of ATS administered intravenously into rats, one kidney of each rat given ATS intravenously 12 h earlier was perfused ex vivo through the renal artery with peroxidase-labeled antirabbit IgG followed by sequential glutaraldehyde and diaminobenzidine perfusions to minimize the ultrastructural damage. The other kidney was removed before the perfusion for histologic study to examine the glomerular injury. The rabbit IgG identified by peroxidase-reaction product was present diffusely in the glomerular mesangium when viewed by light microscopy and exclusively on the surfaces of most mesangial cells by electron microscopy. Immunofluorescence microscopy showed rabbit IgG essentially in the mesangium, and electron microscopy revealed the degeneration of mesangial cells in the kidneys that had been removed before the surgical perfusion. However, no histological abnormalities were found in the kidneys from control rats given ATS absorbed with rat thymocytes. The present study showed that the intravenous administration of ATS into rats induced the extensive mesangial cell damage by the binding of ATS to Thy-1 antigens on the mesangial cells.

Limitations of podocyte adaptation for glomerular injury in puromycin aminonucleoside nephrosis

Glomerular synechiae that occurred in nephrotic rats with a single intraperitoneal injection of puromycin aminonucleoside were analyzed by immunohistochemistry, radiolabeled thymidine (PHI‐thymidine) autoradiography, as well as light, electron and immunoelectron microscopy. To discriminate podocytes from parietal epithelial cells (PEC) and monocytes, monoclonal antibodies (mAb) against podocalyxin and ED1 were used. The cell kinetics of glomerular epithelial cells were autoradiographically assessed with isotope labeling procedures before and during nephrosis (co‐labeled), and a mAb against proliferating cell nuclear antigen (PCNA). All the cell types except the podocyte of normal kidneys were labelled with rHI‐thymidine at different rates. Detachment of degenerated podocytes from the outside of the glomerular basement membrane (GBM) is the first step of synechia, and detached sites are confronted by PEC that were hypertro‐phied and frequently radiolabeled. Evidence that podocytes in glomeruli of nephrotic rats can proliferate was shown by the presence of mitoses, rHt]‐thymidine uptake in the co‐labeled experiment, and by PCNA staining, but reepithelialization over bare segments of the GBM with proliferated podocytes is doubtful. It was concluded that glomerular synechia resulted from the limits of podocyte adaptation to glomerular injuries.

Identification of renal podocytes in multiple species: higher vertebrates are vimentin positive/lower vertebrates are desmin positive

Abstract We sought to characterize podocytes in the kidneys of numerous species from amphibians to mammals because of the pivotal function of these cells in renal diseases. For this purpose, intermediate filament (IF) proteins of podocytes were examined by immunofluorescence microscopy using antibodies against vimentin, cytokeratins, and desmin. These staining patterns were then compared to those of parietal cells of Bowman’s capsule and tubular cells of the first portion of the proximal tubule from the same sources. As a result, podocytes from mammals (rat, rabbit, dog, cow, and human) and birds (chicken) showed intense vimentin staining without exception, but rarely staining for cytokeratins or desmin. Parietal cells from all these animals were highly heterogeneous with respect to cytokeratin or vimentin staining. Of the tubular cells, only those from humans and chickens were reactive and then only with anti-cytokeratin antibodies. In the reptiles (Chrysemys scripta elegans, Chinemys reeveri, Elaphe quadrivirgata, and Anolis carolinensis), podocytes and other epithelial cells were positive for cytokeratins. Vimentin staining differed among the species, but was not characteristic for podocytes. Anti-desmin antibody reacted strongly only with podocytes from Anolis. In amphibians (Rana catesbeiana and Xenopus laevis), anti-desmin antibody stained podocytes more intensely than any other cell. Cytokeratin and vimentin staining did not differentiate podocytes from the other cell types. These findings indicate that podocytes are characterized by intense vimentin staining in the higher vertebrates and by desmin staining in the lower vertebrates denoting potentially distinctive physiological functions of IF proteins in podocytes from each of these groups.

Significance of myeloperoxidase in rapidly progressive glomerulonephritis

Antineutrophil cytoplasmic autoantibodies (ANCAs) are found in the sera of patients with systemic vasculitis and crescentic glomerulonephritis. We developed monoclonal antibodies against myeloperoxidase (MPO), which is a major perinuclear-ANCA antigen, and examined the presence of MPO in renal tissue from patients with rapidly progressive glomerulonephritis (RPGN) using these monoclonal antibodies. Myeloperoxidase was found in the glomeruli in four of five cases of perinuclear ANCA-positive RPGN, and the distribution pattern was different from that of immunoglobulin G. In perinuclear ANCA-negative crescentic glomerulonephritis, MPO also was detected in two of three cases. Myeloperoxidase was not detected in the capillary walls or mesangium in other nephropathies, such as minimal change disease, membranous nephropathy, and mesangial proliferative glomerulonephritis, or in normal controls. Myeloperoxidase activity was elevated and inversely correlated with the titer of MPO-ANCAs in the sera of perinuclear ANCA-associated RPGN. These data suggest that MPO plays an important role in the glomerular injury of RPGN.

In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and dermal fibroblasts

SummaryRat glomerular mesangial cells were cultured and their antigens were compared with those of aortic vascular smooth muscle cells and dermal fibroblasts. Glomeruli, aortic, and dermal expiants were cultured for 3 weeks and subcultured in the same conditions. These cultured cells were evaluated by indirect immunofluorescence studies using antibodies against Thy-1 antigen, desmin, and chicken gizzard actin. Most of mesangial cells were positive for Thy-1, desmin, and actin. On the other hand, fibroblasts were negative for desmin, and smooth muscle cells stained Thy-1 scarcely, and were negative for desmin. In the latter two cells, actin-positive fibrils were thinner and fainter than mesangial cells. These results indicated that mesangial cells could be distinguished in vitro from vascular smooth cells and fibroblasts by immunofluorescence microscopy.

Expression profile of extracellular matrix and its regulatory proteins during the process of interstitial fibrosis after anti‐glomerular basement membrane antibody‐induced glomerular sclerosis in Sprague‐Dawley rats

Anti‐glomerular basement membrane (GBM) nephritis in Sprague‐Dawley (SD) rats was characterized by development of marked glomerular sclerosis and tubulointerstitial fibrosis. To elucidate sequential change of the glomerular sclerosis and tubulointerstitial fibrosis, accumulation and mRNA expression of extracellular matrix (ECM) components and transforming growth factor (TGF)‐β were examined in the glomerulus and cortex during the disease course by histology, immunostaining and ribonuclease protection assay. Mild proliferative and degenerative lesions appeared in the glomeruli by day 15 after anti‐GBM antibody binding to GBM and progressed to glomerular sclerotic lesion thereafter. Conversely, interstitial change was first recognized by infiltration of mononuclear cells after day 20, followed by marked accumulation of ECM and tubular degeneration. The interstitial fibrosis was induced without apparent binding of anti‐GBM antibody to tubular basement membrane. Accumulation of fibronectin, collagen type I and type IV was noted in the interstitium by immunofluorescence microscopy in association with enhanced expression of mRNA for these ECM components and their regulatory molecules such as matrix metalloproteinase (MMP2), tissue inhibitor of metalloproteinase (TIMP)‐1 and TGF‐β1 both in glomeruli and cortex. The glomerular expression of these mRNA increased apparently by day 15 and reached a plateau or a peak at day 20. The expression of the same mRNA increased gradually from day 15 to day 29 in the cortex. These observations show that interstitial fibrosis follows glomerular sclerosis after anti‐GBM antibody injection in SD rats, suggesting that at least a part of the mechanism for ECM accumulation in the glomerulus and interstitium is essentially the same in terms of composition of ECM and expression of its regulatory molecules.