Satoshi Hino

Course and outcome of tubulointerstitial nephritis and uveitis syndrome.

We report here the clinical features and outcomes of 10 patients, aged 11 to 21 years (median, 13.0), with idiopathic tubulointerstitial nephritis (TIN) and uveitis syndrome (TINU syndrome). The initial symptoms were visual impairment in 7 patients and prolonged fever, anemia, or asthenia in 4 patients. An increase in urinary beta(2)-microglobulin was noticed at the initial checkup in all patients, including 2 patients who showed the normal ranges of 24-hour protein excretion. Creatinine clearance was decreased in 8 patients. TIN was found simultaneously with ocular symptoms in 7 patients and preceded these symptoms in the remaining 3 patients. Percutaneous renal biopsy indicated tubulointerstitial lesions in varying degrees. The histological grade of TIN was correlated with urinary beta(2)-microglobulin levels. Systemic steroid therapy was performed in 7 patients because of the progression of uveitis. The 10 patients were followed-up for 16 to 94 months (median, 31.0 months). In all patients, creatinine clearance recovered to the normal ranges (>/=70 mL/min/1.73 m(2)) mostly within 1 year. Urinary beta(2)-microglobulin excretion gradually declined but was slightly elevated in 4 patients at the latest checkup. Uveitis recurred in all 10 patients, which did not affect the renal status. Our findings indicate that early referral of patients from ophthalmologists and determination of beta(2)-microglobulin in the urine is helpful for the early discovery of TINU syndrome. In children and adolescents with this syndrome, TIN spontaneously resolves and its long-term prognosis is good, but uveitis often relapses. Systemic steroids may be required for uveitis, but not for TIN.

Clinical and radiological features in four adolescents with nutcracker syndrome

Abstractu2002We describe four adolescents with the nutcracker syndrome. In three patients, the nutcracker syndrome was detected through mass urinary screening; the other patient was diagnosed after a sudden onset of dark urine. All patients underwent magnetic resonance angiography (MRA) for diagnosis of the nutcracker syndrome, which revealed dilatation of the left renal vein ranging between 7.4 and 13 mm at the hilar portion. A renal biopsy, perfomed in three patients, showed no remarkable abnormalities in the glomerulus or tubulointerstitial tissue. The patients complained of physical discomfort, including headache, abdominal pain, fainting, and tachycardia mimicking clinical symptoms of an orthostatic disturbance. However, no chronic systemic diseases were detected in any of the patients after repeated laboratory examinations. An orthostatic disturbance preceded diagnosis in three patients. This report indicates that the nutcracker syndrome may cause serious physical ailments that clinically mimick an orthostatic disturbance. It may be important to identify the nutcracker syndrome among children who manifest non-specific physical complaints. MRA could be a safe and reliable method for diagnosing the nutcracker syndrome.

Urinary transforming growth factor-β in patients with glomerular diseases

Abstract. We measured the urinary levels of active transforming growth factor-β (TGF-β) by enzyme-linked immunosorbent assay in 12 healthy controls and 42 patients with various glomerular diseases, including mesangial proliferative (IgA nephritis, Henoch-Schönlein purpura nephritis, and IgA-negative mesangial proliferative glomerulonephritis) and non-proliferative (minimal change nephrotic syndrome and focal glomerulosclerosis) types. Urinary TGF-β, expressed as a ratio to urinary creatinine (ng/mg creatinine), was elevated in patients with IgA nephritis and focal glomerulosclerosis, and was significantly higher than in patients with other types of glomerular diseases and healthy controls. There was a significant correlation between urinary TGF-β levels and the grade of interstitial fibrosis. Among patients with proliferative-type disease, urinary TGF-β was significantly correlated with the grade of mesangial matrix increase and the magnitude of proteinuria. The relationship between urinary TGF-β levels and the immunostaining intensity of TGF-β in the glomeruli was not significant. These results indicated that urinary TGF-β reflects the grade of interstitial fibrosis in glomerular diseases and also the mesangial matrix increase in proliferative-type glomerulonephritis. Measuring TGF-β levels in the urine might be helpful in monitoring patients with some types of glomerular disease.

Heparin-binding EGF-like growth factor is expressed by mesangial cells and is involved in mesangial proliferation in glomerulonephritis

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a new member of the EGF family, is mitogenic for several types of cells, through binding to cell surface heparan sulphate proteoglycans. This study has attempted to delineate HB‐EGF expression by mesangial cells and to identify its role in experimental and human glomerulonephritis. Rat mesangial cells, cultured in the presence of phorbol acetate, hydrogen peroxide, interleukin‐1β, and tumour necrosis factor‐α, expressed HB‐EGF mRNA. Recombinant HB‐EGF stimulated rat mesangial cells to proliferate and to express types I and III collagen. In the rat anti‐Thy‐1.1 nephritis, glomerular HB‐EGF mRNA was up‐regulated and peaked at days 5–7; its expression at the protein level in the glomerulus was prominent at days 5–10. By immunofluorescence, HB‐EGF was positive predominantly in the mesangial area of renal tissues from 23 of 45 patients with various types of human glomerulonephritis, showing a significant correlation with the grade of mesangial proliferation; there was no staining in tissues from patients with minimal change nephrotic syndrome and normal kidney tissues. These data provide the evidence that HB‐EGF is synthesized and expressed by mesangial cells and stimulates mesangial cell proliferation and collagen synthesis in vitro. HB‐EGF is a potential mediator in mesangial cell proliferation and matrix expansion in experimental and human glomerulonephritis. Copyright

Proto-oncogene expression in human glomerular diseases

The expression of the protein products and mRNA of c‐fos, c‐myc, p53, and c‐raf was examined in normal renal tissues and biopsy specimens from 73 patients with various glomerular diseases. Immunofluorescent staining showed that there were cell nuclei stained for c‐Fos, c‐Myc, and p53, and cytoplasm positive for c‐Raf, in the glomeruli of patients with proliferative types of glomerulonephritis, including IgA nephritis and lupus nephritis, and in patients with focal glomerular sclerosis. Glomerular expression of c‐fos and c‐myc mRNA was detected by in situ hybridization. The number of proto‐oncogene‐positive glomerular cells was significantly higher in lupus nephritis, IgA nephritis, and focal segmental sclerosis, as compared with minimal change nephrotic syndrome and normal specimens. In IgA nephritis, the population of glomerular cells positive for c‐Fos and c‐Myc and the grade of c‐Raf immunoreactivity were significantly correlated with the proportion of proliferating cell nuclear antigen (PCNA)‐positive glomerular cells, with histological grading of mesangial hypercellularity and matrix increase, and with the magnitude of proteinuria. These data indicate that proto‐oncogene expression is associated with mesangial proliferation and matrix expansion in proliferative types of glomerulonephritis and in focal glomerular sclerosis.

Absence of α6(IV) collagen in kidney and skin of X-linked Alport syndrome patients

Abstract. To identify the abnormalities of the type IV collagen α6 chain, α6(IV), in Alport syndrome, we examined renal and skin tissue using rat monoclonal antibodies against non-consensus amino acid sequences of α6(IV). Immunofluorescence of normal human kidney and skin tissue revealed linear α6(IV) staining in the basement membrane (BM) of Bowman’s capsule, in some tubules, and also in the epidermal BM. Renal specimens from five male patients of four families with X-linked Alport syndrome showed no reactivity for α6(IV) in Bowman’s capsules and tubules. In these patients, α1(IV) and α2(IV) were normal, whereas α3(IV), α4(IV), and α5(IV) were absent from the BMs of the kidney. In skin tissue of male patients, neither α5(IV) nor α6(IV) were detected. The epidermal BM of female heterozygotes with X-linked Alport syndrome showed a mosaic staining for α5(IV) and α6(IV). These findings indicate that, in addition to a disturbed α3(IV)-α4(IV)-α5(IV) network, patients with X-linked Alport syndrome have abnormalities in α6(IV) of the renal and epidermal BMs at the protein level.

The Target Antigen of Anti-Tubular Basement Membrane Antibody-Mediated Interstitial Nephritis

Our previous studies showed that 54 kD and 48 kD tubular basement membrane (TBM) proteins were the major form of the target antigen involved in anti-TBM antibody-mediated tubulo-interstitial nephritis in humans. In those studies, we isolated the 54 kD glycoprotein (named gp54) from collagenase-digested bovine TBM. NH2-terminal amino acid sequencing indicated that gp54 represented a newly defined glycoprotein. In this study, we further characterized the target antigen, using mouse monoclonal antibodies to gp54 and polyclonal anti-gp54 peptide antibody. Two monoclonal antibodies (H79 and H80) were established, and they reacted, by immunofluorescence, predominantly with the proximal TBM of humans, rabbits, and Wistar, Sprague-Dawley, and Brown-Norway rats, but not with that of Lewis rats. They were also fixed by blotting intensely to the 54 kD component and weakly to the 48 kD component of collagenase-digested human TBM. In vivo transfer of H79 to Wistar rats showed extensive linear binding of mouse IgG to the TBM and the basal membrane of the small intestine; however, no pathologic changes were seen by light microscopy. The anti-gp54 peptide antibody reacted with both the 54 kD and 48 kD TBM components of human TBM. mRNA was prepared from rabbit kidneys, and fractionated to enrich mRNA encoding the 54 kD and 48 kD peptides. On in vitro translation experiments with the mRNA fraction, the 54 kD and 48 kD peptides were immunoprecipitated with anti-gp54 antibodies. These findings indicate that the 54 kD and 48 kD components are encoded with different mRNA, but that they share the same antigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of the tubular basement membrane antigen associated with human tubulo-interstitial nephritis

The target antigen, a 54‐kD glycoprotein (gp54). reactive with sera from patients with anti‐tubular basement membrane (anti‐TBM) nephritis, was isolated from collagenase‐digested (CD) bovine TBM. The purified gp54 was shown to be non‐collagenous by amino acid analysis, and to be a unique basement membrane component by amino‐terminal sequencing. The nephritogenicity of gp54 was demonstrated by immunizing strain XIII guineapigs with purified gp54, and producing anti‐gp54 antibody and tubulo‐interstitial nephritis. Anti‐gp54 antibody, affinity‐purified from sera of patients with anti‐TBM nephritis, bound by immunoblotting to 54‐kD and, to a lesser extent, 48‐kD components of partially purified human CD‐TBM. Indirect immunofluorescence showed that gp54 was present in the basement membrane of proximal tubules of the kidneys of normal human, cow, rabbit, guineapig and Brown‐Norway rat but not in Lewis rat. Immunoelectron microscopy revealed localization of gp54 along the interstitial side of the TBM and its association with interstitial collagen fibres. These results indicate that gp54 is the nephritogenic antigen involved in tubulo‐interstitial nephritis, and is unique in chemical characteristics and localization in the kidney.

Glucocorticoid-Induced Apoptosis of Rat Mesangial Cells in Culture

BackgroundApoptosis of glomerular mesangial cells is shown in experimental and human glumerulonephritis. But it is unclear whether or not glucocorticoids can induce apoptosis in mesangial cells.MethodsRat mesangial cells in culture were incubated with dexamethazone and methylprednisolone. Apoptosis was evaluated by DNA-specific staining with fluorescent dye (H33258), in situ nick end labeling, gel electrophoresis of extracted DNA, and electron microscopy.ResultsThe proportion of lysed cells and cells positive for nick end labeling increased at a concentration of 0.2 to 5 mmol/L of dexamethazone and methylprednisolone. Chromatin condensation and DNA ladders in those cells were also seen. Actinomycin D, a transcriptional inhibitor, or cycloheximide, a translational inhibitor, partially blocked glucocorticoid-induced apoptosis of rat mesangial cells.ConclusionsGlucocorticoids induced typical apoptosis in rat mesangial cells. These data provide new information on the pharmacologic action of glucocorticoids on mesangial cells.

Promotion of survival and prevention of apoptosis in rat mesangial cells by a membrane-anchored form of heparin-binding EGF-like growth factor

AbstractBackground. We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) was expressed in rat mesangial cells and was involved in the progression of of glomerulonephritis in human and animal models. Soluble HB-EGF promotes cultured rat mesangial cell proliferation and the synthesis of type I and type III collagen mRNA. However, the precise role of proHB-EGF in mesangial cell function has not yet been clarified. In the present study, we address this question.nMethods. ProHB-EGF-transfected rat mesangial cells (MsC; MsCproHB-EGF) and empty vector-transfected rat MsC (MsCvector) were constructed. Cells were cultured in fetal calf serum (FCS)-containing (10%) or FCS-deficient medium, and the growth rates and numbers of surviving cells were determined. To elucidate the anti-apoptotic effect of proHB-EGF, cells were exposed to hydrogen peroxide (H2O2) or dexamethasone (DEX). Apoptosis was identified by qualitative and quantitative analysis. Expression of Bcl-2 protein in MsCproHB-EGF after exposure to apoptosis inducers was also evaluated.nResults. When cells were plated in a medium containing 10% FCS, the growth rate of MsCproHB-EGF was not different from that of MsC transfected with a plasmid alone (MsCvector). When cultured in the absence of FCS, MsCvector showed decreased cell numbers, indicating apoptotic cell death. In contrast, MsCproHB-EGF formed small colonies, although they did not show increased cell numbers, while cell viability was 90%–100% of the initial cell number after subculture. When quiescent MsC were exposed to DEX or H2O2, MsCvector exhibited significant DNA laddering, whereas MsCproHB-EGF showed resistance to these stimuli. The release of caspase-3 from MsCproHB-EGF after exposure to DEX or H2O2 was significantly lower than that from MsCvector. Bcl-2 protein expression was weak in MsCproHB-EGF at the baseline, but this expression was significantly upregulated after exposure to these stimuli. Confluent MsCproHB-EGF spontaneously expressed high level of p21 protein. Northern blot analysis revealed that MsCproHB-EGF expressed increased levels of type I and type III collagen mRNA and proteins compared with those of MsCvector.nConclusions. These results indicate that proHB-EGF contributes to mesangial cell survival by promoting cell viability and by inhibiting apoptosis. The anti-apoptotic effect of proHB-EGF could be closely related to the upregulation of Bcl-2 and p21 expression.