Deterioration of glomerular endothelial surface layer induced by oxidative stress is implicated in altered permeability of macromolecules in Zucker fatty rats.

AIMS/HYPOTHESIS: The glomerular endothelial layer is coated by the endothelial surface layer (ESL), which is suggested to play a role in regulation of the permselectivity of macromolecules. Production of heparanase, a degrading enzyme of the ESL, is induced by reactive oxygen species (ROS). We hypothesised that oxidative stress could cause deterioration of the glomerular ESL by induction of heparanase, resulting in increased glomerular permeability. METHODS: Male Zucker fatty (ZF) rats with albuminuria and Zucker lean (ZL) rats were used in this study. Some of the ZF rats were treated with the angiotensin II receptor blocker, irbesartan. We determined the amount of ESL by wheat germ agglutinin staining and heparan sulphate proteoglycan production by western blot analysis. Glomerular hyperfiltration of macromolecules was visualised using in vivo microscopy. We used 2',7'-dichlorofluorescein diacetate-derived chemiluminescence staining to assess ROS production, and heparanase production and expression were determined by western blot analysis and quantitative real-time polymerase chain reaction respectively. RESULTS: By 18 weeks of age, ZF rats had developed albuminuria. The glomerular endothelial cell glycocalyx was significantly decreased in ZF compared with ZL rats. Glomerular filtration and the permeability of macromolecules were increased in ZF, but not in ZL rats. Glomerular ROS and heparanase production were significantly increased in ZF compared with ZL rats. These changes in ZF rats were reversed by irbesartan treatment. CONCLUSIONS/INTERPRETATION: Increased oxidative stress induces glomerular ESL deterioration in part through increased heparanase levels, resulting in exacerbation of glomerular permselectivity and development of albuminuria.

Histopathological manifestations of membranoproliferative glomerulonephritis and glomerular expression of plasmalemmal vesicle-associated protein-1 in a patient with polycythemia vera.

Only a few cases of various glomerulonephropathies have been reported in patients with polycythemia vera. We report the case of a 72-year-old female with polycythemia vera in whom renal biopsy examination showed membranoproliferative glomerulonephritis (MPGN)-like lesion and glomerular expression of plasmalemmal vesicle-associated protein-1 (PV-1), a marker of glomerular capillary remodeling after injury. Prior to admission to our hospital for nephrotic syndrome, she had received hydroxyurea and phlebotomy. On admission, she was hypertensive with pretibial edema, hepatosplenomegaly, massive proteinuria (6.14 g/day), low serum albumin (2.9 g/dl), high fibrinogen, fibrin/fibrinogen degradation products and thrombomodulin levels, but with normal serum creatinine and complement levels. Microscopic examination of a renal biopsy demonstrated MPGN-like features with double contour and mesangial interposition. Electron microscopy showed subendothelial deposits, platelets attached to glomerular capillary walls and fibrin deposition. Immunofluorescence study identified IgM deposition along part of the capillary wall and mesangium. CD42b-positive platelets and megakaryocytes were detected in glomerular capillaries, accompanied with increased expression of platelet-derived growth factor receptor b and thrombomodulin in the mesangium and glomerular capillary, respectively. PV-1 was expressed along the glomerular capillary. Anti-platelet and anticoagulant combination therapy, together with the use of anti-hypertensive agents and hydroxyurea, resulted in improvement of the nephrotic syndrome. The findings suggested that activated platelets, enhanced coagulation state and endothelial damage may contribute to glomerulonephropathy associated with polycythemia vera.

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.

Restricted nutrition-induced low birth weight, low number of nephrons and glomerular mesangium injury in Japanese quail.

Insufficient nutrition during the perinatal period causes structural alterations in humans and experimental animals, leading to increased vulnerability to diseases in later life. Japanese quail, Coturnix japonica, in which partial (8-10%) egg white was withdrawn (EwW) from eggs before incubation had lower birth weights than controls (CTs). EwW birds also had reduced hatching rates, smaller glomeruli and lower embryo weight. In EwW embryos, the surface condensate area containing mesenchymal cells was larger, suggesting that delayed but active nephrogenesis takes place. In mature EwW quail, the number of glomeruli in the cortical region (mm2) was significantly lower (CT 34.7±1.4, EwW 21.0±1.2); capillary loops showed focal ballooning, and mesangial areas were distinctly expanded. Immunoreactive cell junction proteins, N-cadherin and podocin, and slit diaphragms were clearly seen. With aging, the mesangial area and glomerular size continued to increase and were significantly larger in EwW quail, suggesting compensatory hypertrophy. Furthermore, apoptosis measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis was higher in EwWs than in CTs on embryonic day 15 and postnatal day 4 (D4). Similarly, plasma glucocorticoid (corticosterone) was higher (P<0.01) on D4 in EwW quail. These results suggest that although nephrogenic activity is high in low-nutrition quail during the perinatal period, delayed development and increased apoptosis may result in a lower number of mature nephrons. Damaged or incompletely mature mesangium may trigger glomerular injury, leading in later life to nephrosclerosis. The present study shows that birds serve as a model for 'fetal programming,' which appears to have evolved phylogenetically early.

A nosocomial parvovirus B19 infection-induced transient aplastic crisis in a patient with chronic renal failure.

A male patient aged 67 years with chronic renal failure (CRF), who had undergone hemodialysis since June 3, complained of dyspnea while walking on June 23, 1998. Rapidly progressive anemia and severe reticulocytopenia were noted. Serological tests showed that parvovirus B19- (B19) specific IgM antibody, but not IgG antibody, was present in the patient's serum. B19 DNA was detected in the patient's serum by the polymerase chain reaction (PCR). Therefore, a definite diagnosis of transient aplastic crisis induced by B19 was made. On June 10, prior to the appearance of this case, a female nurse aged 27 years working in our hemodialysis center, complained of cough, fever and arthralgia. Another female nurse, aged 35 years, developed similar symptoms on July 3. Both nurses had a positive IgM titer against B19, but were negative for IgG, indicating an acute B19 infection. These findings led us to suspect that this series of B19 infection was spread by nosocomial transmission. Although some cases of B19 infection have been reported to occur in laboratory staffs, the B19 nosocomial infection has not been described in the literature. We also suggest that a transient aplastic crisis due to B19 infection could lead to severe anemia in cases of CRF whose erythropoiesis is maintained by a recombinant human erythropoietin.

Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes.

The phenotypic change of the mesangial cell is considered to play a pivotal role in the accumulation of extracellular matrix in diabetic nephropathy. This investigation was undertaken to evaluate the expression of the various isoforms of contractile proteins in the streptozocin (STZ)-induced diabetic rat kidney and in renal biopsy specimens from patients with diabetic nephropathy. Specific antibodies to myosin heavy chain isoforms (SM1, SM2, SMemb), caldesmon, and alpha-smooth muscle actin and cDNAs for SMemb were used. Increased expression of SMemb at the mRNA and protein levels was demonstrated at 1 week after STZ administration in the rat. Both levels were increased at 4 weeks. Mesangial staining of caldesmon was observed at 4 weeks and that of alpha-smooth muscle actin at 24 weeks. Immunohistochemical mesangial staining of the contractile proteins was pronounced in patients with diabetic nephropathy in contrast to the trace mesangial staining in normal control subjects. These results indicate that the phenotypic change in mesangial cells occurs in the early stages of diabetes and that several stages in phenotypic changes may exist. Expression of the contractile protein isoforms, especially SMemb, should serve as a new marker for the subsequent glomerular hypertrophy and sclerosis.

Influence of hypophysectomy on renal proteoglycans and their modulation by insulin-like growth factor-I and its receptor.

Hypophysectomy leads to growth retardation of the animals, which is believed to be related to the deficiency of certain growth factors influencing the metabolism and synthesis of glycoproteins. Conceivably, the extracellular matrix proteins (ECM) are also affected, in particular the sulfated proteoglycans (PGs). In this study, we investigated the status of the ECM proteins and sulfated PGs, and the expression and de novo synthesis of insulin-like growth factor-I (IGF-I) receptor (IGF-IR) in hypophysectomized (Hx) rats. The studies were extended to ascertain the effect of IGF-I on the de novo synthesis of glomerular ECM glycoproteins in Hx rats. An organ perfusion system was used in which isolated rat kidneys were radiolabeled with either [35S]sulfate or [35S]methionine, after which IGF-IR and ECM macromolecules were isolated and characterized by biochemical and tissue autoradiographic procedures. Hypophysectomy resulted in a fall in IGF-I levels in serum and isolated renal glomeruli. Reduced synthesis of ECM proteins, i.e. type IV collagen, laminin, and sulfated PGs, and reduced synthesis and mRNA expression of IGF-IR were observed in the glomeruli of the Hx rats. Tissue autoradiographic studies revealed a reduced grain density (concentration of radiation) over various cell types of the glomerulus. After inclusion of IGF-I in the perfusion medium not only was synthesis restored to normal in Hx rats, but it far exceeded the control basal values in the intact animals. Under the influence of IGF-I, the magnitude of increased synthesis of the ECM proteins, in particular the sulfated PGs, was highly accentuated in the kidneys of the Hx group compared to the controls. Also, a remarkably increased [35S]sulfate incorporation was observed in the glomerular mesangial cells. The analysis of IGF-IR by specific binding studies revealed a decreased concentration of the receptors, but an increased IGF-I-binding affinity, the latter of which probably contributed to the IGF-I-induced accentuated synthesis of renal glomerular PGs in the Hx group.

Expression of the fibroblast growth factor receptor 1-4 genes in glomeruli in anti-Thy1.1 mesangial proliferative glomerulonephritis.

Basic fibroblast growth factor (FGF2) is generally known to induce proliferation of cultured mesangial cells and is expressed in proliferative mesangial cells in anti-Thy1.1 mesangial proliferative glomerulonephritis (anti-Thy1.1 GN). The distribution of the FGF receptor (FGFR) has not been studied in anti-Thy1.1 GN, so we used in situ hybridization to determine whether cells expressing FGFR1-4 mRNAs could be detected. In normal rats, all glomeruli were negative for FGFR1-4 mRNA, but those of the mesangial proliferative phase expressed FGFR1-4 mRNA in proliferative mesangial cells. Proliferation of mesangial cells has not been observed in normal rats injected with FGF2( )but it has been noted in anti-Thy1.1 rats injected with FGF2. These data and our results demonstrate that mesangial cells produce and release FGF2( )after injury and that during the proliferative phase these cells upregulate FGFR in vivo. This study is the first to demonstrate expression of FGFR1-4 mRNAs in pathological glomeruli of anti-Thy1.1 GN. The FGF2 and FGFR1-4 genes were expressed in the proliferative mesangial cells. Upregulation of FGFR is necessary for mesangial proliferation by FGF2.

Apoptosis and extracellular matrix-cell interactions in kidney disease.

Apoptosis and extracellular matrix-cell interactions in kidney disease. Extracellular matrix (ECM)-cell interactions have major effects on phenotypic features such as cell growth, differentiation, and gene expression. Apoptosis is an active form of cell death that is crucial for maintaining an appropriate number of cells as well as tissue organization. Recent reports have implied that ECM can influence survival and apoptosis of several cell lineages including glomerular mesangial cells (MC). Numerous glomerular diseases are associated with the expansion of the mesangial ECM, which may eventually produce glomerular scarring. Glomerular cell apoptosis is associated with the deletion of glomerular cells and the accumulation of ECM in the progression of glomerulosclerosis in rat remnant kidney model induced by 5/6 nephrectomy. Our recent study indicated that basement membrane matrix (a model for normal ECM components) prevented cultured MC from undergoing apoptosis after serum deprivation, thus promoting their survival, compared with type I collagen matrix (a model for abnormal ECM components). Inhibition of matrix-derived signals by antisense oligonucleotides against beta1 integrin increased MC apoptosis. Data suggest that the survival and death of MC are regulated by the surrounding ECM through integrin molecules. The mechanism of regulation of MC apoptosis by ECM requires further in vivo study to gain new insight into the treatment of glomerular diseases as well as the pathophysiology of the mesangium. Diabetic nephropathy is characterized by the abnormal ECM accumulation and the phenotypic change of MC. Some speculations on the possible involvement of apoptosis in diabetic nephropathy are also discussed.

Phenotypic changes of the mesangium in diabetic nephropathy.

In diabetic nephropathy there is accumulation of matrix proteins. Overproduction of these matrix proteins considered to be due to the phenotypic change of mesangial cell. In order to detect the phenotypic change of the mesangial cell, renal biopsy specimens from patients with diabetic nephropathy were stained with antibodies against various types of collagens and contractile-associated protein, caldesmon. Type III collagen was not stained in the glomerulus and type VI collagen showed mesangial pattern from normal controls. In diabetes, mesangial staining of type III collagen and increases in type VI collagen were observed in the mesangium. Increased mesangial staining of caldesmon was noted in the glomerulus from diabetic nephropathy in contrast to only vessel staining from normal controls. These results indicate that phenotypic changes are noted in the mesangium in diabetes. Expression of contractile-associated protein such as caldesmon, would serve as a useful marker to predict glomerulosclerosis.

Clinical significance of necrosis in lupus nephritis.

The significance of necrosis (karyorrhexis), among the most characteristic findings in lupus nephritis, was evaluated by studying the correlation between the existence of necrosis in renal biopsy specimens and laboratory findings. The subjects were 54 patients with diffuse proliferative lupus nephritis and 6 patients with focal proliferative lupus nephritis selected from 143 patients with lupus nephritis. We also compared the clinical course of oral prednisolone and intravenous methylprednisolone pulse therapies after steroid administration. Compared with the non-necrosis group, the necrosis group had significantly lower CH50 levels and more proteinuria. Patients with necrosis were effectively treated with repeated pulse therapy judging by immunological activity and the decrease in proteinuria at an early stage, but responded poorly to oral steroid therapy. As the presence of necrosis in cases of lupus nephritis means high immunological activity of the lesion and there is responsiveness to a large dose of steroids, extensive immunosuppressive therapy including methylprednisolone pulse therapy should be applied to these patients.

Transition of morphologic features in lupus nephritis: does steroid therapy accelerate glomerulosclerosis?

We retrospectively evaluated the morphologic change of 28 follow up biopsies from 24 cases of lupus nephritis according to the classification of the World Health Organization and determined the activity index (AI) and the chronicity index (CI). In the cases with biopsies repeated within 6 months, the AI decreased significantly from 6.7 +/- 1.3 to 3.5 +/- 0.8, while the CI showed no significant change. In cases which were rebiopsied after longer intervals, AI increased significantly from 3.2 +/- 0.7 to 7.5 +/- 1.2; the CI did not change significantly. When AI and CI changes in the cases biopsied again beyond 6 months were compared with respect to therapy, AI showed no significant difference in the methylprednisolone pulse therapy group but was significantly increased in the oral steroid therapy group. The CI tended to be increased in both groups, but not significantly. Steroid pulse therapy was effective in improving active lesions with a high AI. Steroid therapy for lupus nephritis prevented short-term progression of glomerulosclerosis and did not accelerate glomerulosclerosis.

Takayasu's arteritis associated with ulcerative colitis; genetic factors in this association.

Both ulcerative colitis and Takayasu's arteritis are though to be organ-specific immune-mediated inflammatory diseases. We present the rare case of a 23-year-old woman with a 4-year history of ulcerative colitis who developed Takayasu's arteritis one month after giving birth. She was found to carry the human leukocyte antigens (HLA)-B52 and DR2, which were previously noted to be associated with these inflammatory conditions in the Japanese population. The pathogenic relevance of this haplotype to the concomitant development of these two conditions is discussed.

A study on cross-reactivity of anti-DNA antibody with glycosaminoglycans.

To study the pathogenesis of lupus nephritis, the cross reactivity between anti-DNA antibody and glycosaminoglycans (GAGs) was investigated. Monoclonal anti-DNA antibodies were obtained from hybridomas by the fusion of MRL/lpr/lpr splenocytes with murine myeloma cells. Some of these monoclonal anti-DNA antibodies showed cross reactivity with GAGs, such as hyaluronic acid, chondroitin sulfate and heparan sulfate. To elucidate the mechanism of cross reactivity, inhibition assays with propanol and polyethylenimine (PEI), a cationic agent, were carried out. Increase of the concentration of PEI (0.6-2.0% vol/vol) resulted in a dose dependent decrease in the binding ability of anti-DNA antibody to GAGs. Propanol, an organic reagent which disrupts the van der Waals bonds between epitopes and paratopes, showed little inhibitory effect on the binding activity of monoclonal anti-DNA antibody to GAGs. These results indicate that the binding of anti-DNA antibody to GAGs is due to a charge interaction rather than van der Waals forces. Anti-DNA antibody which can react with GAGs in the glomerular basement membrane seems to play an important role in the pathogenesis of lupus nephritis.

[A case of giant prostatic calculi].

A 83-year-old man was admitted because of fever and painful swelling of the right scrotal contents on January 23, 1989. Kidney-ureter-bladder X-ray (KUB) showed multiple big prostatic calculi which were compressed postero-laterally by benign prostate hyperplasia (BPH) according to the CT film. UCG film revealed urethral stricture in the anterior urethra. He was diagnosed as having right epididymitis, urethral stricture, BPH and giant prostatic calculi. He was treated with right orchiectomy, urethral bougie and suprapubic prostatectomy with removal of the calculi. The total weight of the prostatic calculi was 28 g of consisted of 125 pieces. The postoperative course was uneventful and two years after the operation he has been well and free from evident disease.

[Experience in transurethral ureterolithotripsy using a rigid ureteroscope].

We report our experience of transurethral ureterolithotripsy using a rigid ureteroscope on 18 ureteral calculus patients between November, 1985 and July, 1986. The stones could be successfully removed in 3 of the 10 cases of upper ureteral calculi and in all 8 cases of lower ureteral calculi. In this series, transurethral ureterolithotripsy proved to be valuable for the lower ureteral calculus. However, the device still needs to be improved for upper ureteral calculi.

[A case of retroperitoneal teratoma showing high level of CA19-9].

We report a case retroperitoneal mature teratoma showing an abnormally high CA19-9 (carbohydrate antigen 19-9) serum level in a 30-year-old woman. She was hospitalized for an episode of left upper abdominal pain. High CA19-9 tissue level and immunohistochemical findings were found in removed tissue. This case is the third report of a CA19-9-producing teratoma and literature was reviewed and discussed.

[Study of preventive effect of 1-hexylcarbamoyl-5-fluorouracil (HCFU) or combination of HCFU and 2.5-di-O-acetyl-D-glucaro (1-4) (6-3) dilactone (SLA) after preservative operation against bladder cancer].

To treat superficial bladder cancer or invasive bladder cancer presenting as a solitary tumor, conservative therapy such as transurethral resection of bladder tumor or partial cystectomy has long been carried out. We studied the effect of 1-hexylcarbamoyl-5-fluorouracil (HCFU), and the effects of the combination of HCFU and 2.5-di-O-acetyl-D-glucaro-(1-4)(6-3) dilactone (SLA) which is an anti-beta-glucuronidase agent, for preventive therapy. In the patients treated with HCFU, the recurrence rate was 3.6% and 26.6% one year and two years, respectively, after conservative operation. In the patients treated with both SLA and HCFU, the recurrence rate was lower than in those treated with HCFU one year or more after conservative operation, and a long-term preventive effect was expected for nondrinkers.

[Clinical study on cefmenoxime (CMX) in urology].

Basic and clinical studies were made on Cefmenoxime (CMX), a new cephalosporin antibiotic, and the following results were obtained. The serum concentration of CMX was examined in four healthy adults after administration of 250 mg of CMX by intramuscular injection, intravenous injection and one-hour intravenous drip infusion (cross over). In the case of intramuscular injection, the peak value of 5.9 micrograms/ml was obtained 30 minutes after administration, and the half-life in serum was 1.41 hours. In the case of intravenous drip infusion, injection, a concentration value of 11.1 micrograms/ml on the average was obtained after 15 minutes of administration, and the half-life in serum was 1.26 hours. In the case of intravenous drip infusion, the concentration was 12.4 micrograms/ml upon completion of drip infusion, and CMX disappeared from serum at a half-life of 0.94 hour. The urinary recovery up to 6 hours was from 60 to 70% in each The efficacy rate of this preparation was 100% for 4 cases of acute simple cystitis. The efficacy rate of CMX was 70% for 10 cases of complicated urinary tract infection; the 3 cases in which CMX was not effective were patients with a residual catheter and Pseudomonas persisting or appearing as superinfection. It was noted that Serratia, which was resistant to the conventional cephalosporin antibiotics, became negative. No subjective side effects due to the administration of this preparation were observed. As for abnormal laboratory findings, a slight and transient rise in transaminases was observed in one case. On the basis of the above-mentioned results, it was concluded that CMX is an effective preparation for the treatment of urinary tract infections.

[Indications of extracorporeal surgery (autotransplantation) for upper urinary tumors and renal calculi].

Renal preservation and cure of bilateral occurrence of renal tumors or stones as well as occurrence in solitary kidney put all urologists on the thorns of dilemma. Application of extracorporeal surgery resulting in autotransplantation of the kidney for such complicated cases seems useful. Case 1 was a bilateral renal Wilms' tumors in a 17-month-old boy, case 2 and 3 were undiagnosed tumors in renal pelvis and ureter. Extracorporeal surgery enables an accurate partial nephrectomy and yet helps to avoid not only the dissemination of tumor cells, but also undesired nephrectomy in case of benign origin. On the contrary, bilateral nephrectomy followed by dialysis or homo-transplantation will result in an immunosuppressive state and the radicality may only be temporary. Besides, the 5-year survival of dialysis therapy is in the range of 60-65%. Thus the indication of autotransplantation was signified in the tumor situation of case 1. For the 2nd and 3rd cases, this method seems to be the best in satisfying the dilemma in discussion. Likewise, the complicated renal calculi could be completely removed without further deterioration of renal function if they were extracorporeally treated and autotransplanted. We proved that 83% of the difficult stones can be completely removed under hypothermia in situ. For application of autotransplantation to the calculous disease, we feel observation of contraindication is essential for better results at this stage.