Susceptibility quantitative trait loci for pathogenic leucocytosis in SCG/Kj mice, a spontaneously occurring crescentic glomerulonephritis and vasculitis model.

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse, a model of human crescentic glomerulonephritis (CrGN) and systemic vasculitis, is characterized by the production of myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) and marked leucocytosis. This study was performed to identify the specific populations of leucocytes associated with CrGN and susceptibility loci for pathogenic leucocytosis. Four hundred and twenty female (C57BL/6 × SCG/Kj) F2 intercross mice were subjected to serial flow cytometry examination of the peripheral blood (PB). Kidney granulocytes and monocytes were examined histopathologically. Linkage analyses were performed with 109 polymorphic microsatellite markers. Correlation studies revealed that increase of the granulocytes, F4/80(+) cells, CD3(+) CD4(-) CD8(-) T cells and dendritic cells (DCs) in peripheral blood (PB) were associated significantly with glomerulonephritis, crescent formation and vasculitis. In kidney sections, F4/80(low) cells were observed in crescent, while F4/80(high) cells were around the Bowman's capsules and in the interstitium. Numbers of F4/80(+) cells in crescents correlated significantly with F4/80(+) cell numbers in PB, but not with numbers of F4/80(+) cells in the interstitium. Genome-wide quantitative trait locus (QTL) mapping revealed three SCG/Kj-derived non-Fas QTLs for leucocytosis, two on chromosome 1 and one on chromosome 17. QTLs on chromosome 1 affected DCs, granulocytes and F4/80(+) cells, but QTL on chromosome 17 affected DCs and granulocytes. We found CrGN-associated leucocytes and susceptibility QTLs with their positional candidate genes. F4/80(+) cells in crescents are considered as recruited inflammatory macrophages. The results provide information for leucocytes to be targeted and genetic elements in CrGN and vasculitis.

Accelerated tubular cell senescence in SMP30 knockout mice.

An experimental model with accelerated but not drastic renal senescence seemed useful to recognize the mechanisms of how kidney function deteriorates with age. Senescence marker protein-30 (SMP30), whose expression decreased with age and was sex-independent, is mainly expressed in hepatocytes and proximal tubular cells. Therefore, we established a SMP30 deficient strain of mice with a C57BL/6 background by gene targeting to investigate whether this molecule is involved in renal tubular cell senescence. Male SMP30 knockout (SMP30Y/-) mice and male wild-type (SMPY/+) mice (n=5) aged 12 months were examined histologically. Their tubular epithelia showed the deposition of lipofuscin and the presence of senescence-associated beta-galactosidase (SA-beta-GAL). However, no tubular cells were atrophic. In electron microscopy, SMP30-KO mice showed markedly enlarged lysosomes containing an electron dense substance. These are convincing hallmarks of senescence. We recognized the early manifestation of senescence hallmarks in SMP30-KO mice at 12 months old. Thus, this model represents the first report of a mouse strain that manifests accelerated ordinal senescence in a kidney after gene manipulation.

Effects of long-term treatment with mizoribine in patients with proliferative lupus nephritis.

AIM: Mizoribine (MZR) is a purine antimetabolic immunosuppressant agent that has few little severe adverse events. We studied whether maintenance therapy with MZR and prednisolone (PSL) in severe proliferative lupus nephritis patients could improve immunity, reduce proteinuria, prevent renal relapse, and reduce steroid dose. METHOD: Long-term maintenance therapy with MZR and PSL was evaluated in ten patients with biopsy-proven proliferative lupus nephritis. Patients with severe lupus nephritis, who had proteinuria of 0.5 g or more even after treatments with plasma exchange and/or pulse methyl prednisolone, were recruited. MZR at an average dose of 140 +/- 10 (100 - 200) mg was administered two to three times/day in combination with PSL. The average period for the MZR maintenance therapy was 89.7 +/- 5.5 (70 - 126) months. Urine protein excretion, serum hemolytic complement activity (CH50), C3, serum creatinine, general and biochemical blood examinations, anti-ds-DNA antibody were collected at each monthly medical examination. RESULTS: All patients were females, mean age 43.0 +/- 3.3 years. A significant decrease in proteinuria was noted two years after the combination therapy (p = 0.0016). Five patients experienced lupus nephritis relapse. Patients who did not experience relapses had their MZR combination therapy initiated earlier (p = 0.037) when compared with the patients who experienced relapses. Serum creatinine levels remained unchanged in all patients throughout treatment and follow-up, even during renal relapses. Levels of C3 and CH50 normalized as proteinuria decreased. None of the patients developed serious side effects during MZR treatment. A significant steroid-sparing effect was observed three years after initiating MZR (p = 0.0025). CONCLUSION: From our long-term observation, maintenance therapy with low-dose PSL combined with MZR can eliminate proteinuria and have steroid-sparing effect. Early initiation of the therapy can protect against renal relapses among severe proliferative lupus nephritis patients without serious side effects.

Soluble osteopontin and vascular calcification in hemodialysis patients.

BACKGROUND: Vascular calcification often occurs in patients with uremia. As osteopontin (OPN) is not only involved in the physiological but also the pathological calcification of tissues, OPN may be associated with the pathogenesis of aortic calcification in hemodialysis (HD) patients. METHODS: We examined the expression of OPN in atherosclerotic aortas of HD patients. In addition, we performed a prospective longitudinal study by using CT scans to detect aortic calcifications and by measuring the plasma OPN concentration by ELISA in HD patients (20 men, 16 women; mean age 55.2 +/- 21.3 years) and in healthy volunteers (18 men, 17 women; mean age 54.0 +/- 13.2 years). RESULTS: By immunohistochemical staining, OPN was abundantly localized in atherosclerotic plaques of HD patients. The macrophages surrounding the atheromatous plaques were identified as the OPN-expressing cells. We furthermore found that the concentration of soluble plasma OPN was significantly higher in HD patients as compared with the concentrations in age-matched healthy volunteers (837.3 +/- 443.2 vs. 315.1 +/- 117.4 ng/ml, p < 0.01). The OPN concentration was positively correlated with the aortic calcification index in HD patients (r = 0.749, p < 0.01). CONCLUSION: These data suggest that OPN, secreted by macrophages, plays a role in the calcification of atheromatous plaques in HD patients.

Involvement of MAP kinase cascades in Smad7 transcriptional regulation.

Smad7 transcription is known to be regulated by TGF-beta to form a negative-feedback loop of TGF-beta-mediated biological responses. In this study, we sought to determine whether other signaling cascades, especially mitogen-activated protein (MAP) kinases, might be involved in the transcriptional regulation of Smad7. Hyperosmolarity (500 mOsm/kg H(2)O) or anisomycin (10 microg/ml) potentiated TGF-beta-induced increases of Smad7 mRNA abundance in normal rat kidney fibroblasts. SB203580 (10 microM) treatment had no effect on basal and TGF-beta-induced Smad7 mRNA abundance, and the overexpression of kinase-negative ATF2 had no effect on Smad7 promoter activity. On the other hand, overexpression of dominant-negative JNK and dominant-negative c-Jun significantly attenuated the TGF-beta-induced increases of Smad7 mRNA abundance and promoter activity, respectively. Mutations of the AP-1 element near the Smad-binding element in the rat Smad7 promoter also completely abolished TGF-beta-induced Smad7 promoter activity. These results suggested that the JNK cascade, not p38 kinase, cooperated with the Smad signaling to induce Smad7 transcription through the AP-1 element. Serum treatment (10%) attenuated the TGF-beta-induced Smad7 mRNA increase, and PD98059 (30 microM) treatment increased the basal and TGF-beta-induced Smad7 promoter activity. Gel shift analysis revealed that serum treatment decreased the amount of nuclear Smad complex that PD98059 treatment was shown to restore. These results indicated that ERK activation negatively regulated Smad7 transcription possibly by inhibiting translocation of Smad complex to nuclei. In conclusion, JNK cascade and ERK cascade are important positive and negative regulators of Smad7 transcription, respectively.

Identification of T helper cell subsets in continuous ambulatory peritoneal dialysis patients.

BACKGROUND/AIM: End-stage renal failure induces a clinical state of immunodeficiency with a higher incidence of infections and a higher mortality due to infectious complications as compared with the normal population. A crucial element in this immune response is the development of a network of cytokines. CD4-positive T helper (Th) cells differentiate into either Th1 or Th2 cells in response to various stimuli. It is important to examine whether the impaired immune responses in patients with end-stage renal disease are due to an enhanced Th1 response or a reduced number of Th2 cells. METHOD: Using a newly developed immunofluorescence staining of intracellular cytokines for flow cytometric analysis, we studied Th subsets in 24 patients on continuous ambulatory peritoneal dialysis (CAPD). RESULTS: The proportions of Th1 cells did not differ between CAPD patients (18.7 +/- 1.8%) and controls (19.6 +/- 1.5%). Unlike Th1 cells, a significantly higher proportion of CD4 cells in CAPD patients is characterized by a Th2-type cytokine secretion pattern as compared with healthy controls: 2.9 +/- 0.8% versus 1.7 +/- 0.4%. CONCLUSION: These data demonstrate that Th cells from CAPD patients manifest a dysregulated differentiation profile characterized by a major increase in the percentage of Th2 cells and by a normal percentage of Th1 cells.

Analysis of lupus activity in end-stage renal disease treated by hemodialysis.

OBJECTIVE: The activity of systemic lupus erythematosus (SLE) has been reported to decrease in patients who have developed end-stage renal disease (ESRD). However, extrarenal symptoms attributable to the disease activity are noted, especially during the first year of dialysis. We studied the clinical course and evaluate the disease activity of SLE in patients with ESRD on hemodialysis for more than 6 months. SUBJECT AND METHODS: Fourteen patients with SLE who had been initiated on maintenance dialysis at our center between 1982 and 1999 were examined retrospectively. Their clinical details, organ system manifestations, serologic profiles and immunosuppressive treatment regimens were reviewed. Patients with and without postdialysis flaras of SLE were compared statistically. RESULTS: Five patients exhibited 6 SLE flares under treatment with corticosteroids. Two flares occurred within the first year of the initiation of dialysis, and in 1 patient, aggravation of the disease activity was noted 98 months after the initiation of dialysis. Polyarthritis was noted in 5 cases and fever in 4 cases. The serum complement levels decreased in all 6 cases with relapse of SLE activity. Compared with the other 9 patients who did not exhibit SLE relapse, no significant differences were found in 5 patients who did with respect to the demographic and serologic features at the initiation of dialysis. CONCLUSION: We conclude that the disease activity does not always burn out in patients of SLE who show progression to ESRD. SLE flares can sometimes occur even after one year of the initiation of dialysis. SLE patients on dialysis should be carefully followed up by clinical and serological monitoring, and treated by appropriate immunosuppressive therapy.

Evaluation of prognostic factors for myeloperoxidase anti-neutrophil cytoplasmic antibody- (MPO-ANCA) associated glomerulonephritis.

AIMS: To identify prognostic factors for myeloperoxidase anti-neutrophil cytoplasmic antibody- (MPO-ANCA) associated glomerulonephritis. MATERIALS: We analyzed the relations between the clinical and histological features of MPO-ANCA-associated glomerulonephritis and clinical outcome in 14 patients with the disease. The patients were divided into two groups: group 1 consisted of 5 patients with progressive deterioration of renal function leading to end-stage renal disease or chronic dialysis, group 2 consisted of 9 patients in whom the initial deterioration of renal function had improved by the time of the final examination. RESULTS: Creatinine clearance at the time of biopsy was significantly lower in group 1 than in group 2, and urinary protein was higher. The mean interval between onset of symptoms and biopsy in both groups was almost the same. Recovery of renal function was correlated with the percentage of global sclerosis, but patients who had severe crescent formation did not always have a poor response to steroid therapy. There was no statistical difference between the two groups in treatment regimens. Four patients required hemodialysis at the time of biopsy (3 in group 1 and 1 in group 2). Plasmapheresis was performed in 5 patients (1 in group 1 and 4 in group 2). CONCLUSIONS: Degree of proteinuria and renal dysfunction are indicators of a poor prognosis in MPO-ANCA-associated glomerulonephritis. Global sclerosis is a histological feature that is an indicator of a poor prognosis, whereas cellular crescent formation is a predictor of a good response to steroid therapy.

Tubular osteopontin expression in patients with ANCA-associated glomerulonephritis.

OBJECTIVE: To elucidate the role of osteopontin (OPN) in monocyte recruitment in crescentic glomerulonephritis, we investigated immunohistochemical localization of OPN in the kidney and its correlation with clinical and histopathologic parameters in biopsy specimens of patients with myeloperoxidase antineutrophil cytoplasmic autoantibody- (MPO-ANCA) associated glomerulonephritis. METHODS: Twelve patients with MPO-ANCA-associated glomerulonephritis were enrolled in this study. Clinical parameters such as creatinine clearance and urinary protein excretion of each patient were obtained at the time of biopsy. Paraffin-embedded sections were used for immunohistochemical staining using the LSAB method. Five cortical interstitial fields randomly selected at original magnification x 200 were assessed using a computer-assisted color image analyzer. Tubular OPN expression was assessed as the percentage of positive area in the tubulointerstitium. Double immunofluorescent staining using antibodies against OPN and alpha(v)beta3 was performed. RESULTS: In all of the cases studied, OPN was occasionally localized within the glomeruli, and expressed slightly in proximal tubular epithelium and significantly in distal tubular epithelium. Tubular OPN expression tended to be promoted in the interstitium infiltrating by numerous monocytes/macrophages. The extent of tubular OPN expression was positively correlated with serum ANCA titers and urinary OPN concentrations. Enhanced alpha(v)beta3 expression appeared in the distal tubular epithelium expressing OPN. CONCLUSION: These results suggest that inducible expression of OPN and alpha(v)beta3 in the tubular epithelium seems to be associated with interstitial moncyte infiltration and subsequent tubulointerstitial changes in human MPO-ANCA-associated glomerulonephritis.

Serum levels of macrophage colony-stimulating factor and aortic calcification in hemodialysis patients.

Hemodialysis (HD) patients have accelerated atherosclerosis. Recent reports have shown that aortosclerosis is more frequently observed in HD patients than in healthy subjects. Macrophage colony-stimulating factor (M-CSF) secreted by activated macrophages may be involved in the process of aortosclerosis in HD patients. To understand the mechanism behind the increased incidence of aortosclerosis in HD patients, we examined the relationships between serum M-CSF levels and aortic calcification index (ACI) estimated by CT scan. A significant increase in serum M-CSF concentrations was found in HD patients (3.8 +/- 0.2 ng/ml) as compared with controls (1.5 +/- 0.1 ng/ml). No significant differences were observed between chronic glomerulonephritis and diabetes mellitus groups of patients. We also found no significant differences between the groups using different membranes (triacetate 3.8 +/- 0.2 ng/ml vs. polysulfone 3.8 +/- 0.4 ng/ml). There was no correlation between serum M-CSF concentrations and clinical parameters such as age, duration of HD, blood pressure, serum concentrations of nitrogen, creatinine, cholesterol, triglyceride, LDL, Ca x P products, and intact parathyroid hormone. A positive correlation was observed between serum M-CSF levels and ACI in HD patients (r = 0.596, p < 0.01). These results suggest that M-CSF may be involved in the process of aortosclerosis in HD patients.

Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease.

To confirm the significance of excretion of annexin V into the urine and the change of urinary annexin V concentration in kidney disease, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two monoclonal antibodies. Urinary annexin V concentration was measured in healthy individuals and patients with kidney and other diseases. Urinary annexin V did not change over a range of pH between 5.0 and 8.0, and was stable during the course of the study for 24 h at room temperature and for 8 days at 4 degrees C. The mean urinary annexin V concentration in 105 normal healthy individuals was 1.5+/-1.5 ng/ml, while that in patients with nephrotic syndrome and systemic lupus erythematosis (SLE) nephritis was 9.3+/-9.1 and 6.6+/-6.7 ng/ml, respectively, and that in IgA nephropathy and chronic renal failure was 2.6+/-2.1 and 1.3+/-0.7 ng/ml, respectively. Annexin level correlated with urinary protein concentration (r=0. 717), but not the serum creatinine concentration, blood urea nitrogen (BUN) and 24-h creatinine clearance. Mean urinary annexin V concentration in patients with ischemic heart disease, hypertension, and diabetes mellitus was 1.4+/-1.0, 1.4+/-1.1, and 1.7+/-1.3 ng/ml, respectively. In one case of relapsing nephrotic syndrome, the urinary annexin V concentration was markedly increased in the early phase after admission and then decreased. This patient later required hemodialysis. These results suggest that a high urinary annexin V concentration may be an indicator of acute renal injury related to the urinary protein level.

Localization of Smad6 and Smad7 in the rat kidney and their regulated expression in the anti-Thy-1 nephritis.

Under normal conditions, kidney expresses Smad6 and Smad7 most abundantly among the organs of the body. To understand the physiological roles of these Smad expressions in the kidney, we first identified the sites of Smad6 and Smad7 expression in the rat kidney by in situ hybridization. The expression of Smad7 in the rat kidney was only observed in the glomeruli, while Smad6 was expressed in both the glomeruli and thick ascending limb of Henle's loop. In order to investigate whether Smad6 and 7 are also involved in the negative feedback loop of TGF-beta signaling in vivo, we examined the changes of mRNA levels of these Smads in the glomeruli of rat anti-Thy1 (1-22-3) nephritis, a model where the expression of TGF-beta in the glomeruli has been shown to be most up-regulated from day 4 to 14 after the antibody injection. Unexpectedly, 7 days after injection, the levels of Smad6 and Smad7 did not increase but rather decreased to approximately 70% of the levels on day 0. During that period, Smad7 immunostaining was observed in the glomerular endothelial cells (GEN) where Smad3 immunostaining was also observed. This suggested that Smad7 expression was not augmented by the TGF-beta signal in GEN in vivo in anti-Thy-1 nephritis. The absence of up-regulation of these inhibitory Smads may be involved in the pathogenesis of anti-Thy-1 nephritis.

Increased serum levels of vascular endothelial growth factor in human crescentic glomerulonephritis.

OBJECTIVE: To investigate the serum levels of vascular endothelial growth factor (VEGF) in human crescentic glomerulonephritis (CRGN). METHODS: The serum VEGF levels in CRGN patients were compared with those in healthy subjects and in various types of glomerulonephritis. In addition, we investigated the relationship between serum VEGF levels and creatinine levels (Scr) and pathological parameters. RESULTS: The serum VEGF levels of the CRGN patients were significantly higher than in the healthy subjects and patients with MCNS, IgAN, and FGS. No correlation was found between serum VEGF levels and Scr in the RPGN patients. The serum VEGF levels in 6 CRGN patients had significantly decreased after 6 months of corticosteroid therapy. Moreover, there was a significant correlation between the serum VEGF levels and the crescent frequency or the grade of interstitial injury, and the rates of glomerular infiltration by macrophages. CONCLUSIONS: In the CRGN patients, severe glomerular and interstitial damages would result in increased serum VEGF levels and corticosteroid therapy may exert its efficacy through reduction of serum VEGF levels.

Recombinant human erythropoietin stimulates vascular endothelial growth factor release by glomerular endothelial cells.

We designed the present study to address the question of whether recombinant human erythropoietin stimulates DNA synthesis and vascular endothelial growth factor (VEGF) secretion in vitro using cultured bovine glomerular endothelial cells (GENs). Recombinant human erythropoietin dose-dependently stimulated the proliferation of GENs in culture, and this proliferative effect was inhibited by 1 microg/ml anti-VEGF antiserum. The increase in VEGF concentrations in the supernatants containing 10 U/ml rHuEpo was abolished by incubation with 10 microg/ml of anti-human rHuEPO antiserum, 0.2 microg/ml actinomycin D or 10 microg/ml cycloheximide. Taken together, rHuEpo stimulates GEN proliferation in vitro and VEGF release from these cells is associated with stimulation of RNA-dependent DNA and protein synthesis.

Accumulation of advanced glycation end products in the peritoneal vasculature of continuous ambulatory peritoneal dialysis patients with low ultra-filtration.

BACKGROUND: Ultra-filtration failure is a serious complication of long-term continuous ambulatory peritoneal dialysis (CAPD). This complication is related to histological changes of the peritoneum, i.e. severe interstitial fibrosis and microvascular sclerosis. Although their pathogenesis has not been elucidated yet, advanced glycation end products (AGEs) have been shown to accumulate in the peritoneal tissue of CAPD patients. METHODS: Peritoneal biopsy specimens from 14 CAPD patients with low ultra-filtration (n = 9) and high ultra-filtration (n = 5) capacity were immunohistochemically investigated using a monoclonal antibody against AGEs (6D12). The severity of peritoneal fibrosis, microvascular sclerosis and intensity of AGE accumulation were semi-quantitatively evaluated. Peritoneal ultra-filtration capacity was evaluated by calculating daily ultrafiltration volume per body weight (UFV/BW) and D/D0 (glucose) of the peritoneal equilibration test. RESULTS: In all patients with low ultra-filtration, AGE accumulated in the peritoneal fibrous tissue and microvascular walls. Remarkably, AGE accumulated more intensely in hyalinized fibrosis of small venular media. Extent of AGE accumulation in peritoneal interstitium and vascular walls correlated with the progression of interstitial fibrosis (rho = 0.727, P = 0.0088) and vascular sclerosis (rho = 0.915, P = 0.001). UFV/BW was inversely correlated to interstitial fibrosis (rho = -0.660, P = 0.0174), microvascular sclerosis (rho = -0.671, P = 0.0155) and microvascular AGE accumulation (rho = -0.678, P = 0.0145). CONCLUSIONS: In CAPD patients, AGE formation in the peritoneum correlates with the development of severe interstitial fibrosis and microvascular sclerosis, which is associated clinically with impaired peritoneal ultra-filtration.