Renal arteriolar hyalinosis, not intimal thickening in large arteries, is associated with cardiovascular events in people with biopsy-proven diabetic nephropathy.

AIMS: Diabetic nephropathy, a pathologically diagnosed microvascular complication of diabetes, is a strong risk factor for cardiovascular events, which mainly involve arteries larger than those affected in diabetic nephropathy. However, the association between diabetic nephropathy pathological findings and cardiovascular events has not been well studied. We aimed to investigate whether the pathological findings in diabetic nephropathy are closely associated with cardiovascular event development. METHODS: This retrospective cohort study analysed 377 people with type 2 diabetes and biopsy-proven diabetic nephropathy, with a median follow-up of 5.9 years (interquartile range 2.0 to 13.5). We investigated how cardiovascular events were impacted by two vascular diabetic nephropathy lesions, namely arteriolar hyalinosis and arterial intimal thickening, and by glomerular and interstitial lesions. RESULTS: Of the 377 people with diabetic nephropathy, 331 (88%) and 295 (78%) had arteriolar hyalinosis and arterial intimal thickening, respectively. During the entire follow-up period, those with arteriolar hyalinosis had higher cardiovascular event rates in the crude Kaplan-Meier analysis than those without these lesions (P = 0.005, log-rank test). When fully adjusted for clinically relevant confounders, arteriolar hyalinosis independently predicted cardiovascular events [hazard ratio (HR) 1.99; 95% confidence interval (CI) 1.12, 3.86], but we did not find any relationship between arterial intimal thickening and cardiovascular events (HR 0.89; 95% CI 0.60, 1.37). Additionally, neither glomerular nor interstitial lesions were independently associated with cardiovascular events in the fully adjusted model. CONCLUSIONS: Arteriolar hyalinosis, but not intimal thickening of large arteries, was strongly associated with cardiovascular events in people with diabetic nephropathy.

Resolution of mesangial light chain deposits 3 years after high-dose melphalan with autologous peripheral blood stem cell transplantation.

A 52-year-old woman was admitted to our hospital for treatment of nephrotic syndrome. Funduscopic findings showed fundal hemorrhage and soft exudates, and serologic analysis showed a monoclonal serum component that was identified as Bence Jones protein-k type. A bone marrow biopsy showed diffuse proliferation of atypical plasma cells, while a renal biopsy showed diffuse and nodular mesangial proliferation. Immunohistochemical staining confirmed the presence of k chains along the glomerular basement membrane and in mesangial areas. The patient was diagnosed as multiple myeloma (Bence Jones k type) with light chain deposition disease (LCDD). After high-dose melphalan and autologous peripheral blood stem cell transplantation (PBSCT), the multiple myeloma and nephrotic syndrome were in complete remission; her renal function was improved, but a renal biopsy performed 6 months after PBSCT showed the persistence of diffuse and nodular lesions. By contrast, a renal biopsy performed 3 years later showed complete resolution of the diffuse and nodular mesangial proliferation.

Genitalic autogrooming in the male cricket, Gryllus bimaculatus DeGeer.

The genitalia of the male cricket, Gryllus bimaculatus, is automatically maintained. It is carried out by spontaneous undulation of the scaled membrane consisting of the genital chamber floor. To understand the mechanism of that movement, part of the membrane was cut out and examined in vitro with biogenic amines, and the spike activity of neurons innervating muscle fibers of the membrane was analyzed. The esults indicated that the fragment of the membrane, which showed spontaneous twitching in saline, increased its frequency at 5-HT application. In contrast, mianserin (5-HT antagonist) decreased its occurence. Immunocytochemical study indicated that massive 5-HT-positive branchibgs of one main axon nnervated muscle fibers of the genital membrane. Centrally, one of the motoneurons backfilled with ucifer Yellow through the cut end of the nerve 9v of the terminal abdominal ganglion was determined 5-HT positive. These results suggested that the undulatory movement of the genital membrane for genitalic autogrooming is mediated by 5-HT.

A genetic locus controlling aging-sensitive regression of B lymphopoiesis in an autoimmune-prone MRL/lpr strain of mice.

Aging readily affects immune system under the influence of environmental and/or intrinsic factors while accelerating the development of various immune disorders including autoimmune diseases. Little is known about molecular and cellular mechanisms connecting between immune senescence and development of autoimmune diseases. Here, we first show strain-specific and aging-sensitive onset of B-cell abnormality in a lupus-prone MRL/Mp.Fas(lpr) (MRL/lpr) strain of mice. This abnormality was characterized by the regression of B lymphopoiesis in the bone marrow of this strain. We next examined the association between the B-cell regression and onset of autoimmune diseases in aged (MRL/lpr x C3H/He.Fas(lpr)) F2 mice, in which pathologic phenotypes, such as glomerulonephritis, vasculitis, sialoadenitis and arthritis, variously developed. We also searched whole genome to identify genetic loci linked to the B-cell regression by using the same F2 mice. The B-cell regression manifested in the spleen of F2 mice was retrospectively evaluated by reverse transcriptase-based PCR quantification. The results demonstrated that the onset of autoimmune diseases in the F2 mice was not associated with the aging-sensitive B-cell regression. The genetic study identified a significant locus responsible for the B-cell regression in the vicinity of D5Mit233 (29 cM). This is first evidence for the presence of a genetic locus that affects B lymphopoiesis in an aging-sensitive manner.

Molecular characterization of photomixotrophic tobacco cells resistant to protoporphyrinogen oxidase-inhibiting herbicides

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5, 6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5, 6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.

Tubulointerstitial macrophage infiltration in a patient with hypokalemic nephropathy and primary Sjögren's syndrome.

We report a case of hypokalemic nephropathy associated with primary Sjögren's syndrome (SS). The patient presented with profound and persistent hypokalemia secondary to distal renal tubular acidosis (RTA). A renal biopsy exhibited tubular degeneration, marked interstitial fibrosis and intense macrophage infiltration. Hypokalemia has been reported to induce macrophage infiltration in experimental animal models but not in humans. This is the first report of intense tubulointerstitial macrophage infiltration in a patient with hypokalemic nephropathy associated with SS.

Interleukin-6 (IL-6) functions as an in vitro autocrine growth factor in renal cell carcinomas.

Interleukin-6 (IL-6) was found to be a growth factor of renal cell carcinomas Furthermore, renal cell carcinomas freshly isolated from the patients expressed mRNA of IL-6 and secreted biologically active IL-6 under the culture conditions where the tumor cells could grow, but they did not produce IL-6 nor proliferate in the absence of fetal calf serum. The production of IL-6 by the tumor cells was also demonstrated by immunostaining of the IL-6-producing cells utilizing anti-IL-6 antiserum. Moreover, anti-IL-6 antiserum specifically inhibited the in vitro tumor growth. All data indicated that IL-6 functions as an in vitro autocrine growth factor of renal cell carcinomas.

Association of transforming growth factor-beta1 T29C polymorphism with the progression of diabetic nephropathy.

Diabetes mellitus is a leading cause of end-stage renal disease in the Western world. Histologically, mesangial expansion with increased extracellular matrix protein is observed in patients with diabetic nephropathy. Because transforming growth factor (TGF)-beta promotes extracellular matrix production in response to high glucose, TGF-beta is considered to play a central role in the pathogenesis of diabetic nephropathy. We investigated the association of TGF-beta1 T29C polymorphism and the progression of diabetic nephropathy. Forty patients with type 2 diabetes mellitus were enrolled. All patients had had diabetes for more than 10 years. DNA was extracted from peripheral blood cells, and genotype was determined using real-time polymerase chain reaction method. Patients were classified into three groups according to genotype: TT, TC, and CC. Grade of diabetic nephropathy was determined using the amount of urinary excretion of albumin. Demographic characteristics of the patients with each genotype were not statistically different. No differences in the glycemic control and the mode of therapy were observed. Among patients with three genotypes, the severity of diabetic nephropathy was not statistically different. The patients with TT genotype tended to have a higher rate of progression of nephropathy; however, no statistically significant difference was observed among the three groups. Our results suggest that TGF-beta1 T29C polymorphism is not associated with the progression of diabetic nephropathy. Further studies are required to determine the exact role of this polymorphism in the progression of diabetic nephropathy.

Role of interleukin-6 in the progression of mesangial proliferative glomerulonephritis.

Mesangial proliferative glomerulonephritis (mesPGN) is histologically characterized by proliferation of mesangial cells (MC), suggesting the involvement of a growth factor for MC in the pathogenesis of mesPGN. We have previously shown that interleukin-6 (IL-6) induces proliferation of cultured rat mesangial cells, and urine samples from patients with IgA nephropathy contain high level of IL-6 activity. We have also demonstrated that transgenic mice carrying a human IL-6 genomic gene showed severe mesangial proliferation and matrix enlargement. Urinary samples of patients with lupus nephritis as well as IgA nephropathy contain significant IL-6 activity. Over a ten month follow-up, a positive correlation between the urinary IL-6 and pathological score was observed. Hence, measurement of urinary IL-6 can be used as a good indicator for monitoring IgA nephropathy and lupus nephritis. Using RT-PCR methods, IL-6 mRNA was detected in the glomeruli of renal biopsy specimens obtained from patients with IgA nephropathy and lupus nephritis.

Effects of Helicobacter pylori infection on Zollinger-Ellison syndrome.

Both Zollinger-Ellison syndrome (ZES) and Helicobacter pylori infection are major etiologic factors for peptic ulcer. The aim of this study was to investigate the effect of H. pylori infection on ZES with special reference to acid secretion. Sixteen patients with ZES were selected (median age, 59 years; range, 39-66 years; M/F, 9/7), and H. pylori status, ulcer location, gastric acid secretion, serum pepsinogen (PG) I and II concentrations, and PG I/II ratio were determined. The seroprevalence of H. pylori infection was 50%, whereas active H. pylori infection was seen in only 25% of the patients. Thirteen patients had duodenal ulcer (DU), 1 had gastric ulcer (GU), and 2 had both GU and DU. DU was seen in both H. pylori-positive and H. pylori-negative patients, whereas GU was found only in H. pylori-positive patients. Both basal and maximal acid outputs were significantly lower in H. pylori-positive patients than in H. pylori-negative patients (P< 0.05). Moreover, both serum PG I and the PG I/II ratio were significantly lower in H. pylori-positive patients than in H. pylori-negative patients. These results indicate that ZES is an independent risk factor for DU, but H. pylori infection may play some role in the development of GU in ZES. In patients with ZES, H. pylori infection may reduce both hypersecretion from parietal cells and PG I secretion from chief cells, and hyperacidity of the stomach in ZES may have eradicated H. pylori in some patients.

Serum levels of VEGF and basic FGF in the subacute phase of myocardial infarction.

We examined serial changes in serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) measured by ELISAs in 45 patients with acute myocardial infarction (AMI) who received heparin intravenously for 3 to 5 days after the onset and in 30 control subjects with an old myocardial infarction. To evaluate the effect of heparin on these serum levels, heparin was administered intravenously in 10 patients with AMI on day 21. Blood samples were obtained from all AMI patients on days 1, 2, 3, 7, 14, 21, and 28 and from 10 AMI patients before and 1 h after heparin administration. Serum VEGF level was significantly reduced after heparin administration (P<0.001). Serum samples from day 1 to 3 were therefore excluded from the subsequent analysis. Serum VEGF level in AMI patients was significantly higher on day 7 than in the control subjects (P<0.0001), and then decreased over time (P<0.0001). The serum VEGF level on day 7 was independently associated with the peak serum CK level (P<0.05). The serum bFGF level did not differ significantly between the AMI patients and the control subjects. In conclusion, the serum VEGF level may be selectively elevated during the healing process after AMI.

Vascular endothelial growth factor mRNA synthesis by peripheral blood mononuclear cells in patients with acute myocardial infarction.

We studied vascular endothelial growth factor (VEGF) mRNA synthesis by peripheral blood mononuclear cells (PBMCs) in 30 patients with acute myocardial infarction (AMI) and 20 healthy individuals. PBMCs were isolated from all patients on days 3 and 14 after the onset of aMI, and from all of control individuals. To prepare samples containing identical amounts of GAPDH cDNA, competitive PCR was performed by co-amplifying serial dilutions of GAPDH mutant templates. Next, to measure VEGF cDNA quantitatively in the samples containing identical amounts of GAPDH, we also used competitive PCR by co-amplifying mutant templates of VEGF. The serum VEGF concentrations on day 14 in patients with aMI were measured by an ELISA method. Higher levels of VEGF mRNA in PBMCs were present on day 14 than either on day 3 or in the control group. Serum VEGF concentrations correlated with the VEGF mRNA levels of PBMCs on day 14. Peak serum CK levels correlated well with VEGF mRNA levels of PBMCs on day 14. The present findings suggest that PBMCs may be one of the candidates responsible for elevated circulatory VEGF protein following aMI. In addition, VEGF mRNA may be overexpressed in PBMCs in response to cardiac muscle damage.

The prognostic significance of urinary interleukin 6 in IgA nephropathy.

Our previous study has shown that interleukin 6 (IL-6), a multifunctional cytokine, is closely associated with the pathogenesis of mesangial proliferative glomerulonephritis (mesPGN). To investigate whether urinary IL-6 can be used as an indicator in the prognosis of patients with IgA nephropathy, we monitored IL-6 activity in the urine of patients with IgA nephropathy for 10 months and compared IL-6 activity with clinical data as well as the histological changes of the kidneys obtained from the patients. It was found that among the patients who had continuously high urinary IL-6 activity, histological progression of IgA nephropathy was observed. On the other hand, among the patients whose urinary IL-6 became undetectable during the 10-month follow-up, histological improvement of IgA nephropathy was observed. These data suggest that the measurement of urinary IL-6 is a helpful tool for monitoring the progression of IgA nephropathy.

Urinary levels of IL-6 in patients with active lupus nephritis.

Using the IL-6 dependent hybridoma, MH60.BSF2, we measured urinary levels of interleukin 6 (IL-6) in 29 patients with active lupus nephritis. We detected IL-6 activity in the urine of 24 (83%) of 29 patients before the initiation of therapy. The median value of urinary IL-6 levels in patients with a histologic diagnosis of WHO class IV on renal biopsy was significantly higher than that in patients with other classes (p < 0.01). After treatment, urinary levels of IL-6 decreased significantly (p < 0.001). These data suggest that urinary levels of IL-6 may be a valuable tool for monitoring the progression of lupus nephritis.

Clinical and morphological predictors of renal outcome in adult patients with focal and segmental glomerulosclerosis (FSGS).

In this retrospective study, we examined 35 adult patients with biopsy-proven, primary focal and segmental glomerulosclerosis (FSGS) and nephrotic syndrome to determine whether any of the clinical and morphological features of FSGS were associated with a higher risk of a poor renal outcome. Clinical factors assessed were the age, sex, amount of urinary protein, and presence of microscopic hematuria, hypertension and renal dysfunction at onset in each patient. Morphological parameters included the number of segmental sclerosis and global sclerosis, sclerosis score, location of segmental sclerosis, mean glomerular diameter, grade of tubulo-interstitial changes, and presence of vascular lesions. Twenty-three patients (66%) were in complete or incomplete (partial) remission, and 12 (34%) were non-responders at the end of follow-up. On univariate analysis, the age at onset, sclerosis score, mean glomerular diameter, and grade of tubulo-interstitial changes in no response were significantly greater than those parameters in remission. Multivariate logistic regression analysis revealed that the degree of tubulo-interstitial changes and mean glomerular diameter were independent risk factors for a poor renal outcome. These findings suggest that the estimation of these latter two parameters allows the nephrologist to predict the probable course and prognosis of an adult with FSGS. Intensive and prolonged therapy is recommended for patients without these two morphological features.

Association of an insertion polymorphism of angiotensin-converting enzyme gene with the activity of lupus nephritis.

BACKGROUND: Lupus nephritis is a common manifestation of systemic lupus erythematosus (SLE). The pathogenesis of lupus nephritis has not been fully understood; however, immunological abnormalities have been considered in the development and activity of lupus nephritis. As angiotensin-converting enzyme (ACE) is implicated in various immunological phenomena, we investigated the correlation between insertion (I)/ deletion (D) polymorphism of the ACE gene and the activity of lupus nephritis. PATIENTS AND METHODS: Eighty-four patients with SLE and 100 healthy subjects were enrolled in this study. Following the extraction of genomic DNA from the peripheral blood, the ACE genotype was determined by the polymerase chain reaction. The patients were classified by the histological findings according to the WHO classification. In addition, the activity index and chronicity index were used to assess the severity of renal involvement. RESULTS: Individuals with II genotype showed a significantly increased activity of lupus nephritis. The allelic frequency was I/D = 0.84/0.16 in patients with WHO class IV renal lesions, and I/D = 0.36/0.64 in those with WHO class I lesions and 0.61/0.39 in patients with WHO class I or WHO class II. The difference in the allelic frequency between patients with WHO class IV and those with WHO class I or WHO class I + WHO class II was statistically significant (p = 0.00016 or p = 0.027, respectively). Moreover, lupus nephritis patients with II genotype showed significantly higher activity index than those with DD genotype (p = 0.0009). CONCLUSION: These results suggest that the insertion polymorphism of the ACE gene may correlate with the activity of lupus nephritis.

Inhibitory effects of ginseng on proliferation of cultured mouse mesangial cells.

To evaluate the pharmacological action of ginseng, its effects on the proliferative activity of mesangial cells, which are thought to play an important role in the regulation of renal function, were determined in terms of [3H]thymidine uptake. When the extract was added to the medium of mesangial cell cultures, it suppressed the proliferation of mesangial cells, and similar proliferation-inhibitory activity was found in the total saponin and ginsenoside-Rd fractions, consistent with the renal effects observed in our previous in vivo studies. The inhibition of mesangial cell proliferation by the extract can thus be explained by the action of ginsenoside-Rd.

Inhibitory effects of crude drug components on the proliferation of cultured human mesangial cells.

The effects of a component of an Oriental medicine and green tea plants utilized for the traditional beverage on the proliferation of mesangial cells were determined in terms of the 3H-thymidine uptake. When magnesium lithospermate B was added to the medium of the mesangial cell culture, it suppressed the proliferation of mesangial cells at a concentration of 50 micrograms/ml. The 3H-thymidine uptake began to display an inhibitory action from the 6.25 micrograms/ml (-)-epicatechin 3-O-gallate, (-)-epigallocatechin 3-O-gallate or green tea tannin mixture, showing a more conspicuous inhibition at 50 micrograms/ml that did the magnesium lithospermate B at the corresponding concentration. In particular, (-)-epigallocatechin 3-O-gallate, the predominant component of the green tea tannin mixture, exerted an inhibitory effect at a relatively low concentration.

Rapid measurement of urinary IL-6 by ELISA: urinary IL-6 as a marker of mesangial proliferation.

To determine whether the urinary level of interleukin 6 (IL-6) measured by enzyme-linked immunosorbent assay (ELISA) can be used as a marker of mesangial proliferation, we studied urinary levels of IL-6 in 124 patients with primary and secondary glomerulonephritis, using ELISA. Although urinary levels of IL-6 were correlated with the degree of mesangial proliferation, there was no correlation between urinary levels of IL-6 and urinary protein excretion or renal function. Urinary levels of low-molecular-weight proteins, which are parameters of tubular dysfunction, were not correlated with the urinary excretion of IL-6. These results suggest that the urinary level of IL-6 may be a useful marker for mesangial proliferation.

[Expression of Fc epsilon receptor 2 in minimal change nephrotic syndrome].

In order to clarify the mechanism for elevation of serum IgE level in minimal change nephrotic syndrome (MCNS), we investigated Fc epsilon receptor 2 (Fc epsilon R2/CD23) expression, using fluorescein isothiocyanate (FITC) labeled anti-CD20 monoclonal antibody, phycoerythrin (PE) labeled anti-CD23 monoclonal antibody and two-color flow cytometry. Moreover, serum IgE level was examined by radio-immunosorbent test. The subjects included 25 cases of MCNS, 17 cases of focal glomerular sclerosis (FGS), and 25 healthy volunteers as controls. The patients in the nephrotic stage of MCNS demonstrated significantly elevated levels of serum IgE, while those in the remission stage of MCNS showed no change in their serum IgE levels. In patients with nephrotics of MCNS, CD23+ cells, CD20+ CD23+ cells and CD23/CD20 ratio were significantly increased compared to normal controls. Furthermore, CD23/CD20 ratio in MCNS was significantly correlated with serum IgE level. Concerning FGS, there were no differences in serum IgE level and Fc epsilon R2 expression compared to those of normal controls. These results suggest that Fc epsilon R2 expression may be important in the mechanism of elevated serum IgE level in MCNS.