Caldesmon isoform associated with phenotypic modulation of mesangial cells.

Caldesmon (CaD) is a major calmodulin- and actin-binding protein distributed in smooth muscle cells (SMC) and nonmuscle cells. There are at least two high-molecular-weight CaD (h-CaD) isoforms and four low-molecular-weight CaD (l-CaD) isoforms produced by alternative splicing. Isoformal interconversion is associated with phenotypic modulations of vascular SMC. We investigated the CaD isoform in human and rat glomerular mesangial cells (MC) to characterize the phenotypic changes of MC involved in glomerular diseases. A Western blot analysis and reverse-transcription analysis using exon-specific primers revealed that one l-CaD isoform lacking exons 1, 3b and 4 was predominantly expressed in human cultured MC. The expression of this isoform was markedly enhanced in anti-Thy1.1 nephritis rats and streptozotocin-induced diabetic rats, while little expression was observed in the normal glomerulus. Isoformal interconversion did not occur during the phenotypic changes of MC. These data suggested that the activated MC resembled dedifferentiated SMC in terms of the CaD expression pattern, and that CaD is a useful marker of the phenotypic modulations of MC.

Regulation of survival and death of mesangial cells by extracellular matrix.

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.

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.

Expression of interleukin-8 in human glomerulonephritis.

Interleukin-8 (IL-8) is a cytokine, which possesses both chemotactic and activating properties for neutrophils, lymphocytes and basophils. Various evidence has indicated IL-8 to be implicated in the pathophysiology of immune-mediated renal diseases. We thus examined the expression of IL-8 in renal diseases. We detected the expression of IL-8 both in mRNA and the protein levels in renal biopsy specimens obtained from patients with IgA nephropathy and lupus nephritis. A significant correlation was found between the expression of IL-8 mRNA and the number of neutrophils in the glomerulus. We also found a negative correlation between the expression of IL-8 mRNA and creatinine clearance. Our study thus suggested IL-8 to be involved in the pathophysiology of proliferative glomerulonephritis.

Bcl-2 expression and apoptosis in nephrotoxic nephritis.

The product of the Bcl-2 proto-oncogene has been shown to prolong cell survival by preventing apoptosis in several cell lineages. To investigate the regulatory mechanisms of apoptosis in glomerulonephritis, we examined the expression pattern of the Bcl-2 protein together with cellular events in rat nephrotoxic nephritis. Bcl-2 protein and proliferating cell nuclear antigen were detected in glomeruli by immunohistochemistry. Morphologic changes of apoptosis were identified by electron and light microscopy and an in situ DNA nick end labeling method. The first (heterologous) phase began with significant neutrophil infiltration shortly after the injection of nephrotoxic serum. Both Bcl-2 expression and the number of proliferating cells in the glomeruli were at maximum at 24 h in the heterologous phase. Glomerular hypercellularity with an influx of macrophages and the number of apoptotic glomerular cells peaked on day 14 in the second (autologous) phase. Glomerulonephritis resolved after that. These results suggest that overexpression of the Bcl-2 protein may play a role in glomerular cell survival and exacerbation of glomerulonephritis. Apoptosis may occur as an active mechanism in the resolution of the autologous phase in nephrotoxic nephritis.

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.

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.

[Evaluation of physical fitness and exercise performance in patients with chronic pulmonary emphysema].

Physical fitness was studied in patients with chronic pulmonary emphysema using Kraus-Weber methods in addition to pulmonary function and exercise tolerance. In Kraus-Weber tests, explosive strength of abdominal muscles in these patients were within the normal range, but both abdominal and back muscle endurance were significantly diminished compared to age-matched controls. On the other hand, flexibility was not different between the patients and the controls, although large variation was present. Exercise performance as assessed by 6 minutes' walk distance in patients was significantly correlated with FEV1.0, DLco and maximal inspiratory mouth pressure, as well as explosive strength of abdominal muscles and abdominal and back muscle endurance capacity. Treadmill walking training for 20 minutes with a load greater than 80% VO2max, twice a week for 2 months was performed in 11 patients with mild to moderate pulmonary emphysema. Six minutes' walk distance (6MD) was significantly prolonged with improvement of back muscle endurance and flexibility. Another walking training consisting of five repetitions of two minutes' near maximal walking and a two minute interval of rest was performed in 6 patients with severe pulmonary emphysema. 6MD tended to increase with improvement of both back and abdominal muscle endurance. However, pulmonary function tests and VO2max showed no significant changes after both types of training. Improved walked distance after the training was significantly correlated with improved VO2 at AT. Furthermore VO2, VE, HR and lactate production during exercise at the same load were significantly decreased compared to pre-training. Dyspnea sensation measured by modified Borg scale during exercise was improved after the training. It is concluded that a physical training program adapted to the condition of the individual patients could improve exercise performance, and should be prescribed in addition to medication.

[Evaluation of a radioimmunoassay of alpha 1-microglobulin (alpha 1-m) with simplified procedures].

A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25 microliters, and thus 200 mg/l of alpha 1-m standard was prepared using 50 microliters of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intra-assay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about alpha 1-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of alpha 1-m were less than 25 mg/l in serum and less than 10 mg/l in urine. The present results indicate that the determination of alpha 1-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function.

Low testosterone levels in diabetic men and animals: a possible role in testicular impotence.

Poorly controlled NOD spontaneously diabetic mice were proven to have significantly less plasma and testicular testosterone than well-controlled diabetic mice (489 +/- 15 ng/dl and 3.89 +/- 0.79 micrograms/100 g tissue, vs. 176 +/- 24 and 9.00 +/- 1.24, respectively), and these in turn had significantly less than NOD non-diabetic control mice. These data were consistent with our previous observation of a decrease in total plasma and testicular testosterone levels in streptozotocin diabetic rats. A greater difference between total plasma testosterone levels and free testosterone levels was found in streptozotocin diabetic rats (17 +/- 4 ng/dl vs. 91 +/- 7 ng/dl) than in control rats (660 +/- 141 vs. 352 +/- 77). Fat droplets, depicting blocked testosterone synthesis, were found in the testicular Leydig cells of streptozotocin diabetic rats and disappeared with insulin treatment. No difference was found among plasma total testosterone concentrations in people in different stages of diabetes, as had been previously reported. However, human diabetic males, free of complications but poorly controlled, had less free testosterone than those without complications but well controlled (18.0 +/- 2.0 pg/ml vs. 22.8 +/- 1.3), who in turn had significantly less than age-matched controls (25.3 +/- 1.1 pg/ml). These data suggest gonadal dysfunction in diabetes mellitus.

[Basic studies on an immunoradiometric assay system for human parathyrin (intact PTH1-84)].

Two-site immunoradiometric assay for human parathyrin (PTH1-84) is specific for the intact, secreted, biologically active 84 amino peptide. This system incorporates two-different polyclonal antibodies to human intact PTH and has several technical advantages for use. This assay could detect a wide range of PTH in patients with hypo-, hyperparathyroidism, chronic renal failure and hypercalcemia with malignancy, especially distinguishing the level of human intact PTH in hypoparathyroidism from in normal.

[Studies on an antibody-coated tube method of aldosterone].

We studied on the basic performance of a non-extraction coated tube RIA kit, DPC Aldosterone kit (Nippon DPC Corporation). 1. The average binding CVs of the calibrators (25 to 1,200 pg/ml) were stable in a range from 1.4 to 1.9%. 2. The minimum sensitivity was 17 pg/ml from 2SD method of 0 calibrator. 3. The intra-assay reproducibility test showed an average CV as 9.0%, and the inter-assay test showed as 8.8%. 4. The recovery ratios of a recovery test were 85.4 to 121.6%. 5. The correlation test with another maker's kit showed a correlation curve as y = 0.970 x + 19.68 and a correlation coefficiency as r = 0.970. 6. The normal range, the correlation between fasting recumbency and daytime sitting, and the correlation between walking and resting, showed similar results as former reports.

Plasma and testicular testosterone in experimental diabetic rats.

Testicular and plasma testosterone levels were found to be decreased markedly in streptozotocin diabetic rats compared with those of controls. Treatment with 6 units NPH insulin daily for one week almost normalized plasma testosterone levels parallel to the increase in body and liver weights in diabetic rats, while testosterone levels in testicles were not significantly changed. Plasma prolactin and LH levels were unchanged among control, diabetic and diabetic insulin-treated rats. Thus, testosterone reduction in the testis might play a role in diabetic impotence.