Plasma and blood vessel endothelin-1 concentrations in hypertensive uremic rats treated with erythropoietin

The present study was designed to evaluate whether changes in plasma and blood vessel endothelin-1 (ET-1) concentrations may play a role in the enhanced blood pressure response to recombinant human erythropoietin (r-HuEPO) replacement therapy in uremia. Renal failure was induced by 5/6 nephrectomy (Nx). Uremic rats received either r-HuEPO (100 u s.c. three times a week) or the vehicle for 5 weeks. They were compared to control rats receiving the vehicle. Systolic blood pressure (tail cuff method), hematocrit, serum creatinine, plasma and tissue ET-1 were measured at the end of the protocol. Immunoreactive ET-1 (ir-ET-1) was determined by radioimmunoassay of acid-extracts from the plasma, thoracic aorta and mesenteric arterial bed. Creatinine increased about three fold in Nx animals. Blood pressure in control rats was 120+/-3 mmHg compared to 161 +/-6 mmHg in the Nx + vehicle group (p <0.01) and 199+/-9 mmHg in the Nx + r-HuEPO group (p <0.01 vs Nx + vehicle). Hematocrit in control rats was 41.3+/-0.4% vs 32.6+/-1.8% in the Nx + vehicle group (p <0.01) and 47.6+/-1.5% in the Nx + r-HuEPO group (p <0.01). Plasma ir-ET-1 levels were similar in the Nx + vehicle and Nx + r-HuEPO groups (7.9+/-1.0 and 7.8+/-0.8 pg/ml). In contrast, thoracic aorta ir-ET-1 content was significantly higher in the Nx + r-HuEPO group than in the Nx + vehicle group (20.3+/-2.9 vs 13.4+/-1.9 pg, p <0.05). Similar results were obtained in the mesenteric arterial bed. There were significant correlations between blood pressure and ir-ET-1 content in the thoracic aorta (r= 0.45, p<0.05) and in the mesenteric arterial bed (r= 0.41, p<0.05). Vascular ET-1 content but not plasma levels are increased in uremic rats treated with r-HuEPO suggesting an increase in blood vessel ET-1 production which may play a role in the pathogenesis of r-HuEPO-induced hypertension.     

Intermittent versus continuous administration of 1,25-dihydroxyvitamin D3 in experimental renal hyperparathyroidism

Conflicting results have been reported regarding the efficacy of intermittent versus continuous administration of 1,25(OH)2D3 in renal secondary hyperparathyroidism. To address this issue we examined sham-operated control rats and hyperparathyroid rats with subtotal (5/6) nephrectomy (Nx). The Nx animals (20 to 22 animals per group) were subjected to three treatment protocols: (i) solvent treatment (Nx-solvent); (ii) two i.p. injections of 35 pmol 1,25(OH)2D3 on days 0 and 4 (Nx-bolus); and (iii) continuous infusion of 70 pmol 1,25(OH)2D3 over six days via osmotic minipump (Nx-infusion). All measurements were performed six days after start of treatment. As compared to sham-operated controls, the pre-pro-PTH/beta-actin mRNA ratio was 2.04-fold higher in Nx-solvent. Both modes of administration of 1,25(OH)2D3 resulted in inhibition of PTH mRNA concentrations relative to Nx-solvent. The pre-pro-PTH/beta-actin mRNA ratio was, however, significantly lower (P < 0.05) in Nx-bolus than in Nx-infusion (Nx-bolus 1.26 higher than sham-operated controls; Nx-infusion 1.65 higher than sham-operated controls). Aminoterminal PTH (N-PTH) serum concentrations were higher in Nx-solvent (52 +/- 4 pg/ml) than in sham-operated controls (32 +/- 3 pg/ml, P < 0.01). N-PTH concentrations in Nx-bolus (38 +/- 4 pg/ml) were significantly lower than in Nx-solvent (P < 0.01) and in Nx-infusion (46 +/- 4 pg/ml, P < 0.05). Parathyroid gland weight (microgram/g body wt) was higher in Nx-solvent (1.30 +/- 0.08 pg/ml) than in sham-operated controls (0.79 +/- 0.04 pg/ml, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular apoptosis and proliferation in experimental renal fibrosis

BACKGROUND: The progression of chronic renal failure (CRF) is associated with the progressive deletion of renal cells along with the fibrosis of the kidney. We have studied the role of programmed cell death (apoptosis) in the progression of experimental CRF and renal scarring. METHODS: The sub-total (5/6th) nephrectomy (SNx) model of CRF was studied in adult male Wistar rats, with renal tissue collected from experimental and control animals on days 7, 15, 30, 60, 90, and 120 post SNx (n = 6 per group). These were examined for morphological signs of apoptosis by light and electron microscopy. Further, we stained the nuclear chromatin by the acridine orange fluorescent method and detected signs of DNA cleavage by endonucleases via the principal of TUNEL staining (ApopTag). Rates of cellular proliferation were measured simultaneously by immunohistochemical staining for the proliferating cell nuclear antigen (PCNA). In addition, cell division was monitored by counting of morphologically mitotic motifs detectable by light microscopy. RESULTS: Progressive renal insufficiency associated with glomerulosclerosis and tubulointerstitial fibrosis took place in the majority of SNx rats. In these animals, we noted a marked and progressive increase in the number of apoptotic glomerular, tubular as well as interstitial cells. The most significant apoptotic changes were seen in the tubules of remnant kidneys peaking at day 120 post-SNx. At this stage, the increase in apoptosis compared to controls was 10.33+/-2.67 (M+/-SEM) fold for glomerular cells (P< or =0.006), 26.20+/-4.56 fold for tubular cells (P < 0.0001) and 4.66+/-0.81 fold for interstitial cells (P< or =0.001). Parallel changes in the number of PCNA positive renal cells were observed. Maximal PCNA staining was seen at day 120 when the increase with respect to controls was 14.00+/-4.93 fold (P< or = 0.05) for glomerular cells, 60.01+/-12.20 fold (P< or =0.05) for tubular cells and 28.59+/-4.45 fold (P< or = 0.05) for interstitial cells. As expected, the number of cells undergoing division and detectable by conventional light microscopy was lower at any time point to those expressing PCNA. We also observed a close correlation between the severity of tubular atrophy and tubulointerstitial fibrosis with the rate of tubular apoptosis (r=0.970, R2 =0.941, P< or =0.001). CONCLUSIONS: We have shown a time-dependent increase in apoptosis and PCNA antigen positive staining in the sub-total nephrectomy model of chronic renal failure correlating with the progression of renal fibrosis. PCNA staining did not match analysis for mitosis and was considered to overestimate the number of proliferating cells in the tissue. With this reservation in mind and taking into account the relative time-frames in vivo of apoptosis and proliferation; apoptosis potentially outweighs proliferation by a factor of 2 8-fold, when examined over the same time period. Consequently, even small changes in the finite numbers of apoptotic cells become highly significant. Our results have shown the definite role of apoptosis within progression of renal damage and highlighted how it may contribute to the progression of tubular atrophy and play a role in the pathogenesis of tubulo-interstitial scarring.     

Influence of dose range on degree of nonlinearity detected in dose-proportionality studies for drugs with saturable elimination: single-dose and steady-state studies

Macrophages may modulate mesangial expansion following renal injury via secretory products. We undertook the present study to determine the effects of macrophage supernatants on mesangial cell proliferation. Macrophage supernatants collected in serum-free media after 24 hours caused significantly enhanced mesangial cell proliferation in long-term culture at concentrations up to 50% but caused suppression at higher concentrations (control, 122,000 +/- 14,000 cells/well; 50% supernatant, 188,000 +/- 15,100 cells/well, P < 0.02 compared to control, n = 4; 80% supernatant, 52,000 +/- 3,500 cells/well, P < 0.01 compared to control, n = 4). In short-term culture [3H]thymidine incorporation, a measure of DNA synthesis, was significantly enhanced compared to control at supernatant concentrations up to 30% (30% supernatant, 4,120 +/- 310 cpm/well; control, 3,210 +/- 97 cpm/well, P < 0.5, n = 4), but uptake was reduced at high concentration (80% supernatant, 2,900 +/- 74 cpm/well; control, 3,210 +/- 97 cpm/well, P < 0.05, n = 4). When macrophage supernatants were collected after 48 hours incubation and incubated with mesangial cells, mesangial cell thymidine uptake was significantly suppressed compared to control (48-hour supernatant, 4,060 +/- 260 cpm/well; control, 5,890 +/- 270 cpm/well, P < 0.01, n = 4) and compared to 24-hour supernatants, which enhanced uptake (24-hour supernatant, 8,080 +/- 340 cpm/well; control, 5,890 +/- 270 cpm/well, P < 0.01, n = 4). Our results suggest that macrophage supernatants can directly enhance mesangial cell proliferation in vitro in both short-term and long-term culture, though this effect is lost at high concentrations of supernatant. These data lend support to the potential role of the macrophage in mediating mesangial expansion following renal injury.     

Progress in diagnosis of gastric cancer and improvement of treatment results]

It is known that growth hormone (GH) contributes to glomerulosclerosis and that this probably occurs via insulin-like growth factor-I (IGF-I). However, the manner by which GH and nephrectomy (Nx) alter IGF-binding protein (IGFBP) mRNA in the kidney has not been fully explained. The effects of GH on renal IGF-I and IGFBP-1, 2, 3, 4, and 5 following Nx were examined in spontaneous dwarf rats (SDRs) which have a complete and specific lack of GH among pituitary hormones. In normal Sprague-Dawley rats (SDs), Nx resulted in significant decreases in levels of IGFBP-1 mRNA and IGFBP-5 mRNA to 62.7 +/- 4.9 and 56.5 +/- 5.0% those of sham-operated kidneys, respectively. Nx did not alter the IGF-I mRNA level in SDs. The levels of IGFBP-2, IGFBP-3, and IGFBP-4 mRNAs were likewise unchanged following nephrectomy. In SDRs, Nx significantly decreased the levels of IGFBP-1, IGFBP-4, and IGFBP-5 mRNA, to 57 +/- 2.6, 46 +/- 12, and 64 +/- 8.1% of sham-operated animals. However, Nx did not alter the levels of IGF-I, IGFBP-2, and IGFBP-3 mRNA. GH injections of nephrectomized SDRs fully normalized the decreased IGFBP-4 mRNA levels, whereas levels of IGFBP-1 and IGFBP-5 mRNA were not reversed. The altered expression of IGFBP-4 mRNA following Nx of SDRs compared to that of SDs appears highly significant since it is known that, unlike SDs, glomerulosclerosis does not fully develop in SDRs following renal ablation. The change in the IGFBP-4 mRNA level might be related to the development of glomerulosclerosis in SDs.     

Survey of echinococcosis in Hokkaido and measure against it

Exposure to hyperoxia results in lung injury and a decrease in lung collagen. Retinol is known to influence collagen gene expression, and retinol deficiency has been shown to potentiate hyperoxic lung injury. To investigate the combined effects of retinol deficiency and hyperoxia on lung collagen expression, retinol-deficient rats were exposed to acute hyperoxia, and expression of the alpha-1 chains of type I procollagen [pro alpha 1 (I)] and type III procollagen [pro alpha 1 (III)] were determined using Northern hybridization analyses and immunohistochemical staining. Hyperoxia alone reduced pro alpha 1 (I) mRNA by 60 +/- 4% (p < .05) and pro alpha 1 (III) mRNA by 30 +/- 5% (p < .05), and retinol deficiency alone reduced pro alpha 1 (I) mRNA abundance by 49 +/- 8.8% (p < .05) and pro alpha 1 (III) mRNA abundance by 14 +/- 7.5% (p = not significant), respectively. Retinol deficiency plus hyperoxia did not cause any further reduction in procollagen mRNA than that seen with oxygen exposure alone. Immunohistochemical staining demonstrated decreased staining for type I collagen in retinol-deficient animals. Hyperoxic exposure resulted in decreased connective tissue staining and increased alveolar wall staining for type I collagen. Retinol deficiency and hyperoxia together resulted in a marked increase in alveolar exudates staining for type I collagen. No changes in type III collagen staining were seen. These findings demonstrate that while retinol deficiency does not potentiate hyperoxia-induced reductions in procollagen mRNA, it is associated with alterations in collagen staining in distal lung and immunohistologic evidence of collagen fragments in alveolar exudates.     

Reduced expression of the renal calcium-sensing receptor in rats with experimental chronic renal insufficiency

Chronic renal insufficiency is associated with elevated serum parathyroid hormone (PTH) levels (2 degrees HPT), deficiency of 1,25-dihydroxyvitamin D (1,25(OH)2D), and hypocalciuria. In chronic renal insufficiency, the 2 degrees HPT may result from reduced expression of the parathyroid gland extracellular Ca(2+)-sensing receptor (CaSR). Since the CaSR was cloned from rat and human kidney, this study examined in rats whether expression of the renal CaSR is altered in experimental chronic renal insufficiency. Four weeks after chronic renal insufficiency was induced by 5/6 nephrectomy (Nx) in Sprague Dawley rats, the serum creatinine concentration was 0.96+/-0.06 mg/dl compared with 0.35+/-0.02 mg/dl in sham-operated animals (P < 0.05). The serum total Ca2+ and phosphorus concentrations were not different. In the Nx group, the serum concentration of amino-PTH was higher (65+/-8 pg/ml), and the concentration of 1,25(OH)2D was significantly lower (47+/-5 pg/ml) compared with 45+/-5 pg/ml and 61+/-4 pg/ml (P = 0.05) in the sham group, respectively. In a subset of rats studied, the Nx group was hypocalciuric (1.4+/-0.5 mg/kg per d) compared with the sham group (3.7+/-0.5 mg/kg per d) (P < 0.05). In the Nx rats, CaSR mRNA expression and CaSR protein levels were found to be reduced by 35 and 38%, respectively, than those observed in controls. These results suggest that reduced renal CaSR expression in chronic renal insufficiency may play a role in disordered mineral ion homeostasis, including hypocalciuria.     

Increase by tri-iodothyronine of endothelin-1, fibronectin and von Willebrand factor in cultured endothelial cells

Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T3) for 13 +/- 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T3 significantly increased vWF secretion (50 ng T3/ml: 117 +/- 5%, P < 0.01; 100 ng T3/ml: 127 +/- 26%, P < 0.01) as well as vWF mRNA expression (50 ng/ml: 116 +/- 13%, P < 0.001; 100 ng/ml: 136 +/- 30%, P < 0.002) (results are means +/- S.D. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 +/- 42% of control, P < 0.05) and 100 (116.8 +/- 16% of control, P < 0.05) ng T3/ml, and FN mRNA expression by 50 ng T3/ml (123 +/- 20%, P < 0.05). Long-term incubation with T3 increased both ET-1 secretion (25 ng/ml: 124 +/- 25%, P < 0.001; 50 ng/ml: 165 +/- 53%, P < 0.05; 100 ng/ml: 116 +/- 17%, P < 0.05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 +/- 16%, P < 0.05; 50 ng/ml: 134 +/- 43%, P < 0.02; 100 ng/ml: 120 +/- 20%, P < 0.02). Protein kinase C (PKC) isoforms epsilon and beta II were not significantly affected by T3, whereas PKC alpha was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T3/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [3H]thymidine assays, remained unaffected in HUVECs after exposure to T3. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC alpha-, beta II- and epsilon-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.     

Abrogation of glomerular injury in nephrotoxic nephritis by continuous infusion of interleukin-6

Interleukin-6 (IL-6) has been reported to have pro- and anti-inflammatory effects. It has also been shown to cause mesangial cell proliferation in vitro and has been suggested as a mediator of injury in proliferative nephritis. We have assessed the effects of continuous infusion of human recombinant (hr) IL-6, by osmotic minipump, on the degree of glomerular injury, and on glomerular and interstitial cell proliferation, in the accelerated autologous phase of nephrotoxic nephritis. Two groups of rats were pre-immunized with 1 mg of normal rabbit IgG in Freund's complete adjuvant. One week later, nephritis was induced by an intravenous injection of 1 ml of rabbit nephrotoxic serum. One day before the induction of nephritis, group 1 (N = 9) was subcutaneously implanted with osmotic minipumps filled with 50 micrograms (200 microliters) of IL-6 (equivalent to a dose of 6 micrograms/day), while in group 2 (N = 11) the minipumps were filled with 200 microliters of normal saline. In group 3 (N = 6) normal rats were infused with 50 micrograms of IL-6 alone. The rats were killed seven days after implantation of minipumps. The administered hrIL-6 was detectable in the circulation within the pathophysiological range, and induced a hepatic acute phase response, as assessed by alpha 2-macroglobulin levels. Continuous treatment with IL-6 resulted in a significant reduction in albuminuria (from 195 +/- 37 mg/20 hr to 60 +/- 15 mg/20 hr on day 1, and from 494 +/- 52 mg/20 hr to 238 +/- 30 mg/20 hr on day 7, P < 0.002) and in the prevalence of glomerular capillary thrombosis (from 19 +/- 3% to 5 +/- 1%, P < 0.002). There was also a reduction in macrophage infiltration (ED1 + ve cells from 524 +/- 34 to 466 +/- 14 per 50 glomeruli, P < 0.02) and activation (ED3 + ve cells from 106 +/- 13 to 42 +/- 5 per 50 glomeruli, P < 0.002). Immunohistology showed fewer interstitial Ia + ve cells (OX3 and OX4) in the IL-6 treated group. Similar results were obtained in a second set of experiments in which the IL-6 treatment was extended until day 14. Kidney sections taken from nephritic rats infused with IL-6 showed no increase in glomerular or interstitial cell proliferation when stained with antibodies to proliferating cell nuclear antigen. There was no difference in the deposition of rabbit IgG or rat IgG along the glomerular basement membrane (GBM), and the titer of rat anti-rabbit IgG was similar in the IL-6 and control treated rats. Infusion of IL-6 alone in normal rats had no functional or pathological effects. In conclusion, these results show that IL-6 has powerful anti-inflammatory effects in a rat model of anti-GBM nephritis, and does not induce mesangial cell proliferation in vivo.     

Enhanced proliferation, apoptosis, and matrix accumulation by mesangial cells derived from HIV-1 transgenic mice

BACKGROUND: Mice, transgenic for HIV-1 genes, have been demonstrated to develop renal lesions mimicking HIV-associated nephropathy. Focal glomerulosclerosis (FGS) has been reported to be the predominant glomerular lesion in these animals. In the other models of FGS, the accumulation of mesangial matrix and mesangial cell proliferation have been shown to be the preceding abnormalities. We evaluated the proliferation, apoptosis, and matrix accumulation by mesangial cells derived from mice transgenic for HIV-1 genes as well as from nontransgenic mice. METHODS: Mesangial cells were cultured from mice transgenic for HIV-1 genes (HTrMC) and nontransgenic mice (NTrMC) of the same age and sex. The growth rate of HTrMC and NTrMC was determined under identical conditions. Morphologic evaluation of apoptosis was performed by staining cells with Hoechst (H)-33342 and propidium iodide. Accumulation of mesangial cell collagen type IV, laminin, and fibronectin was measured by the dot blot assay. Total RNA was extracted from HTrMC and NTrMC and Northern blots were generated. These blots were probed with specific probes for TGF-beta, proteoglycan (P16), and GAPDH. RESULTS: Mesangial cells (HTrMC) derived from transgenic mice had greater (P < 0.004) proliferation when compared to mesangial cells (NTrMCs) from nontransgenic mice (HTrMCs, 4.2 +/- 0.3 vs NTrMCs, 3.0 +/- 0.2 x 10(4) cells/well). HTrMCs also showed enhanced (P < 0.0001) apoptosis compared to NTrMCs (HTrMCs, 13.2 +/- 1.5% vs NTrMCs, 3.1 +/- 0.5% apoptotic cells/field). HTrMCs accumulated an increased (P < 0.02) amount of collagen type IV (HTrMCs, 5659.7 +/- 472.8 vs NTrMCs, 3882.2 +/- 339.7 ng/well); whereas NTrMCs accumulated a greater amount of laminin when compared to HTrMCs (HTrMCs, 12.8 vs NTrMCs, 29.6 +/- 2.9 ng/well). HTrMCs also showed an enhanced mRNA expression of TGF-beta and an attenuated expression of proteoglycan (P16). CONCLUSIONS: These results suggest that mesangial cells derived from mice transgenic for HIV-1 genes have enhanced proliferation and collagen accumulation. The enhanced expression of TGF-beta may have contributed to enhanced HTrMC proliferation and the accumulation of collagen. The present study provides the basis for a hypothesis that mesangial cells may be contributing to the development of focal glomerulosclerosis in mice transgenic for HIV-1 genes.     

Proximal tubular basement membrane width in insulin-dependent diabetes mellitus

Although glomerular structure has been studied, careful evaluation of tubular basement membrane (TBM) structure in diabetes in humans has not been done. We measured proximal TBM width, glomerular basement membrane (GBM) width, mesangial fractional volume [Vv(Mes/glom)], mesangial matrix fractional volume [Vv(MM/glom)], and cortical interstitial fractional volume [Vv(Int/cortex)] in 35 insulin-dependent diabetic (IDDM) patients and 20 controls. The patients' mean age was 28 +/- 10 years (X +/- SD) and IDDM duration was 17 +/- 8 years. Twenty-five patients were normoalbuminuric, four microalbuminuric, and six had overt proteinuria. Tubular basement membrane and GBM widths were measured by the orthogonal intercept method and mesangial and interstitial parameters by point counting. The TBM width was 915 +/- 320 nm in IDDM patients and 558 +/- 116 nm in controls (P = 0.0005); the TBM width was also increased in normoalbuminuric patients (849 +/- 297 nm, P = 0.0005). The TBM width was strongly directly related to GBM width (r = 0.67, P < 0.001), Vv(Mes/glom) (r = 0.52, P < 0.01), and Vv(MM/glom) (r = 0.61, P < 0.001), but only weakly to Vv(Int/cortex) (r = 0.29, NS). The TBM width (r = 0.65, P < 0.001) and GBM width (r = 0.65, P < 0.001) were strongly related to hemoglobin A1C (HbA1C), while the Vv(Mes/glom) (r = 0.35, P < 0.05) and Vv(Int/cortex) (r = 0.30, NS) were only weakly related to HbA1C. Thus, increased proximal TBM width is an integral component of early nephropathology in IDDM patients. This study suggests that the metabolic disturbances of diabetes are strong determinants of the constellation of structural abnormalities occurring in human diabetic nephropathy.     

PAF-antagonists with lipid structure. 6. Alkylpropandiol lipids with acyl- and ether- structures at the C-3 position and heterocyclic head groups; synthesis, characterization and structure-activity relationship

OBJECTIVE: To investigate whether nonenzymatic glycated end products (AGEs) have effects on the expression and bioactivity of plasminogen activator inhibitor-1 (PAI-1), one of the seripin proteinases, which lead to extracellular matrix (ECM) degradation in cultured human mesangial cells. METHODS: Human mesangial cells (HMC) were cultured. Cell proliferation, fibronectin production, mRNA expression and bioactivity of PAI-1 were determined after exposure to AGE-BSA for 24 hours and 48 hours in vitro. RESULTS: HMC stimulated by AGE-BSA exhibited inhibition in HMC proliferation, increase in fibronectin production, and PAI-1 bioactivity. These changes were pronounced with prolongation of experimental time. PAI-1 mRNA expression increased significantly at 24 hr (0.45% +/- 0.06% vs 0.65% +/- 0.08%, P < 0.05), however more marked increase of PAI-1 mRNA expression at 48 hr (0.51 +/- 0.08% vs 0.92 +/- 0.10%, P < 0.01). CONCLUSIONS: Increase of mRNA expression and bioactivity of PAI-1 induced by AGEs decreased ECM degradation and play an important role in the pathogenesis of ECM accumulation and glomerulosclerosis.     

Native and oxidized low density lipoproteins modulate mesangial cell apoptosis

Hyperlipidemia has been demonstrated to contribute to hypercellularity of the mesangium in experimental animal models of glomerulosclerosis. We studied whether it also has the potential to convert a hypercellular mesangium into a hypocellular one by inducing mesangial cell (MC) apoptosis. Low density lipoprotein (LDL) enhanced (P < 0.001) mouse mesangial cell (MMC) proliferation at lower concentrations (control, 10.3 +/- 0.3 vs. LDL 100 micrograms/ml, 24.2 +/- 0.3 x 10(4) cells/ml) but augmented (P < 0.001) apoptosis at higher concentrations (control, 5.6 +/- 0.5% vs. LDL, 500 micrograms/ml 26.2 +/- 3.4% apoptotic cells/field). Oxidized (OX) LDL enhanced MMC apoptosis in concentrations of 50 to 200 micrograms/dl. There was a direct relationship between MMC apoptosis and oxidation of LDL as judged by measuring thiobarbituric acid reactive species (TBARS). Since superoxide dismutase (SOD) attenuated (P < 0.001) LDL-induced MMC apoptosis, it seems to be mediated through the generation of free radicals by mesangial cells (control, 4.3 +/- 1.5%; LDL, 200 micrograms/ml, 19.4 +/- 0.5%; LDL + SOD, 8.1 +/- 1.3% apoptotic cells/field). LDL also induced a similar effect on human mesangial cells. These studies were further confirmed by DNA fragment assays and ELISA for programmed cell death. LDL treated cells also showed enhanced mRNA expression for RSG-2, a marker for active cell death. These in vitro results provide a basis for the speculation that LDL has the potential to cause an initial hypercellular and subsequent hypocellular mesangium in the course of the development of glomerulosclerosis.     

Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal beta-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, O3, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7.5 microM, compared with a scrambled (MSO3) control oligo (n = 3, P < 0.01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1.78 +/- 0.13 (MSO3) vs 0.19 +/- 0.14 (O3) pmol/10(6) cells per 240 min, n = 7, P < 0.01). A significant increase in insulin mRNA (100 +/- 10% (MSO3) vs 124 +/- 8% (O3), n = 3, P < 0.05) and insulin content (13.0 +/- 0.9 (MSO3) vs 17.4 +/- 1.4 (O3) pmol/10(6) cells, n = 7, P = 0.028) was observed following treatment with O3 at 7.5 microM. O8, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than O3, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated beta-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.     

Bradykinin potentiates the chemoresponsiveness of rat cutaneous C-fibre polymodal nociceptors to interleukin-2

Endothelin-1 (Et-1) is a 21-amino acid peptide primarily synthesized by endothelial cells. It was originally classified as a potent vasoconstrictor but recent evidence suggests that it also possesses a wide variety of non-vascular actions. It stimulates fibroblast and smooth muscle cell proliferation and it has been shown to stimulate fibroblast collagen metabolism. However, studies on its ability to regulate collagen production remain incomplete, and its effect on post-translational processing of procollagen has not been studied. This report details the effect of Et-1 on the rates of procollagen synthesis and degradation in two fibroblast cell lines; human foetal lung (HFL-1) and whole foetal rat fibroblasts (Rat 2). Fibroblast cultures were incubated for 24 hr in the presence or absence of Et-1 before procollagen metabolism was determined by measuring hydroxyproline. Non-collagen metabolism was also determined in these cultures from the uptake of tritiated phenylalanine. Et-1 stimulated procollagen synthesis in HFL-1 fibroblasts and reduced synthesis in Rat 2 cells. The response was dose dependent with the greatest effect at 1.10(-6) M Et-1 for both cell types (155 +/- 6% of control (mean +/- SD, n = 6, P < 0.01) and 61 +/- 4% of control (n = 4, P < 0.01) for HFL-1 and Rat 2 fibroblasts, respectively). Non-collagen protein synthesis was increased to 148 +/- 5% of control (P < 0.05) at 1.10(-6) M Et-1. Non-collagen protein synthesis remained unaffected in the HFL-1 fibroblast cultures. Procollagen degradation, expressed as a proportion of total procollagen synthesis, was decreased in HFL-1 fibroblasts (control, 29 +/- 2%; Et-1, 1.10(-6) M; 21 +/- 2%; P < 0.01), and increased in Rat 2 fibroblasts (control 42 +/- 1%; Et-1, 1.10(-6) M; 49 +/- 1%; P < 0.01). Blocking of the EtA receptor for Et-1, using the receptor antagonist-BQ123, abolished the effect of Et-1 on procollagen metabolism in both cell types. These results suggest that different populations of fibroblasts exhibit heterogeneous responses to Et-1. It is concluded that Et-1 may play an important role in the extent and distribution of fibrosis seen in diseases associated with the overproduction of Et-1.     

The impact of early normalization of haematocrit by erythropoietin on renal damage in the remnant kidney model

BACKGROUND: Correction of anaemia in moderate to advanced renal failure is still a matter of debate because of postulated detrimental effects of erythropoietin on the progression of renal damage. METHODS: The renal effects of early normalization of haematocrit (Htc) by erythropoietin (rHuEpo) were investigated from the time of 5/6 nephrectomy up to 8 weeks post-intervention in three groups of remnant kidney model rats: untreated controls (CON), rats receiving 100 UI/kg body-wt of rHuEpo i.p. twice a week (EPO), and rats receiving rHuEpo in which periodic phlebotomies maintained Htc similar to the value of the control group (PHL). The latter group was included to evaluate the direct effects of rHuEpo on renal damage, i.e. independent from Htc correction. RESULTS: Two weeks after renal ablation (basal), Htc decreased in CON and PHL rats (from 49.3+/-1.4% to 43.2+/- 1.1, P < 0.05 and from 49.6+/-1.1 to 43.3+/-1.5%, P<0.05 respectively), while it remained consistently normal in EPO rats (48.9+/-1.2% to 48.9+/-1.50/%, P<0.05 vs other groups). Thereafter Htc did not change throughout the remaining period in all groups. At the end of the study, with respect to basal, resting blood pressure increased significantly by the same extent in CON (+ 13+/-2%) and EPO rats (+ 15+/-5%), while it remained constant in PHL rats. Notably, creatinine clearance significantly decreased in CON (-53+/-8% 8 vs basal) and EPO (-38+/-8% vs basal), while it did not change in PHL rats. Likewise the degree of proteinuria as well as renal morphologic alterations and glomerular hypertrophy/sclerosis was similar in CON and EPO rats, and was significantly more severe than in the phlebotomized group. The only difference detected between CON and EPO group was the greater mesangial hypercellularity in rHuEpo-treated rats. CONCLUSION: In uraemic rats, chronic treatment with rHuEpo aimed at normalization of Htc beginning the early stage of renal failure does not inevitably account for a rise in systemic blood pressure. In addition, neither erythropoietin per se nor the correction of haematocrit accelerates the progression of renal damage when blood pressure remains constant.     

Effects of elevated concentrations of prostaglandin F2 alpha on pregnancy rates in progestogen supplemented cattle

An experiment was performed to determine the effect of elevated prostaglandin F2 alpha (PGF2 alpha) on pregnancy rates of progestogen-treated bred cows in the presence or absence of luteal tissue. Ninety-one beef cows were bred (Day 0) and assigned randomly to receive either 3 mL saline (CON), 15 mg PGF2 alpha, or 15 mg PGF2 alpha + lutectomy (P + L) administered intramuscularly (i.m.) at 8 h intervals on either Days 5-8, 10-13, or 15-18 postbreeding. Lutectomies were performed by transrectal digital pressure before initiation of treatment on Day 5, 10, or 15 for the respective treatment groups. All cows were fed 4 mg/day of melengesterol acetate from two days prior to initiation of treatment until Day 30 postbreeding. Mean concentrations of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were increased in cows administered PGF2 alpha and P + L treatments (398 +/- 23 and 413 +/- 22 pg/ml, respectively; p < 0.01) compared to the CON group (80 +/- 29 pg/ml) regardless of treatment group. Mean concentrations of oxytocin (OT) were increased in cows given PGF2 alpha on Day 10 and 15 (p < or = 0.0001) and tended to be increased on d 5 when compared to CON and P + L treatment groups on Day 5. Pregnancy rates were reduced (p < or = 0.03) in the PGF2 alpha treatment group (23%) and by Day 5-8 compared to CON (72%). Lutectomy tended to improve pregnancy rate in P + L (5-8; 55%) compared to PGF2 alpha (5-8; p = 0.1). Pregnancy rates tended (p < or = 0.07) to increase in the PGF2 alpha treatment groups on Days 5-8 treatment (23%, 50%, and 60% for Days 5-8, 10-13, and 15-18, respectively). The later the treatments were initiated pregnancy rates did not differ between treatments given on Days 10-13 and 15-18. In conclusion, the most susceptible period of embryonic growth to the negative effects of PGF2 alpha was during morula to blastocyst development. Removal of luteal tissue diminishes the negative effects of PGF2 alpha through interruption of the luteal oxytocin-uterine PGF2 alpha feedback loop.     

Glomerular endothelial cells synthesize collagens but little gelatinase A and B

Mesangial sclerosis is a major feature of progressive renal disease. The mesangium contains mesangial cells and is bounded by the peripheral glomerular basement membrane and endothelial cells. Mesangial cells synthesize and degrade extracellular matrix. Whereas both mesangial and endothelial cells synthesize extracellular matrix components, the degradative pathway, well studied in the former, has not been investigated in endothelial cells. This study examines lines of all three glomerular cell types derived from female B6SJLF1/J mice, as well as mRNA levels for collagens alpha1(I), alpha1(IV), alpha3 (IV), alpha5 (IV), and alpha1 (VI), laminin, tenascin, matrix metalloproteinase-2 (MMP-2), and MMP-9. Type I and IV collagen synthesis was confirmed by enzyme-linked immunosorbent assay. MMP-2 and MMP-9 enzyme activity was measured by zymography. It was found that glomerular endothelial cells are a significant source of collagens, laminin, and tenascin. However, they express only low levels of MMP-2 and no detectable MMP-9. Stimulation with exogenous transforming growth factor-beta1 leads to a significant increase in collagen I, tissue inhibitors of metalloproteinase-1, and MMP-9 in conditioned media. These data suggest that glomerular endothelial cells may play an active role in extracellular matrix remodeling in glomerular disease.