Heterozygosity for factor V Leiden in a haemodialysis patient with recurrent shunt thrombosis.
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Catheters, Indwelling↗
Biomedical subjects
R M Schaefer
Publications and source records attributed to R M Schaefer.
Tubular gelatinase A (MMP-2) and its tissue inhibitors in polycystic kidney disease in the Han:SPRD rat.
Thickening of the tubular basement membrane is one of the hallmarks of the polycystic kidney disease (PKD). The present study was conducted to investigate the potential role of the matrix metalloproteinase-2 (MMP-2) and its specific tissue inhibitors (TIMP-1 and TIMP-2) in the accumulation of matrix components in PKD. As a model of PKD, two-month-old heterozygous Han:SPRD rats, which are at an early stage of cystogenesis, were used. MMP-2, but not MMP-9 (gelatinase B) nor MMP-3 (stromelysin) could be detected in proximal tubules of the normal rat kidney. The presence of the inhibitors TIMP-1 and TIMP-2 was confirmed on the mRNA level. In tubules from PKD rats MMP-2 activity was lower (31 +/- 8 vs. 58 +/- 7 U/prep., N = 9, P < 0.05), mRNA of MMP-2 was reduced 4.2 +/- 0.6-fold (N = 4, P < 0.05) and enzyme protein was depressed 3.8 +/- 0.8-fold (N = 4, P < 0.05). By contrast, TIMP-1 mRNA was 9.0 +/- 1.1-fold and TIMP-2 mRNA 3.8 +/- 0.7-fold (N = 4, P < 0.05) elevated over controls. Cyst fluid from homozygous rats contained MMP-2 protein and activity. These findings indicate that tubular MMP-2 activity is reduced in PKD, due to down-regulation of MMP-2, up-regulation of TIMP-1 and TIMP-2, and luminal secretion of the enzyme. It is conceivable that these alterations relate to the enhanced matrix accumulation observed in the evolution of PKD.
Animals↗
Alterations of cathepsins B, H and L in proximal tubules from polycystic kidneys of the Han:SPRD rat.
Abnormalities of tubular matrix metalloproteinases have been shown recently to occur early in the course of polycystic kidney disease (PKD). The present study was conducted to determine whether lysosomal cysteine proteinases were altered in proximal tubules from 2-month-old, heterozygous Han:SPRD rats. The activities of cathepsins B (-45%), H (-39%) and L (-37%) were significantly lower in proximal tubules from PKD rats as compared to healthy offspring. Enzyme proteins were also decreased (cath. B, 2.4 +/- 0.7-fold; cath. H, 1.9 +/- 0.6-fold; N = 4, P < 0.05), while mRNA levels for cathepsins B, H and L were not different. Tubular cystatin C, a major inhibitor of cathepsins, was normal with regard to protein and mRNA levels in PKD animals. The decrease in cathepsins in PKD was specific for tubules, as enzyme activities in glomeruli and liver tissue were unchanged and limited to the lysosomal compartment, since marker enzymes for cytoplasm, endoplasmatic reticulum and mitochondria were all normal. Intralysosomally, soluble enzymes like cathepsins and beta-NAG were decreased, while membrane-bound acid phosphatase was unchanged. The presence of cathepsins could be demonstrated in cyst fluid from homozygous PKD rats and urinary excretion of cathepsins was enhanced in heterozygous animals. Taken together, these findings indicate that the reduction in tubular cathepsins B, H and L was neither due to decreased gene expression nor to upregulation of specific inhibitors, but was likely due to enhanced apical secretion of these enzymes.
Animals↗
Treatment with erythropoietin and loss of dialyser clearance.
Hyperkalaemia as well as increased predialysis values for creatinine and phosphate have been reported in many clinical recombinant human erythropoietin (rHu-EPO) trials in haemodialysis patients. These problems were overcome in most cases by increasing the dose of dialysis. With the exception of urea clearance, solute clearances should decrease with increased haematocrit values. Urea in red blood cells is in diffusion equilibrium with plasma water and it can be assumed that urea clearance is only minimally affected by an increasing haematocrit. Equilibration between red cells and plasma for larger solutes (creatinine) is slow compared to dialyser transit time, so that intracellular creatinine is effectively prevented from participating in the dialytic exchange. Therefore, as the haematocrit increases, the amount of creatinine removed should decrease. These considerations are particularly important for red cell potassium and phosphate, which virtually do not equilibrate with plasma water. In fact, loss of clearance has been reported to range from 5% to 10% for urea and 15% to 20% for creatinine, potassium and phosphate after correction of anaemia. This is particularly relevant in the setting of rapid, high efficiency dialysis with treatment times of 2 h, where more frequent episodes of hyperkalaemia and increased requirements of phosphate binders have been reported. For those who prescribe dialysis by using urea modelling (Kt/V), the dialyser clearance K may have to be corrected for effective blood water clearance and the dose prescribed should be increased, because urea modelling will overestimate the clearance of other uraemic solutes.
Erythropoietin↗
How to diagnose and correct iron deficiency during r-huEPO therapy--a consensus report.
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Anemia, Iron-Deficiency↗
Role of lysosomal cathepsin activities in cell hypertrophy induced by NH4Cl in cultured renal proximal tubule cells.
An increase of renal ammoniagenesis has been implicated in renal hypertrophy associated with various clinical disorders such as metabolic acidosis, diabetic nephropathy, and renal insufficiency. In vivo and in vitro studies have shown that ammonia promotes hypertrophy in tubular epithelial cells. To elucidate its role on protein turnover, the effects of NH4Cl on the activities of cathepsins B, H, and L+B, as well as on protein synthesis and degradation in LLC-PK1 cells, were investigated. The results show that NH4Cl (20 mM) induced cell hypertrophy, as defined by an increase in both cell protein content and cell volume (+25.5 +/- 1.3 and +10.4 +/- 0.1% after 48 h). This hypertrophy was associated with the suppression of the activities of cathepsins B and L+B (-57.0 +/- 0.9 and -54.5 +/- 1.5% after 48 h) and a reduction of protein degradation rate (-59.7 +/- 4.1% after 48 h), but without enhanced protein synthesis. The findings were further supported with an additional experiment, showing that the protein synthesis inhibitor cycloheximide (10 microM) did not blunt NH4Cl-induced cell hypertrophy. Moreover, NH4Cl (20 mM) resulted in a persistent elevation of the lysosomal pH, whereas the rise in the cytosolic pH was only transient. This alkalinization in lysosomes may be causatively involved in the impairment of the activities of cathepsins B and L+B. In conclusion, the suppression of the activities of cathepsins B and L+B and the subsequent reduction of protein breakdown due to intralysosomal alkalinization contribute to NH4Cl-induced hypertrophy in LLC-PK1 cells.
Ammonium Chloride↗
Protein restriction influences glomerular matrix turnover and tubular hypertrophy by modulation of renal proteinase activities.
Following renal ablation, there is marked compensatory renal growth, which is associated with alterations in the activities of renal proteinases. In the present study, rats underwent 5/6 nephrectomy (5/6-NX). Sixteen weeks after surgery, glomeruli and tubules were isolated and proteinase activities were determined using fluorogenic peptidyl substrates. Following 5/6-NX, there was considerable compensatory renal growth resulting in a final weight of 1,923 +/- 46 mg for the remnant kidney as compared to 1,402 +/- 63 mg for the left kidney of SHAM animals. This hypertrophic response was associated with lower activities of tubular cysteine proteinases (cathepsin L & B: -43%; cathepsin B: -61%; cathepsin H: -53%). Significantly reduced activities were also observed for glomerular collagenase (20.2 +/- 6.2 vs. 53.4 +/- 5.7 mU/micrograms DNA) and gelatinase (24.1 +/- 5.0 vs. 130.8 +/- 8.4 mU/micrograms DNA) activities. Protein restriction (5 vs. 20% casein) considerably attenuated compensatory renal growth after surgical ablation (790 +/- 45 vs. 1,923 +/- 46 mg) and partially prevented the fall in tubular cathepsin activities. In terms of glomerular enzymes, protein restriction caused a significant increase in the activity of gelatinase from 24.1 +/- 5.0 to 66.7 +/- 9.2 mU/micrograms DNA, while collagenase remained unchanged. From these data, we conclude that compensatory renal growth is strongly influenced by the amount of protein ingested. It appears that this effect is mediated by modulation of renal proteinase activities.
Animals↗
Effects of parathyroid hormone on renal tubular proteinases.
Parathyroid hormone (PTH) has been implicated to exert detrimental effects on remnant nephrons in chronic renal failure. The present investigation addressed the influence of PTH on the proteolytic capacity of isolated proximal tubules both from normal (SHAM) and partially nephrectomized rats (5/6-NX). Proteolytic activities were measured either against azocasein (pH 5.4) or with specific fluorogenic peptidyl substrates for individual cysteine proteinases. Azocaseinolytic activity was enhanced 6 weeks after 5/6-NX in tubules (SHAM 19.0 +/- 1.0 vs. 5/6-NX 24.4 +/- 1.5 U/mg protein), while thereafter activities declined progressively with time (5/6-NX 16 weeks: 12.9 +/- 1.2 U/mg protein). This loss in proteolytic activity could almost completely be prevented by parathyroidectomy (PTX) (5/6-NX + PTX 16 weeks: 18.6 +/- 1.1 U/mg protein). By contrast, severe hyperparathyroidism (induced by a low calcium/high phosphorus diet fed for 6 weeks) in 5/6-NX animals resulted in a significant decline in proteolytic activities in remnant tubules (5/6-NX 24.4 +/- 1.5 vs. 5/6-NX+diet 16.4 +/- 1.9 U/mg protein). When specific activities of tubular cathepsins were measured in healthy rats who had received exogenous PTH, each individual cysteine proteinase (cathepsin L: -42%; cathepsin B: -27%; cathepsin H: -51%) was suppressed. This effect of PTH could readily be abolished by the simultaneous administration of verapamil. These results suggest that chronic PTH excess exerts a suppressive effect on tubular proteinase activities both in normal and partially nephrectomized rats. This PTH effect seems to be mediated by an increase of cytosolic calcium.
Animals↗
Low-density lipoprotein suppresses cathepsins B and L activity in rat mesangial cells.
Disturbances of lipid metabolism are considered to play a pathogenetic role in glomerulosclerosis. Since intraglomerular have been proposed to be involved in the pathogenesis of the glomerulosclerosis, we have investigated the influence of LDL on the activity of the cellular proteases. Cathepsins B and L were measured with the aid of fluorometry, and 7-amido-4-methylocoumarin derivates were used as substrates; Z-Arg-Arg-AMC for cathepsin B, Z-Phe-Arg-AMC for cathepsins B and L together. Rat mesangial cells cultured 24 h in medium supplemented with LDL revealed inhibition of cathepsin B activity at concentrations of 250 micrograms LDL/ml medium, lower LDL concentrations were without apparent effect. Since the glomerular accumulation of structural and nonstructural proteins plays an important role in glomerulosclerosis, we conclude that the augmented proteolytic activity of mesangial cells might be one of the pathways located by which hyperlipidemia causes an increased susceptibility to glomerular damage.
Animals↗
Dose-dependent stimulation/inhibition effects of cyclosporin A on lysosomal cathepsin activities in cultured proximal tubule cells.
The effects of cyclosporin A on the activities of lysosomal cysteine proteinases (cathepsin B, H, L+B) in LLC-Pk1 cells were investigated to elucidate their potential role in cyclosporin A-induced nephrotoxicity. Cyclosporin A at lower doses (0.1--1,000 ng/ml) stimulated cathepsin B, H, L+B. In contrast, at a higher dose (10,000 ng/ml), it inhibited these proteinase activities associated with a reduction in protein degradation. In line with the altered proteinase activities, cellular protein content was decreased at the lower dose (10 ng/ml) and increased at the higher dose. The higher dose of cyclosporin A also enhanced cellular lipid peroxide content after an exposure of 4 and 10 h. Co-incubation with superoxide dismutase (40 U/ml) did not ameliorate the inhibition of cathepsin B activity induced by the high dose of cyclosporin A. On the contrary, the calcium channel blocker verapamil (10(-6) M) prevented this inhibition. In conclusion, cyclosporin A exerts a dose-dependent biphasic effect on lysosomal cysteine proteinase activities. A rise in cytosolic Ca2+ concentration, but not an enhanced lipid peroxidation, may be involved in the suppression of cathepsin B activity induced by the higher dose of cyclosporin A. These studies raise the possibility that alterations of tubular proteinase activity may play a role in the cyclosporin A-induced nephrotoxicity.
Calcimycin↗
Prevention of cardiac hypertrophy in experimental chronic renal failure by long-term ACE inhibitor administration: potential role of lysosomal proteinases.
The pathogenesis of cardiac hypertrophy in chronic uremia is poorly understood. In the present study, the long-term effects of chronic uremia on cardiac morphology and various cysteine proteinases of the heart were investigated in rats with and without antihypertensive therapy by the angiotensin converting enzyme inhibitor enalapril or by the calcium channel blocker verapamil. 16 weeks after subtotal nephrectomy considerable uremia had developed associated with arterial hypertension, rise in heart weight and heart weight/body weight ratio. Morphologically myocardial cells developed marked hypertrophy. Determination of various cysteine proteinases by fluorometry revealed a significant decline of cathepsin B activity while the activities of cathepsin H and L were unchanged. Antihypertensive treatment with enalapril and verapamil normalized the blood pressure and improved renal function significantly. Myocardial cell hypertrophy and the enhanced heart weight/body weight ratio were normalized under treatment with enalapril but not with verapamil. Simultaneously, the impaired cathepsin B activity returned to the normal range after enalapril treatment. It is concluded that the cardiac hypertrophy in uremia is at least partly caused by an activation of the circulating and/or cardiac renin-angiotensin system. Impaired proteinase activity in the uremic state may be involved in the development of cardiac hypertrophy.
Animals↗
Insulin-like growth factor I induced reduction in cysteine proteinase activity in freshly isolated proximal tubule cells of the rat.
The potential effects of insulin-like growth factor I (IGF-I) on lysosomal cysteine proteinases (cathepsin B, H and L+B activities) were investigated in the freshly isolated proximal tubule cells of rats. IGF-I significantly inhibited these enzyme activities after an incubation time of 80 min. This effect was associated with a dose-dependent increase in cellular protein content. The study suggests that, besides the established enhanced protein synthesis, IGF-I-induced cellular hypertrophy is mediated by a suppression of the proteolytic enzyme activity in proximal tubular cells.
Animals↗
The hypochromic red cell: a new parameter for monitoring of iron supplementation during rhEPO therapy.
Bone marrow iron supply may become rate limiting for hemoglobin synthesis during rhEPO-stimulated erythropoiesis. In the present study we followed the occurrence of hemoglobin-deficient red cells as a parameter of iron-deficient erythropoiesis in rhEPO-treated dialysis patients. rhEPO-treated patients with iron overload displayed very low numbers of hypochromic red cells (1%), while those with iron-deficiency had a hypochromic subpopulation of 22% (normal range < 2.5% of circulating red cells). Prior to rhEPO treatment, 10 dialysis patients showed normal numbers of hypochromic red cells (2.1%), despite mild iron deficiency (transferrin saturation: 17%). Once rhEPO (150 U/kg/week) was started, the percentage of hypochromic red cells rose significantly to 15.3% within 4 weeks of therapy. This was readily reversed when intravenous iron (750 mg/4 weeks) was added to the therapeutic regimen (5.5% after 4 weeks of i.v. iron). Taken together, quantitative red cell analysis seems to be a reliable tool to detect iron-deficient erythropoiesis in rhEPO-treated dialysis patients.
Anemia↗
Role of proteinases in renal hypertrophy and matrix accumulation.
Graded compensatory renal growth was induced either by unilateral (UNX) or 5/6 nephrectomy (5/6-NX). Over the experimental period of 16 weeks, kidney weight increased by 59% in SHAM animals, while the remaining kidney in UNX rats more than doubled its initial weight. The hypertrophic response was most pronounced in the remnant kidney after 5/6-NX with a four fold increment in kidney weight. Morphologically glomerular volume increased moderately after UNX (+27%), while 5/6-NX was associated with marked glomerular hypertrophy (+87%). Significant focal sclerosis was found in 11% of glomeruli in the remaining kidney after UNX. By contrast 83% of glomeruli wre sclerosed in the remnant kidney after 5/6-NX. In parallel, there was a significant increase in the glomerular protein/DNA ratio (+23%) in 5/6-NX but not in UNX animals. These glomerular alterations were associated with lower glomerular cysteine and metalloproteinase activities (collagenase, -57%; gelatinase, -49%) in 5/6-NX rats, while UNX rats had normal glomerular proteinase activities. In terms of tubular proteinases, cathepsin activities were significantly lower in UNX rats (cath. L+B, -38%; cath. B, -37%; cath. H, -27%) and more so after 5/6-nephrectomy (cath. L+B, -72%; cath. B, -73%; cath. H, -73%), while metalloproteinase activities were only reduced in 5/6-NX rats (collagenase, -35%; gelatinase, -58%). These findings demonstrate that kidney hypertrophy is associated with reduction in renal proteinase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
Acid Phosphatase↗
Antihypertensive treatment with a vasodilating beta-blocker, carvedilol, in chronic hemodialysis patients.
Carvedilol is an antihypertensive agent which displays unselective beta-blocking, alpha 1-blocking and antioxidant properties. It is primarily metabolized by the liver and excreted via the biliary system. The compound is highly lipophilic and strongly bound to plasma proteins. Consequently, there is no elimination during hemodialysis. The efficacy, safety, and pharmacokinetic profile of carvedilol titrated to effect were investigated in an open clinical trial in 15 long-term hemodialysis patients with arterial hypertension over a period of 12 weeks. The drug was administered only on days without dialysis. After a wash-out phase of one week, carvedilol was started in a dose of 12.5 mg per day. All 15 patients were titrated according to the antihypertensive effect to a daily dose of 25 mg of carvedilol. Carvedilol was effective in lowering blood pressure in hemodialysis patients (RR systolic: 170 +/- 11 vs. 144 +/- 9 mmHg; RR diastolic: 98 +/- 10 vs. 85 +/- 10 mmHg). The pharmacokinetic parameters of carvedilol and its active metabolite M2, assessed in 12 of the 15 patients, were not influenced by the lack of renal function or intermittend haemodialysis. In particular, there was no accumulation of carvedilol or its metabolite M2. In terms of side effects, three patients had to be withdrawn from the trial, because of hypoglycemia (n = 1), insufficient blood pressure control (n = 1) and prolonged hypotension (n = 1). Taken together, these results indicate that carvedilol is a safe and efficacious antihypertensive agent which can be used in patients maintained by maintenance dialysis treatment.
Adrenergic beta-Antagonists↗
Angiotensin-II-induced cell hypertrophy: potential role of impaired proteolytic activity in cultured LLC-PK1 cells.
BACKGROUND: Angiotensin II-induced hypertrophy of both mesangial and tubular cells has been shown to be caused by enhanced protein synthesis. There are no data about its role on protein breakdown. Therefore, protein turnover and proteolytic activities were investigated in LLC-PK1 cells. METHODS: Protein turnover was measured by determining the incorporation and release of [14C]phenylalanine; collagenolytic and gelatinolytic activities were assayed by using fluorogenic peptidyl substrates. RESULTS: Angiotensin II (10(-8)-10(-6) M) exerted a dose-dependent inhibition of collagenolytic and gelatinolytic activities, associated with reduction of protein degradation rate. In addition angiotensin II stimulated protein synthesis in the cells. These combined effects on protein turnover resulted in an increase in both cell size and cell protein content (31.7% after 48 h). However, the rise of cell protein content was only partly (48.0%) prevented by the protein synthesis inhibitor cycloheximide (10(-5)M), which supports the role of decreased protein degradation in the angiotensin-II-induced cell hypertrophy. The angiotensin-II-induced effects on proteolytic activities as well as on cell protein content could be abolished by coincubation with the angiotensin II type I-receptor antagonist DuP 753 (10(-6)M). The calcium-channel blocker verapamil (10(-6)M) ameliorated the impairment of collagenolytic activity. On the contrary the calcium ionophore A23187 (10(-6)M) mimicked the action of angiotensin II on this enzyme activity (control 34.5 +/- 1.9; angiotensin II 24.0 +/- 2.0; A23187 23.0 +/- 2.2 and angiotensin II+verapamil, 33.8 +/- 2.6 pmol/min/micrograms DNA). The role of cytosolic [Ca2+] in the actions of angiotensin II could be finally shown by a dose-dependent rise which was partly blunted by verapamil. CONCLUSION: The angiotensin-II-induced hypertrophy in LLC-PK1 cells is caused not only by enhanced protein synthesis but also by reduced protein degradation. The concomitant decline of collagenolytic and gelatinolytic activities may contribute to the accumulation of extracellular matrix, and presumably also to cell hypertrophy. These effects are obviously mediated via angiotensin II type I receptors and seem to be [Ca2+] dependent.
Analysis of Variance↗
Renal proteinases and kidney hypertrophy in experimental diabetes.
IDDM is associated with an increase in kidney size, which is due to cellular hypertrophy and progressive matrix accumulation within the glomerulus and throughout the tubulo-interstitium. The present study addressed the potential role of cysteine and metalloproteinases in renal hypertrophy of short-term diabetes. Three weeks after induction of streptozotocin diabetes in rats, intraglomerular gelatinase activity (streptozotocin: 23 +/- 4 vs control: 44 +/- 3 mU/microgram DNA) and cathepsin L+B activity (streptozotocin: 6.7 +/- 0.8 vs control: 9.3 +/- 0.7 U/microgram DNA) were significantly decreased. Insulin treatment completely prevented the decline in glomerular proteinase activity (gelatinase: 37 +/- 6 mU/microgram DNA; cathepsin L+B: 9.6 +/- 0.9 U/microgram DNA). In isolated proximal tubules a similar pattern of enzyme activity could be observed. Three weeks of diabetes caused a significant decline in cathepsin L+B activity (streptozotocin: 28 +/- 2 vs control: 37 +/- 3 U/microgram DNA). Insulin treatment again prevented the decline in these tubular proteinase activities. In parallel, kidney weight increased by 22% and glomerular protein/DNA ratio rose by 17% in untreated diabetic rats. Diabetic rats receiving insulin displayed a normal glomerular protein/DNA ratio and the kidney weight was increased by only 5%. These results show that renal hypertrophy of early diabetes is closely associated with a decline in both glomerular and tubular proteinase activity. Adequate insulin substitution prevented renal hypertrophy and the reduction in proteinase activity.
Animals↗
Lovastatin ameliorates depressed intraglomerular proteolytic activities in experimental nephrotic syndrome.
Lipid abnormalities have been implicated in the pathogenesis of glomerulosclerosis in experimental models of kidney disease. In previous studies it has been shown that Adriamycin-induced nephropathy is associated with reduced activities of glomerular proteinases. This observation led to the hypothesis that reduced proteolytic activities may be responsible for mesangial protein accumulation, which ultimately leads to global sclerosis of the glomerular tuft. The aim of the present study was to investigate whether lovastatin treatment, which prevents progressive glomerulosclerosis in experimental nephrotic syndrome, would also have an effect on glomerular proteinase activities. Adriamycin administration resulted in a persistent nephrotic syndrome with gross proteinuria (377 +/- 26 mg/24 h), hypoalbuminemia (2.1 +/- 0.12 vs. 2.8 +/- 0.02 g/dl), hypercholesterolemia (575 +/- 74 vs. 68 +/- 1.5 mg/dl) and elevated triglyceride levels (1,155 +/- 78 vs. 57 +/- 8 mg/dl). Glomerular azocaseinolytic activities both at pH 5.4 (-21%) and 7.4 (-37%) were significantly reduced. In contrast to human subjects, nephrotic rats that were treated with lovastatin displayed reduced triglyceride levels (767 +/- 134 mg/dl); their serum cholesterol, however, remained unchanged. In terms of glomerular proteolytic enzyme activities, the decline in azocaseinolysis at both pH values was, at least partly, prevented by lovastatin. On the basis of these data, it appears that the beneficial effect of lovastatin on the evolution of glomerulosclerosis in the nephrotic rat is associated with the conservation of glomerular proteolytic activities.
Albuminuria↗