Immunosuppression enhances atherogenicity of lipid profile after transplantation.

BACKGROUND: Patients after renal transplantation exhibit high cardiovascular morbidity and mortality because of the accumulation of cardiovascular risk factors such as hypertension or dyslipidemia. To elucidate the influence of immunosuppressive therapy on hyperlipidemia, we studied serum lipids and lipoproteins in renal transplant patients who received prednisone and either azathioprine or cyclosporine or triple immunosuppressive therapy. METHODS: Serum lipids and lipoprotein levels were measured in 216 renal transplant patients (81 female and 135 male) with stable graft function of 4.8 +/- 2.3 years (range six months to eight years) after transplantation. Patients were divided into three groups according to one of the following immunosuppressive regimens: (a) prednisone and azathioprine, (b) prednisone and cyclosporine, or (c) prednisone, azathioprine, and cyclosporine. Healthy, age- and sex-matched subjects served as controls. In addition to measurement of total serum lipids, lipoproteins were isolated by preparative ultracentrifugation, and lipids were determined in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) density classes. RESULTS: Total serum triglycerides, VLDL, and LDL triglycerides, as well as VLDL cholesterol were elevated in all renal transplant patients, but elevation was pronounced in female patients. In contrast to total serum cholesterol, which was significantly increased in only female patients, elevation of LDL-triglyceride/apo B ratio was more marked in male patients. Patients in group A exhibited only mild hypertriglyceridemia, whereas triglyceride enrichment in VLDL and LDL was more distinct in group B and was most pronounced in patients of group C. Furthermore, hypertriglyceridemia increased with the dose of administered prednisone. CONCLUSIONS: Immunosuppressive therapy in renal transplant patients leads to accumulation of triglyceride-enriched VLDL and LDL. Triglyceride enrichment in LDL indicates the accumulation of small, dense LDLs, which are known to bear enhanced atherosclerotic risk. This study provides data that underline the use of individually adjusted immunosuppressive therapy and steroid-sparing protocols in renal transplant patients to improve their atherogenic lipoprotein profile.

Abnormalities in lipoprotein metabolism in hemodialysis patients.

BACKGROUND: Patients on chronic hemodialysis treatment are at elevated atherogenic risk, and dyslipidemia appears to be one of the major risk factors. However, most of these patients exhibit elevated serum triglycerides, whereas serum cholesterol and low-density lipoprotein (LDL) cholesterol levels are in the normal range. This study was therefore designed to examine the influence of hypertriglyceridemia under the condition of hemodialysis and diabetes mellitus on LDL metabolism. METHODS: LDL was isolated from healthy controls, hypertriglyceridemic diabetic patients, and nondiabetic hemodialysis patients (N = 30, 10 in each group), which were separated into six subfractions by density gradient ultracentrifugation and were characterized concerning lipid/protein composition, degree of glycation, and oxidation. Uptake of 125I-labeled LDL was examined via LDL receptors of HepG2 cells and scavenger receptors of mouse peritoneal macrophages. RESULTS: In hemodialysis patients, serum triglycerides were significantly elevated, whereas cholesterol levels were within the normal range. Triglyceride enrichment occurred in the very low-density lipoprotein (VLDL) class and LDL class, and an accumulation of a highly atherogenic small dense LDL subfraction could be detected predominantly in patients with non-insulin-dependent diabetes mellitus. LDL of hemodialysis patients also contained elevated levels of lipid peroxidation products, which were even higher in diabetic patients. Alterations in composition, size, and configuration of LDL from diabetic and nondiabetic patients on hemodialysis impaired LDL receptor-mediated degradation and enhanced the uptake of these modified LDL particles via nonsaturable scavenger receptors. CONCLUSION: Diminished LDL receptor-mediated uptake of modified, triglyceride-rich, small dense LDL most likely leads to accumulation of these lipoproteins in vivo, favoring the development of atherosclerotic lesions. Future clinical studies must demonstrate whether patients will benefit from reducing these atherogenic particles by lipid-lowering intervention.

Non-insulin-dependent diabetes mellitus and hypertriglyceridemia impair lipoprotein metabolism in chronic hemodialysis patients.

Patients with diabetes mellitus undergoing chronic hemodialysis treatment have the worst outcome on dialysis due to an increased rate of cardiovascular complications. Nearly all patients present with dyslipidemia, a prominent vascular risk factor, probably responsible for the high rate of vascular injury. Since both uremia and diabetes predispose to hypertriglyceridemia, the present study was conducted to investigate the influence of diabetes mellitus and/or hypertriglyceridemia on lipoprotein metabolism in hemodialysis patients. LDL was isolated and characterized from hyper- and normotriglyceridemic diabetic and nondiabetic hemodialysis patients (n = 40; 10 in each group); also, LDL-receptor-dependent uptake and intracellular cholesterol metabolism were studied in HepG2 cells. In addition, scavenger-receptor-mediated uptake was examined in mouse peritoneal macrophages. LDL isolated from nondiabetic normotriglyceridemic hemodialysis patients exhibited impaired cellular uptake via the LDL receptor. Additionally, intracellular sterol synthesis was less inhibited and cholesterol esterification was reduced compared with LDL from healthy control subjects. Reduction of catabolic capacities was more marked in hemodialysis patients who were either diabetic or hypertriglyceridemic and even more pronounced in patients presenting with a combination of both diabetes and hypertriglyceridemia. Hypertriglyceridemic and diabetic patients showed reduced lipase activity and increased LDL oxidation. Furthermore, they accumulated a fraction of small, dense LDL, and LDL was predominantly taken up via the scavenger-receptor pathway in peritoneal macrophages. This study elucidates the distinct influence of diabetes and/or hypertriglyceridemia in hemodialysis patients on cellular LDL metabolism via specific and nonspecific metabolic pathways. Furthermore, it underscores the cumulative impact of these pathologic entities on impairment of lipoprotein metabolism and increase of cardiovascular risk.

Effect of osmolarity on LDL binding and internalization in hepatocytes.

The present study has been performed to elucidate a possible role of cell volume in low-density lipoprotein (LDL) binding and internalization (LDL(b+i)). As shown previously, increase of extracellular osmolarity (OSMe) and K+ depletion, both known to shrink cells, interfere with the formation of clathrin-coated pits and thus with LDL(b+i). On the other hand, alterations of cell volume have been shown to modify lysosomal pH, which is a determinant of LDL(b+i). LDL(b+i) have been estimated from heparin-releasable (binding) or heparin-insensitive (internalization) uptake of 125I-labeled LDL. OSMe was modified by alterations of extracellular concentrations of ions, glucose, urea, or raffinose. When OSMe was altered by varying NaCl concentrations, LDL(b+i) decreased (by 0.5 +/- 0.1%/mM) with increasing OSMe and LDL(b+i) increased (by 1.2 +/- 0.1%/mM) with decreasing OSMe, an effect mainly due to altered affinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 micro/ml at 219, 293, and 435 mosM, respectively. A 25% increase of OSMe increased cytosolic (by 0.46 +/- 0.03) and decreased lysosomal (by 0.14 +/- 0.02) pH. Conversely, a 25% decrease of OSMe decreased cytosolic (by 0.28 +/- 0.02) and increased lysosomal (by 0.17 +/- 0.02) pH. Partial replacement of extracellular Na+ with K+ had little effect on LDL(b+i), although it swelled hepatocytes and increased lysosomal and cytosolic pH. Hypertonic glucose, urea, or raffinose did not exert similar effects despite a shrinking effect of hypertonic raffinose. Monensin, which completely dissipates lysosomal acidity, virtually abolished LDL(b+i). In conclusion, the observations reveal a significant effect of ionic strength on LDL(b+i). The effect is, however, not likely to be mediated by alterations of cell volume or alterations of lysosomal pH.

Corticotropin increases the receptor-specific uptake of native low-density lipoprotein (LDL)--but not of oxidized LDL and native or oxidized lipoprotein(a) [Lp(a)]--in HEPG2 cells: no evidence for Lp(a) catabolism via the LDL-receptor.

To understand the interaction of corticotropin (ACTH) and lipid catabolism, we analyzed the influence of ACTH on receptor-mediated lipoprotein uptake and compared the uptake and degradation of human native (N-LDL) and oxidized (Ox-LDL) low-density lipoprotein and native (N-Lp(a)) and oxidized (Ox-Lp(a)) lipoprotein(a) by human hepatoma (HepG2) cells. The receptor affinity of N-LDL, Ox-LDL, N-Lp(a), and Ox-Lp(a) was comparable (Kd, 33, 13, 24, and 13 micrograms/mL medium), whereas the maximum degradative capacity was 10.5-fold higher in N-LDL (Vmax, 1,978 ng/mg cell protein) compared with Ox-LDL (189 ng/mg). In N-LDL, it was 4.5-fold higher than in N-Lp(a) (442 ng/mg) and eightfold higher than in Ox-Lp(a) (246 ng/mg) (P < .05). Addition of ACTH to the cell cultures increased receptor-specific degradation of N-LDL by 44% (2,866 v 1,978 ng/mg, P < .05), whereas changes in Ox-LDL, N-Lp(a), and Ox-Lp(a) showed no significant increase. No differences in uptake specificity were observed with or without ACTH. In addition, a 12-hour preincubation of liver cells with LDL increased Lp(a) uptake by 40% to 50% with (411 v 620 ng/mg) and without (393 v 558 ng/mg) ACTH administration. These data indicate that ACTH elevates receptor-specific uptake of N-LDL, but only to a low extent versus Ox-LDL, N-Lp(a), or Ox-Lp(a). These results support the hypothesis that catabolism of oxidized lipoproteins and Lp(a) through the LDL receptor pathway is only a minor route of lipid metabolism, whereas LDL receptor activity itself can be stimulated by ACTH.

Uptake and metabolism of lipoproteins from patients with diabetes mellitus type II by glomerular epithelial cells.

BACKGROUND: Recent studies suggest that dyslipidaemia accelerates the progression of diabetic nephropathy, but the various pathomechanisms underlying such abnormalities are not completely delineated. METHODS: We isolated, radiolabelled, and characterized very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) from eight diabetic patients with moderate impairment of renal function and dyslipidaemia and studied their interaction with LDL receptors in human glomerular epithelial cells. RESULTS: While diabetic VLDL showed no compositional changes, LDL particles contained a higher proportion of triglycerides at the expense of cholesterol in comparison with healthy controls. Despite differences in composition, both VLDL and LDL from patients exhibited reduced receptor affinity and cellular uptake capacity by glomerular epithelial cells. Since LDL composition was altered intracellular cholesterol homeostasis was investigated. Due to reduced cholesterol content and lower uptake capacity, diabetic LDL were less effective in suppressing intracellular sterol synthesis and in activating acylcholesterol acyltransferase than LDL from controls. Electrophoretic mobility of apoB from diabetic patients was enhanced as compared to controls, most probably due to the higher degree of glycation (17 + 1.7 versus 11 + 1%, P < 0.05) but not to oxidation (TBARS 0.5 + 0.2 versus 0.2 + 0.1 mumol/1). Oxidized LDL was not taken up in significant amounts, indicating no scavenger receptor activity in glomerular epithelial cells. CONCLUSION: The receptor-specific uptake of diabetic VLDL and LDL by glomerular epithelial cells is impaired. Compositional changes of the LDL particle and glycation of the protein moiety may contribute to altered glomerular uptake. However, glycation of the protein moiety may be superior to compositional changes. Because glomerular structures like mesangial matrix and endothelial cells are known for preferential binding of modified lipoproteins, further studies are required to elucidate their potential role in the progression of diabetic glomerulosclerosis.

UTP and ATP induce different membrane voltage responses in rat mesangial cells.

UTP and ATP induce different membrane voltage responses in rat mesangial cells. Recent studies have indicated that UTP and ATP might modulate mesangial cell function in a different manner. Here we compared the effect of UTP and ATP on membrane voltage (Vm) and ion currents in mesangial cells in primary culture, and we examined whether different nucleotide receptors are involved. In patch-clamp experiments in the fast whole cell configuration, UTP (in contrast to ATP) caused a sustained and concentration-dependent depolarization (half-maximal effective dose, 10(-5) M), but ATP caused only a transient depolarization. During the depolarization, UTP induced a sustained increase of the whole cell conductance (Gm), whereas ATP induced only a transient increase of Gm. When cells were dialyzed with Cs2SO4 and extracellular Cl- was replaced by 145 mM sodium gluconate, addition of UTP or ATP (both 10(-4) M) did not significantly increase Gm. Addition of ATP in the presence of UTP caused an additional depolarization by 5 mV, which was followed by a hyperpolarization by 21 mV. Repetitive application of ATP led to an attenuation of the ATP-induced depolarization. Then, in the presence of ATP, UTP still induced a significant depolarization by 10 mV. Suramine and reactive blue 2 did not inhibit the depolarization induced by UTP, but these inhibited the Vm response to ATP. In microfluorescence experiments, UTP and ATP caused a concentration-dependent increase of the intracellular calcium activity ([Ca2+]i) in mesangial cells. Application of both UTP and ATP had no additive effect on [Ca2+]i. The results suggest that mesangial cells possess, in addition to P2y purinoceptors, separate nucleotide receptors for UTP.

Structural and compositional modifications of diabetic low-density lipoproteins influence their receptor-mediated uptake by hepatocytes.

Dyslipoproteinaemia is an important risk factor for the development of atherosclerosis in noninsulin-dependent diabetes mellitus (NIDDM). This study shows that the uptake of low-density lipoproteins (LDLs) prepared from the plasma of patients with NIDDM in cultured human hepatoma cells is largely reduced. In addition, diabetic LDL was less effective in suppressing intracellular cholesterol synthesis. This is because of physicochemical and biochemical differences between lipoproteins from diabetic and from normal individuals. LDL from patients with NIDDM was abnormal with regard to charge, the degree of glycation, the lipid composition and the conformation of the apolipoprotein B receptor-binding domain. The diminished receptor-mediated uptake of apolipoprotein B-containing lipoproteins in diabetic individuals most probably leads to the accumulation of these lipoproteins in vivo and may be of great importance to the pathogenesis of atheroclerosis in these patients.

Lipids and progression of renal disease: role of modified low density lipoprotein and lipoprotein(a).

Atherogenic lipoproteins accumulate in the arterial wall as well as within the glomerulus and may accelerate vascular and glomerular injury. We therefore assessed whether oxidized low density lipoprotein (LDL) and lipoprotein(a) [Lp(a)] influence three major systems: (i) endothelium-dependent vasodilation, (ii) renin release of juxtaglomerular (JG) cells, and (iii) proliferation and viability of mesangial cells (MC). Lipoproteins were prepared from human plasma. Renal arteries were obtained from rabbits and JG as well as MC cells from mouse, rat and human kidneys. Dilator responses were detected in isolated arterial segments by a photoelectric device. Renin activity of JG cells was measured in culture supernatants and cells and DNA synthesis by 3H-thymidine incorporation in MC. Acetylcholine-induced, endothelium-dependent dilator responses of renal arteries were not significantly attenuated after incubation with native Lp(a). However, exposure to in vitro oxidized Lp(a) suppressed dilator responses in a dose-dependent manner. Using a chemiluminescence assay, we could detect increased O2- production by arteries pretreated with oxidized Lp(a), which suggested that enhanced nitric oxide (NO) inactivation by O2- might be the underlying mechanism of impairment of endothelium-dependent dilations. In general, oxidized Lp(a) was far more potent than oxidized LDL in this effect. In JG cells, both oxidized LDL and Lp(a) dose-dependently stimulated renin release. Coincubation with HDL significantly suppressed oxidized LDL and Lp(a) stimulated renin release and O2- production. In MC native and oxidized Lp(a) were poor ligands for the LDL receptor, but bound more tightly to extracellular matrix than native LDL. Native and oxidized Lp(a) elicited proliferation or toxicity of MC in a dose-dependent fashion. Stimulation of DNA synthesis in MC or renin release in JG cells was partly blunted or eliminated when cells were incubated with oxidized LDL and Lp(a) in the presence of superoxide dismutase and catalase, enzymes removing O2- and H2O2. These dat suggest a common underlying mechanism. Atherogenic lipoproteins induce formation of oxygen radicals not only in arteries, but also in glomeruli and JG cells, causing an inhibition of nitric oxide mediated vasodilation, stimulation of renin release, and modulation of mesangial cell growth and proliferation. The damaging effect of the lipoproteins can be prevented by antioxidative enzymes and HDL.

Use of HMG-CoA Reductase Inhibitors after Kidney and Heart Transplantation: Lipid-Lowering and Immunosuppressive Effects.

Hypercholesterolaemia is common in patients treated with cyclosporin after kidney and heart transplantation; coronary vasculopathy, graft atherosclerosis or cardiovascular complications are the most frequent causes of mortality. Coronary heart disease has been attributed to hypercholesterolaemia and has been identified as a major risk factor of long term graft outcome in patients after kidney transplantation. HMG-CoA reductase inhibitors have been proven to be effective in lowering serum cholesterol concentrations in kidney and heart graft recipients receiving long term cyclosporin immunosuppression, and are therefore the drugs of choice in patients requiring treatment for hypercholesterolaemia after organ transplantation. The hydrophilic HMG-CoA reductase inhibitors, such as pravastatin and fluvastatin, should be distinguished from the lipophilic agents, lovastatin and simvastatin, with regard to toxicity and accumulation. Maximal doses of drugs in the latter group should be avoided, whereas the former have been administered at high dosages over prolonged periods of time without adverse effects. Recent preliminary data indicate that treatment with pravastatin not only decreases serum cholesterol but may have beneficial effects on the incidence, recurrence and severity of rejection episodes after kidney and heart transplantation.

Receptor-mediated lipoprotein uptake by human glomerular cells: comparison with skin fibroblasts and HepG2 cells.

BACKGROUND: Currently the mechanisms of glomerular lipid accumulation are not completely understood. The present study characterizes the mechanisms of lipid uptake by glomerular cells. Since renal diseases are frequently associated with an accumulation of apoE-containing triglyceride-rich lipoproteins, we were interested to investigate whether glomerular epithelial or mesangial cells possess VLDL receptors besides the well established LDL receptors. METHODS: Uptake kinetics of 125I-labelled very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) in human glomerular epithelial and mesangial cells were compared to lipid uptake in cells with established receptor status, i.e. human skin fibroblasts and HepG2 cells. RESULTS: Glomerular epithelial cells, mesangial cells, and skin fibroblasts as well as hepatocytes express VLDL receptor mRNA, indicating that they exhibit VLDL receptors. VLDL uptake in glomerular epithelial cells, mesangial cells and skin fibroblasts occurred with a lower specificity than in HepG2 cells (-25%). No differences were found for the specificity of LDL uptake. VLDL uptake in HepG2 cells was inhibited more effectively with VLDL than with LDL. In skin fibroblasts, glomerular epithelial and mesangial cells, VLDL and LDL were equally effective inhibitors of VLDL uptake. The degradation-uptake ratio of VLDL in glomerular cells was elevated 50% compared to HepG2 cells, suggesting highly efficient intracellular lipoprotein turnover in these cells. CONCLUSION: We conclude that glomerular epithelial and mesangial cells as well as skin fibroblasts and HepG2 exhibit VLDL receptors additionally to their LDL receptors, even though the regulation of the VLDL receptor in HepG2 cells seems to differ from the regulation in glomerular epithelial and mesangial cells. The high degradation-uptake-ratio in these renal cells suggests the presence of an effective clearance pathway which might serve as protection against lipoprotein accumulation.

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively.

We have separated and quantified two new minor hemoglobins named HbA1d3a and HbA1d3b. The level of HbA1d3a was significantly higher in uremic than in non-uremic patients (3.00 +/- 0.50% vs. 1.28 +/- 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r = 0.80, n = 81, p < 0.002) and with plasma urea concentration (r = 0.78, n = 81, p < 0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA1d3a is an alpha-chain modified by carbamylation. The HbA1d3b level an diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 +/- 0.49 vs. 1.90 +/- 0.33). This was attributed to HbA1d3 modified by glycation. Indeed HbA1d3b correlated significantly with HbA1c (r = 0.71, p < 0.002) and with serum glucose level (r = 0.62, p < 0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averaged long-term uremia and glycemia in uremic and diabetic patients.

Is the determination of LDL cholesterol according to Friedewald accurate in CAPD and HD patients?

Lipid abnormalities are a major cause of accelerated atherosclerosis in patients with end-stage renal disease. In many clinical laboratories, the concentration of low density lipoproteins (LDL), the most atherogenic lipoprotein fraction, is estimated by calculating LDL cholesterol according to Friedewald. Hypertriglyceridemia, a common finding in uremic patients, is a main limitation to the use of the Friedewald formula, and the estimation of LDL cholesterol may, therefore, not be reliable in these patients. As accurate quantitation of LDL cholesterol is needed to decide on the initiation of lipid lowering therapy, we have evaluated the accuracy of the Friedewald formula in 171 patients on continuous ambulatory peritoneal dialysis (CAPD), 136 hemodialysis (HD) patients and 887 clinically healthy individuals by comparing it with a combined ultracentrifugation and precipitation 'reference' method. When we excluded sera with total triglycerides above 400 mg/dl [4.56 mmol/l], the Friedewald formula correlated excellently with the reference method; non-parametric correlation coefficients were 0.976, 0.971, and 0.956 in clinically healthy individuals, CAPD and HD patients, respectively. In the control individuals, the Friedewald formula produced slightly lower concentrations of LDL cholesterol than the reference method (means: 142 +/- 40 mg/dl vs 150 +/- 39 mg/dl or 3.68 + 1.04 mmol/l vs. to 3.89 + 1.01 mmol/l, respectively). This was also true in HD patients (means: 145 +/- 51 vs. 146 +/- 49 mg/dl or 3.76 +/- 1.32 vs. 3.78 +/- 1.27 mmol/l, respectively), but not in CAPD patients (means: 165 +/- 50 vs. 162 +/- 47 mg/dl or 4.27 +/- 1.30 vs. 4.20 +/- 1.22 mmol/l). Our data show that, unlike in other forms of secondary dyslipoproteinemia, the Friedewald formula is sufficiently reliable in patients with end-stage renal disease. Much the same, however, as in control individuals, other methods to quantify LDL cholesterol like ultracentrifugation or lipoprotein electrophoresis are recommended when serum triglycerides exceed 400 mg/dl [4.56 mmol/l].

Potential role of lipids in the progression of diabetic nephropathy.

Dyslipoproteinemia in non-insulin-dependent diabetes mellitus (NIDDM) is an important risk factor in the development of atherosclerosis and glomerulosclerosis. The lipid profile of NIDDM patients is characterized by elevated serum triglycerides and VLDL levels and reduced HDL cholesterol levels. Serum LDL levels may be elevated as well in some patients with NIDDM, but several alterations in the biochemical and physical properties of LDL particles are more characteristic resulting in reduced receptor specific uptake of these lipoproteins. Non-enzymatic glycosylation of LDL and augmented oxidation is common in diabetic patients making lipoproteins susceptible for uptake by the macrophage scavenger receptors and thus leading to foam cell formation and further glomerular damage. A reduction in the progression of diabetic nephropathy by lowering proteinuria and thereby serum cholesterol during treatment with ACE-inhibitors demonstrates the importance of such a therapy. The multiple factors involved in the pathogenesis of diabetic nephropathy are difficult to evaluate in regard to their individual contribution. Nevertheless antiproteinuric and lipid lowering therapy can be expected to reduce vascular damage and the progression of diabetic nephropathy.

Effects of lipoprotein(a) on mesangial cell proliferation and viability.

BACKGROUND: Lipoprotein abnormalities are considered to accelerate glomerular injury in various forms of renal disease, probably affecting mesangial proliferation. Serum levels of the atherogenic Lipoprotein(a) (Lp(a)) are elevated in patients with nephrotic syndrome and Lp(a) deposits have been identified in diseased glomeruli. So far, the influence of Lp(a) on mesangial cell function has not been defined. METHODS: The influence of Lp(a) on mesangial cell proliferation was assessed in a rat mesangial cell culture model by direct measurement of cell growth as well as analysis of DNA-synthesis and mRNA levels of c-fos and c-myc, two growth-associated 'immediate early response genes'. Results. Lp(a) triggered a biphasic response on DNA synthesis: 3H-thymidine uptake was increased when cells were incubated with Lp(a) (2.5-10 microg/ml) for 24 h. The response was dose dependent, a maximal effect was seen for Lp(a) 5 microg/ml. The stimulatory properties of Lp(a) were comparable to 10% fetal calf serum (FCS). No additive effect of 10% FCS and Lp(a) on DNA synthesis was observed. Cell proliferation was moderately stimulated (120+/-9% of control) by low levels of Lp(a) in the presence of small amounts of FCS. Messenger RNA levels for c-fos and c-myc were upregulated as early as 15 min after incubation with Lp(a) 5 microg/ml, a maximum response was observed after 20 and 240 min respectively. Stimulation of DNA synthesis was partly blunted when cells were incubated with Lp(a) in the presence of catalase 100 U/ml and superoxide dismutase 10(-7)M (SOD) but not in the presence of SOD alone. Lp(a) in concentrations above 10 microg/ml depressed DNA-synthesis and elicited signs of cytotoxicity. The cytotoxic effects of Lp(a) were not blunted by oxygen radical scavengers. The stimulatory and cytotoxic effects of Lp(a) were not restricted to specific isoform. CONCLUSION: Low concentrations of Lp(a) stimulated growth of mesangial cells, whereas higher concentrations had antiproliferative or toxic effects. The stimulation on mesangial cell proliferation as well as the cytotoxic effects caused by Lp(a) are both likely to have a negative impact on the course of renal disease.

Interaction of native and oxidized lipoprotein(a) with human mesangial cells and matrix.

The trapping of apolipoprotein(a) and apolipoprotein B-100 in glomeruli of patients with the nephrotic syndrome seems to be linked to a less favorable course of renal disease. To evaluate the potential role of lipoprotein(a) as a mediator of glomerular injury, we measured uptake of native lipoprotein(a) [Lp(a)] and oxidatively modified Lp(a) by cultured human mesangial cells and matrix and studied the effects of Lp(a) on mesangial cell DNA-synthesis and cellular proliferation. Uptake of Lp(a) by mesangial cells occurred at a significantly lower affinity (KD 16 micrograms/ml vs. 39 micrograms/ml) and a lower maximum degradative capacity (6.7-fold) than for LDL. Specificity of receptor mediated uptake was 50% for Lp(a) compared to 84% for LDL. Oxidative modification of both Lp(a) and LDL was accompanied by a significant decrease in uptake and degradative capacities. Due to the limited uptake, native and oxidatively modified Lp(a) had only marginal effects on intracellular cholesterol metabolism, which was measured as inhibition of sterol synthesis and stimulation of cholesterol esterification. However, binding of Lp(a), oxidized Lp(a) and oxidized LDL to extracellular mesangial matrix was enhanced compared to LDL. Furthermore, incubation of mesangial cells with Lp(a) and oxLp(a) in concentrations of 2.5 micrograms/ml and higher resulted in a decrease of DNA synthesis. Regardless of the oxidative status, a maximal suppression of DNA synthesis was observed at 20 micrograms/ml Lp(a). Native Lp(a) also blunted the stimulatory effects of PDGF on mesangial cell DNA-synthesis. Lp(a) did not alter basal TGF-beta transcription in human mesangial cells. The avid interaction of Lp(a) and modified lipoproteins with mesangial matrix provides a concept for the enhanced entrapment of these lipoproteins in the diseased glomerulum. Native Lp(a) is a poor ligand for the LDL receptor; oxidation of Lp(a) even lowers the affinity towards this receptor. Further studies must be carried out to clarify the pathophysiological significance of Lp(a) trapping in the mesangial matrix.

Cholesterol metabolism in glomerular cells: effect of lipoproteins from nephrotic patients.

Although hyperlipidemia is a well recognized complication of the nephrotic syndrome, the precise metabolism of human lipoproteins by human glomerular cells and the effects of abnormalities in lipid and protein composition on this process have not been defined. This study examined the effects of apoB-100 containing low-density-lipoprotein (LDL) and apo B,E, containing intermediate-density lipoprotein (IDL), isolated from patients with the nephrotic syndrome (n = 6), on intracellular sterol synthesis and cholesterol esterification by human glomerular epithelial and mesangial cells. For comparison studies, human skin fibroblasts and Hep G2 cells were used. In the patients, serum LDL cholesterol level was increased threefold and IDL tenfold as compared to healthy subjects. LDL of nephrotic patients showed no differences in lipid/protein composition as compared to control LDL but IDL contained 58% more cholesterol than IDL from healthy controls. Therefore, nephrotic and control LDL showed identical inhibition of intracellular sterol synthesis and similar cholesteryl ester formation in all the four cell types. In contrast, cholesterol-rich IDL of nephrotic patients suppressed intracellular sterol synthesis more effectively than control IDL. The cholesterol esterification rate of IDL from patients was enhanced three fold on average as compared to control IDL. The various cell types differed in their rate of LDL esterification. The data indicate that the enhanced inhibition of intracellular sterol synthesis and cholesterol esterification by apo E-containing cholesterol-ester-rich IDL, which accumulate in nephrotic patients, may render these lipoproteins possible candidates for glomerular lipid deposition and progressive renal injury.

VLDL and LDL metabolism in human and rat mesangial cells.

Human low-density lipoprotein (LDL) is taken up by rat mesangial cells in a receptor-dependent manner, although lipoprotein metabolism and lipoprotein receptors differ substantially between rodents and humans. We therefore compared binding and uptake kinetics as well as intracellular cholesterol metabolism of apoB- and apoB,E-containing lipoproteins in rat and human mesangial cells. Uptake of very-low-density lipoprotein (VLDL) and LDL in both human and rat mesangial cells occurred in a receptor-specific, concentration-dependent manner and the process was saturable. However, LDL uptake specificity, receptor affinity and maximal degradation capacity was remarkably lower in rat than in human mesangial cells. Exposure of cells to LDL suppressed intracellular sterol synthesis more effectively in the human than in the rat cell line (87 vs. 36%, respectively). Additionally, cholesteryl ester formation was enhanced 23-fold by LDL in human as compared to rat mesangial cells. In contrast, degradation capacities of VLDL were higher in rat and uptake specificity as well as receptor affinity were similar. Inhibition of intracellular cholesterol synthesis and oleate formation rate by VLDL occurred to a similar extent in both cell lines. The data demonstrate that mesangial cell uptake of apoB-containing lipoproteins depends on the species investigated, whereas apoE-rich lipoprotein particles are taken up independent of species. Therefore, human mesangial cells should be preferred over rat mesangial cells when investigating lipoprotein metabolism to elucidate the potential role of lipoproteins in mediating glomerular injury and progression of renal disease in man.

Preferential uptake of intermediate-density lipoproteins from nephrotic patients by human mesangial and liver cells.

Hyperlipidemia of nephrotic origin could potentially cause glomerular injury as well as increase the risk of atherosclerosis. The precise interaction of human lipoproteins abnormal in lipid and protein composition, with lipoprotein receptors has not been clearly defined. This study examines receptor-mediated uptake and intracellular cholesterol metabolism of apolipoprotein (apo)B,E containing intermediate-density lipoprotein (IDL) and apoB-100 containing low-density lipoprotein (LDL), isolated from patients with the nephrotic syndrome (N = 6), in human glomerular mesangial and HepG2 cells. In the patients, serum IDL and LDL cholesterol levels were significantly increased as compared with those of healthy subjects. The IDL of nephrotic patients contained 80% more cholesterol than the IDL of healthy controls. No differences in lipid/protein composition were found in the LDL density range. Therefore, nephrotic and control LDL showed identical affinities for receptor-mediated uptake. In contrast, the IDL of nephrotic patients was taken up by mesangial cells and HepG2 with higher affinity than the LDL. Intracellular sterol synthesis was suppressed more effectively and cholesterol esterification rate was enhanced 2.2-fold by nephrotic IDL as compared with control IDL. These data indicate that hypercholesterolemia of nephrotic origin cannot be explained by reduced ligand binding for LDL. ApoE-containing IDL of patients with the nephrotic syndrome were avidly taken up by glomerular mesangial cells and could therefore play the predominant role in the development of glomerulosclerosis and atherosclerosis associated with this disorder.