Down-regulation of hepatic LDL receptor-related protein (LRP) in chronic renal failure

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1 Kidney International, Vol. 67 (2005), pp Down-regulation of hepatic LDL receptor-related protein (LRP) in chronic renal failure CHOONG KIM and NOSRATOLA D. VAZIRI Division of Nephrology and Hypertension, and Department of Physiology and Biophysics, University of California, Irvine, California Down-regulation of hepatic LDL receptor-related protein (LRP) in chronic renal failure. Background. Chronic renal failure () is associated with premature atherosclerosis, impaired high-density lipoprotein (HDL)-mediated reverse cholesterol transport, depressed clearance, and elevated plasma concentrations of very lowdensity lipoprotein (VLDL), chylomicrons, and their atherogenic remnants. LDL receptor-related protein (LRP) is a member of the LDL receptor gene family that is heavily expressed in the liver, and mediates removal of at least 30 different ligands, including VLDL remnants (IDL) and chylomicron remnants. This study was conducted to test the hypothesis that the well-known defect in clearance of IDL and chylomicron remnants in may be indicative of diminished hepatic LRP abundance. Methods. Hepatic tissue LRP mrna abundance [reverse transcription-polymerase chain reaction (RT-PCR)] and protein abundance (Western blot analysis) were determined in rats 8 weeks after 5/6 nephrectomy ( group) or sham operation (control group). Results. The group exhibited hypertension, diminished creatinine clearance, increased plasma triglyceride concentration, and elevated total cholesterol-to-hdl cholesterol concentration ratio compared to the corresponding values found in the control group. This was associated with a significant downregulation of hepatic LRP mrna expression and immunodetectable LRP protein. Conclusion. results in down-regulation of hepatic LRP. This abnormality can, at least in part, account for the previously documented elevation of plasma concentration and depressed clearance of chylomicron remnants and IDL in. Chronic renal failure () is associated with increased risk of atherosclerotic cardiovascular disease [1, 2] and profound alterations of plasma lipid profile [3 5]. -associated dyslipidemia is marked by impaired high-density lipoprotein (HDL) maturation, Key words: chronic kidney disease, lipid disorders, atherosclerosis, lipoprotein remnants, cardiovascular disease. Received for publication June 15, 2004 and in revised form August 24, 2004 Accepted for publication September 28, 2004 C 2005 by the International Society of Nephrology hypertriglyceridemia, elevated plasma concentration, and defective clearance of very low-density lipoprotein (VLDL), chylomicrons (CM), and their highly atherogenic remnants (IDL and CM remnants) [3 8]. Intermediate-density lipoprotein (IDL) and CM remnants are the by-products of lipolysis of VLDL and CM, respectively, by lipoprotein lipase in the circulation. CM remnants are removed from the circulation by the liver via hepatic LDL receptor-related protein (LRP) [9, 10]. IDL particles are either converted to LDL via further lipolysis by hepatic triglyceride lipase or are removed from the circulation by the liver via receptor-mediated endocytosis. LRP is a member of the LDL receptor gene family that is heavily expressed in the liver [11]. This highmolecular-weight (600 kd) receptor has substantial structural similarity to LDL receptor, and recognizes a wide array of diverse ligands, including ApoB 48- and ApoEenriched lipoproteins (CM remnants, IDL, HDL), hepatic triglyceride lipase, coagulation factors, fibrinolytic enzymes, protease inhibitors, and protease-protease inhibitor complexes [12 15]. In view of the critical role of hepatic LRP in clearance of CM remnants, IDL, and proteinase-inhibitor complexes, we hypothesized that the reported increased in plasma concentration of CM remnants, IDL [6 8], and inactivated coagulation enzymes [16] in may be indicative of hepatic LRP deficiency. Accordingly, hepatic tissue LRP gene expression and protein abundance were determined in rats 8 weeks after 5/6 nephrectomy or sham operation. The results showed significant down-regulation of hepatic LRP mrna and protein expressions in the animals, thus confirming the original hypothesis. METHODS Animal models Male Sprague-Dawley rats weighing 225 to 250 g were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA). They were housed in a climate-controlled, light-regulated facility with 12:12-hour day-night cycles. The animals were fed regular rat chow (Purina Mills, 1028

2 Kim and Vaziri: LRP in chronic renal failure 1029 Table 1. Primers used for measurements of LRP mrna and 18s rrna by RT-PCR Product Gene DNA sequences bp LRP Forward 5 -GGCTGCTGATGGCTCCCGAC Reverse 5 -TCTCGTCCGTGCTGGCCAGG-3 18s Forward 5 -AGGAATTGACGGAAGGGCAC rrna Reverse 5 -GTGCAGCCCCGGACATCTAAG-3 Brentwood, MO, USA) and water ad libitum, and were randomly assigned to the and control groups. The animals assigned to the group were subjected to 5/6 nephrectomy by surgical resection using a dorsal incision, as described previously [17]. The animals assigned to the control group were subjected to sham operation. All surgical procedures were carried out under general anesthesia (Na pentobarbital 50 mg/kg IP). Strict hemostasis and aseptic techniques were observed. The animals were observed for 8 weeks. At the conclusion of the 8-week observation period, the animals were placed in metabolic cages for a 24-hour urine collection. They were then killed by exsanguination via cardiac puncture between the hours of 9 and 11 a.m. Liver was removed immediately, snap-frozen in liquid nitrogen, and stored at 70 C until processed. Serum triglyceride, VLDL, HDL, and creatinine concentrations and urinary creatinine were determined using standard laboratory methods. The protocol employed in this study was approved by the Institutional Committee for Care and Use of Animals at the University of California, Irvine. RNA isolation and RT-PCR RNA was isolated from frozen liver using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified by RNeasy kit (Qiagen, Valencia, CA, USA). One tenth microgram total RNA from each sample was reverse transcribed to cdnas by using MuLV reverse transcriptase (Applied Biosystems, Foster City, CA, USA) with a mixture of 1 mmol/l dntp and 2.5 lmol/l random primers in a 10 ll volume for 10 minutes at 25 C and 30 minutes at 48 C. The reaction was stopped by heating at 94 C for 5 minutes. Expression of LRP mrna was assessed by reverse transcription-polymerase chain reaction (RT-PCR) using 18s ribosomal RNA as internal control. The primer sequences used are depicted in Table 1. The primers were designed with Primer3 program purchased from Invitrogen. For 18s rrna amplification, we used alternate 18s primers (Ambion, Austin, TX, USA), which yields a 324-bp product. In each PCR reaction, 18s ribosomal RNA was coamplified with the target cdna. The primers were tested for their compatibility with the alternate 18s primer. The cdnas were amplified using standard PCR buffer, 0.2 mmol/l dntp, 1 lmol/l LRP primer set, 0.5 lmol/l 18s primer/competimer mix, 2 mmol/l MgCl 2, Table 2. Creatinine clearance (Ccr), plasma concentrations of creatinine, triglycerides, total cholesterol, and VLDL cholesterol, and plasma total cholesterol-to-hdl cholesterol ratio in the sham-operated control rats () and rats with chronic renal failure () Group a P value Ccr ml/min 2.6 ± ± 0.09 <0.01 Creatinine ml/dl 0.4 ± ± 0.2 <0.01 Triglycerides mg/dl 47 ± ± 17 <0.01 Total cholesterol 59 ± ± 11 <0.01 VLDL cholesterol mg/dl 9.3 ± ± 3.4 <0.01 Total/HDL cholesterol, ratio 2.7 ± ± 0.5 <0.05 a N = 6ineach group. and 1.25 U of Taq DNA polymerase (Applied Biosystems) in 50 ll oftotal volume for 30 cycles. Each cycle consisted of 1-minute denaturation at 94 C and 45 seconds annealing at 60 C, and 45 seconds extension at 72 C. PCR products were separated on a 1.2% agarose gel with ethidium bromide by eletrophoresis. Signal intensity was determined by laser scanning densitometry. On each occasion, the LRP mrna abundance was normalized to the corresponding 18s ribosomal RNA. Measurements of LRP protein LRP receptor protein abundance in the liver tissue was measured by Western blot analysis using a monoclonal antihuman LRP antibody prepared in our laboratory using the IgG-11H4 hybridoma cell line purchased from ATCC (Manassas, VA, USA). Data analysis Student t test was used in statistical analysis of the data, which are given as mean ± SEM. P values less than 0.05 were considered significant. RESULTS General data Creatinine clearance in the group (0.56 ± 0.9 ml/min) was significantly lower than that found in the control group (2.6 ± 0.2 ml/min, P < 0.001). In contrast, systolic arterial pressure was significantly higher in the group (165 ± 4mmHg) than in the sham-operated control rats (123 ± 2mmHg,P < 0.01). Body weight obtained at the conclusion of the study was significantly lower in the group (366 ± 6g)than in the control group (389 ± 16 g). Compared with the control group, the animals showed a marked increase in plasma triglycerides, VLDL cholesterol, and total cholesterol-to-hdl cholesterol ratio (Table 2). Liver LRP data Data are shown in Figures 1 and 2. Compared with the sham-operated control rats, the group exhibited a

Representative reverse transcription-polymerase chain reaction (RT-PCR) and group data depicting hepatic LDL receptor-related protein (LRP) mrna and 18s ribosomal RNA abundance in chronic renal

3 1030 Kim and Vaziri: LRP in chronic renal failure LRP LRP 18s rrna Actin Hepatic LRP expression, LRP mrna/18s rrna * Hepatic LRP protein, LRP/actin * 0 0 Fig. 1. Representative reverse transcription-polymerase chain reaction (RT-PCR) and group data depicting hepatic LDL receptor-related protein (LRP) mrna and 18s ribosomal RNA abundance in chronic renal failure () and sham-operated control rats. N = 6ineach group, P < Fig. 2. Representative Western blot and group data depicting hepatic LDL receptor-related protein (LRP) and actin abundance in the chronic renal failure () and sham-operated control groups. N = 6, P < significant reduction of LRP mrna abundance normalized against 18s ribosomal RNA. Similarly, immunodetectable LRP protein abundance was significantly lower in the liver of the rats than the corresponding value found in the sham-operated control group. DISCUSSION The main features of the -induced dyslipidemia include hypertriglyceridemia, elevated plasma concentration, and impaired clearance of triglyceride-rich lipoproteins (VLDL and CM) and their remnants, coupled with a defective HDL maturation [3 8]. Hypertriglyceridemia in this model is not caused by increased hepatic triglyceride synthesis because acyl-coa: diglycerol acyltransferase (DGAT), which is the final step in triglyceride biosynthesis, is down-regulated in the liver [18]. Instead, it is primarily caused by downregulations of skeletal muscle and adipose tissue lipoprotein lipase [17, 19 21], which is essential for the lipolysis of VLDL and CM, and of VLDL receptor [22, 23], which is involved in the peripheral uptake of VLDL. This is accompanied by hepatic triglyceride lipase deficiency [21, 24], which plays a critical role in the removal of the remaining triglycerides in the IDL particles and their eventual conversion to LDL. The role of lipoprotein lipase, hepatic triglyceride lipase, and VLDL receptor in metabolism of endogenous and exogenous plasma lipids is depicted in Figure 3. The effects of lipoprotein lipase, VLDL receptor, and hepatic triglyceride lipase deficiencies on metabolism of triglyceride-rich lipoproteins are compounded by impaired maturation of HDL-3 to HDL- 2in [5]. The latter is due to down-regulation of lecithin: cholesterol acyltransferase (LCAT), the key enzyme involved in HDL-mediated cholesterol uptake in [25]. HDL-2 serves as the ApoE (lipoprotein lipase and VLDL receptor ligand) and ApoC-II (lipoprotein lipase cofactor) donors to the nascent VLDL and chylomicrons. Consequently, diminished HDL-2 level in contributes to the defective lipoprotein lipase-mediated lipolysis of VLDL and CM and VLDL-receptor mediated uptake of VLDL by adipocyte and myocytes. The animal studies outlined above provided the molecular basis of clinical studies that have demonstrated depressed plasma LCAT activity, diminished plasma post heparin lipolytic activity, and elevated plasma prebeta HDL (lipid-poor HDL) in patients [26 29].

4 Kim and Vaziri: LRP in chronic renal failure 1031 Intestine Exogenous LPL* CM CMR Liver LDL-r LRP* LDL Extrahepatic tissues Endogenous VLDL IDL HTL* VLDL-r* LPL* Fig. 3. Pathways of metabolism of endogenous and exogenous lipids. Very low-density lipoprotein (VLDL) and chylomicrons (CM) are produced and secreted by the liver and intestine, respectively. In the circulation, VLDL and CM undergo lipolysis by lipoprotein lipase (LPL) to become VLDL remnants (IDL) and CM remnants (CMR). CMR are removed by the liver via LRP. IDL undergo further lipolysis by hepatic triglyceride lipase (HTL) to become LDL, which is, in turn, removed by either the liver via LDL receptor (LDLr) or by extrahepatic tissues. In addition, IDL can be directly removed from the circulation by a receptor-mediated process. A novel pathway has been recently identified by which VLDL can be removed via VLDL receptor (VLDLr). These proteins are down-regulated in. The animals employed in the present study exhibited a significant reduction of immunodetectable LRP in the liver tissue. This was accompanied by a parallel reduction of LRP mrna abundance. Thus, results in down-regulation of LRP at the level of gene transcription. The observed hepatic LRP deficiency, shown for the first time here, provides the molecular mechanism of the elevated plasma concentration of the atherogenic CM remnants in [6 8]. In addition, hepatic LRP deficiency can, in part, contribute to elevation of plasma IDL in. As noted earlier, results in down-regulation of hepatic triglyceride lipase [21, 24], which, in turn, impedes conversion of IDL to LDL via the lipolytic pathway. This leads to a rise in the available supplies of IDL for clearance via the receptor-mediated endocytosis, commonly known as the shunt pathway. LRP plays an important part in hepatic clearance of IDL through the shunt pathway [11, 12]. Consequently, the combined down-regulations of the hepatic triglyceride lipase and LRP work in concert to impair IDL clearance and raise plasma concentration of this oxidation-prone, atherogenic lipoprotein remnant by limiting the lipolytic and shunt pathways of its metabolism. By promoting accumulation of highly atherogenic CM remnants and IDL in the plasma, hepatic LRP deficiency can contribute to the atherogenic diathesis in. In addition, hepatic LRP deficiency has been shown to promote atherosclerosis in LRP knockout mice, independent of its action on lipoprotein metabolism [30]. This phenomenon has been attributed to the accumulation of other proatherogenic ligands of LRP. Thus, acquired hepatic LRP deficiency can be added to many previously identified risk factors for cardiovascular disease in. CONCLUSION results in acquired hepatic LRP deficiency, which can contribute to the atherogenic diathesis and elevated plasma lipoprotein remnants in chronic renal failure. Reprint requests to N.D. Vaziri, M.D., MACP, Division of Nephrology and Hypertension, UCI Medical Center, 101 The City Drive, Bldg 53 Rm 125 Rt 81, Orange, CA ndvaziri@uci.edu REFERENCES 1. FOLEY RN, PARFREY PS, SARNAK MJ: Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 32:S , RAINE AE, MCMAHON SH, SELWOOD NH: Mortality from myocardial infarction in patients on renal replacement therapy in the UK. Nephrol Dial Transplant 6: , ATTMAN PO, SAMUELSSON O, ALAUPOVIC P: Lipoprotein metabolism and renal failure. Am J Kidney Dis 21:573 92, SAHADEVAN M, KASISKE BL: Hyperlipidemia in kidney disease: Causes and consequences. Curr Opin Nephrol Hypertens 11: , SHOJI T, NISHIZAWA Y, NISHITANI H, et al: Impaired metabolism of high density lipoprotein in uremic patients. Kidney Int 41: , RON D, OREN I, AVIRAM M, et al: Accumulation of lipoprotein remnants in patients with chronic renal failure. Atherosclerosis 46:67 75, NORBECK HE, CARLSON LA: Increased frequency of late pre-beta lipoproteins (LP beta) in isolated serum very low density lipoproteins in uraemia. Eur J Clin Invest 10: , NESTEL PJ, FIDGE NH, TAN MH: Increased lipoprotein-remnant formation in chronic renal failure. N Engl J Med 307: , ROHLMANN A, GOTTHARDT M, HAMMER RE, HERZ J: Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants. J Clin Invest 101: , HUSSAIN MM, MAXFIELD FR, MAS-OLIVA J, et al: Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. J Biol Chem 266: , KRIEGER M,HERZ J: Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptorrelated protein (LRP). Annu Rev Biochem 63: , KUCHENHOFF A, HARRACH-RUPRECHT B, ROBENEK H: Interaction of apo E-containing lipoproteins with the LDL receptor-related protein LRP. 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5 1032 Kim and Vaziri: LRP in chronic renal failure 16. VAZIRI ND, GONZALES EC, WANG J, SAID S: Blood coagulation, fibrinolytic and inhibitory proteins in end-stage renal disease: Effect of hemodialysis. Am J Kidney Dis 23: , VAZIRI ND, WANG XQ, LIANG K: Secondary hyperparathyroidism downregulates lipoprotein lipase expression in chronic renal failure. Am J Physiol Renal Physiol 273:F925 F930, VAZIRI ND, DANG B, ZHAN CD, LIANG K: Downregulation of Acyl- CoA: diglycerol acyltransferase (DGAT) in chronic renal failure. Am J Physiol Renal Physiol 287:F90 F94, VAZIRI ND, LIANG K: Downregulation of tissue lipoprotein lipase expression in experimental chronic renal failure. Kidney Int 50: , SATO T, LIANG K, VAZIRI ND: Downregulation of lipoprotein lipase and VLDL receptor in rats with food glomerulosclerosis. Kidney Int 61: , SATO T, LIANG K, VAZIRI ND: Dietary protein restriction and oral absorbent, AST-120 ameliorates lipoprotein lipase, hepatic lipase and VLDL receptor deficiencies in rats with focal glomerulosclerosis. Kidney Int 64: , VAZIRI ND, LIANG K: Downregulation of VLDL receptor expression in chronic experimental renal failure. Kidney Int 51: , LIANG K, OVEISI F, VAZIRI ND: Role of secondary hyperparathyroidism in the genesis of hypertriglyceridemia and VLDL receptor deficiency in chronic renal failure. Kidney Int 53: , KLIN M, SMOGORZEWSKI M, NI Z, et al: Abnormalities in hepatic lipase in chronic renal failure: Role of excess parathyroid hormone. J Clin Invest 97: , VAZIRI ND, LIANG K, PARKS JS: Downregulation of hepatic lecithin: Cholesterol acyltransferase gene expression in chronic renal failure. Kidney Int 59: , GUARNIERI GF, MORACCHIELLO M, CAMPANACCI L, et al: Lecithincholesterol acyltransferase (LCAT) activity in chronic uremia. Kidney Int (Suppl 8):S26 30, CHAN MK, PERSAUD J, VARGHESE Z, MOORHEAD JF: Pathogenic roles of post-heparin lipases in lipid abnormalities in hemodialysis patients. Kidney Int 25: , AKMAL M, KASIM SE, SOLIMAN AR, MASSRY SG: Excess parathyroid hormone adversely affects lipid metabolism in chronic renal failure. Kidney Int 37: , CHEUNG AK, PARKER CJ, REN K, IVERIUS PH: Increased lipase inhibition in uremia: Identification of pre-beta-hdl as a major inhibitor in normal and uremic plasma. Kidney Int 49: , ESPIRITO SANTO SM, PIRES NM, BOESTEN LS, et al: Hepatic lowdensity lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol. Blood 103: , 2004

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