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NDT Advance Access originally published online on November 28, 2007
Nephrology Dialysis Transplantation 2008 23(2):751-756; doi:10.1093/ndt/gfm675
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


A novel variant apolipoprotein E Okayama in a patient with lipoprotein glomerulopathy

Masaru Kinomura1, Hitoshi Sugiyama1, Takao Saito2, Akira Matsunaga3, Ken-ei Sada1, Motoko Kanzaki1, Yuki Takazawa1, Yohei Maeshima1, Hiroyuki Yanai4 and Hirofumi Makino1

1Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan, 2Division of Nephrology and Rheumatology, Fukuoka University School of Medicine, Fukuoka, Japan, 3Division of Cardiology, Department of Internal Medicine, Fukuoka University School of Medicine, Fukuoka, Japan and 4Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Correspondence and offprint requests to: Hitoshi Sugiyama, Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-86-235-7235; Fax: +81–86-222-5214; E-mail: hitoshis{at}md.okayama-u.ac.jp

Keywords: apolipoprotein E; fibrate; hyperlipidemia; proteinuria; renal lipidosis



   Introduction
Top
 Introduction
Case report
 Supplementary Data
 Acknowledgements.
 References
 
Lipoprotein glomerulopathy (LPG) is a rare disorder characterized by lipoprotein thrombi in the intraglomerular capillaries and high serum concentrations of apolipoprotein E (apoE). In 1989, Saito et al. [2,3]. Patients with LPG usually present with nephrotic syndrome and a relatively rapid progression to end-stage renal failure.

Human apoE is composed of 299 amino acids and mediates tissue uptake of triglyceride-rich lipoproteins through both low-density lipoprotein (LDL) receptor and LDL-receptor-related protein pathways. Genetic variations at the apoE gene locus code for three different major isoforms designated E2, E3 and E4, representing their migration characteristics of isoelectric focusing (IEF). The wild-type allele is apoE3, while apoE2 (Arg158Cys) and apoE4 (Cys112Arg) are less common. ApoE3 and apoE4 are thought to bind equally to the lipoprotein receptors, whereas apoE2 is defective in its lipoprotein receptor binding [4]. Homozygosity for apoE2 (Arg158Cys) results in the development of type III hyperlipoproteinemia [5]. ApoE4 may be a risk factor for atherosclerosis and Alzheimer's disease [6].

Recently, novel variants of apoE have been identified in patients with LPG, including apoE Sendai (Arg145Pro) [7], apoE Kyoto (Arg25Cys) [8], apoE Tokyo (Leu141 to Lys143del) [9], an 18-amino acid deletion in apoE1 [10], apoE Maebashi (Arg142 to Leu144del) [11] and apoE Chicago (Arg147Pro) [2]. In this report, we identified an apoE phenotype 1/2 in a 20-year-old woman with LPG. Restriction isotyping using HhaI resulted in an apoE genotype 2/2. DNA sequence analysis further revealed a G to C point mutation at the amino acid residue 150, as predicted by replacement of arginine to glycine (Arg150Gly). We therefore propose apoE Okayama as a novel and unique mutation in LPG.



   Case report
Top
 Introduction
 Case report
Supplementary Data
 Acknowledgements.
 References
 
The 20-year-old woman was in good health until October 2005, when she was presented with proteinuria during the annual health checkup in her company. In January 2006, she was referred to our hospital for evaluation of the proteinuria. Her brother did not have any illness. Her mother had history of preeclampsia but currently had no urinary abnormalities. Her father was alive and well. None of the members of her family showed hyperlipidemia, blind deafness or symptoms of renal dysfunction.

At the time of admission, her blood pressure was 117/59 mm Hg. There was no edema. Urinalysis revealed proteinuria with moderate protein of 100 mg/dL; mild occult blood, with 5–10 red blood cells per high-power field; and two granular casts per whole field. Twenty-four-hour urinary protein excretion was 0.9 g. Blood examination revealed iron deficiency anaemia, red blood cell counts: 5.12 millions/µL; hemoglobin: 10.1 g/dL (101 g/L); Fe: 10 µg/dL; and ferritin: 3.4 ng/mL. Laboratory findings showed blood urea nitrogen: 8.5 mg/dL (3.0 mmol/L); serum creatinine: 0.57 mg/dL (50 µmol/L); 24 h creatinine clearance: 66.1 mL/min; albumin: 4.1 g/dL (41 g/L); total cholesterol: 261 mg/dL (6.76 mmol/L); and triglycerides: 152 mg/dL (1.72 mmol/L). Plasma lipoproteins, separated by sequential ultracentrifugation [Table 1. The antinuclear antibody test was slightly positive (24.0 index), but antidouble stranded DNA autoantibodies were negative. C3 level was 82 mg/dL (0.82 g/L) and C4 level was 10 mg/dL (0.1 g/L). Hepatitis B surface antigen and hepatitis C antibody tests were negative. The level of plasma {alpha}-galactosidase was within normal range.


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  		Table 1. Apolipoprotein profiles before and after lipid-lowering therapy

 
Pathologic findings
Renal biopsy specimens were processed using standard methods and were composed of two fragments of renal cortex containing 23 glomeruli; none of these were hyalinized. The glomerular architecture was distorted by a widespread amorphous glomerular capillary thrombi that stained slightly with periodic acid-Schiff (PAS) (Figure 1A). Staining with oil red O showed a number of lipid droplets in the capillary lumina of the glomeruli (Figure 1B). Thirty-five percent of glomeruli had segmental mesangial sclerosis surrounded by dilated capillary lumen filled with lipid thrombi. Twenty-two percent of glomeruli had small synechiae between the glomerular capillaries and Bowman capsule (Figure 1A). Foam cells were not present in either the glomeruli or the tubulointerstitium. There was mild and limited interstitial fibrosis and tubular atrophy involving approximately less than 10% of the renal cortical tissue. Routine fluorescent microscopy showed trace to 1+ linear deposits of immunoglobulin A and M (not shown). Immunofluorescence studies using goat antihuman apoB or apoE antibodies (Chemicon, Temecula, CA, USA) demonstrated strong segmental staining for both apoE and apoB within capillary lumen, although staining for apoB was more patchy than that of apoE staining (Figure 1C and D). A few mesangial stainings of apoE and apoB were also observed. Two glomeruli were examined by electron microscopy. Almost all of the dilated capillary lumina were occluded by numerous lipid granules and lamellate vacuoles in the capillary lumens (Figure 1E). A myelin-body-like structure was partly observed in the capillary lumen (Figure 1F).


Figure 1
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  		Fig. 1. Glomerular findings from renal biopsy specimens in a patient with LPG: Light (A, B), immunofluorescent (C, D) and electron micrographs (E, F) are shown; (A) capillary lumen are markedly dilated and possibly occupied by lipoprotein thrombi (asterisks), (B) massive pale-stained deposits are observed in the capillary lumen. Segmental stainings of apoE (C) or apoB (D) are present in the glomeruli. Lamellate thrombi (E) or zebra body-like lesion (F) occupy the dilated capillary lumen. A, Periodic acid-Schiff stain; B, Oil-red-O stain. Scale bars are 50 µm (A–D), 10 µm (E) and 3.5 µm (F).

 
Phenotype, genotype and DNA sequence analysis of apoE
Serum apoE phenotypes were analyzed using isoelectric focusing polyacrylamide gel electrophoresis as previously described [7,8]. In brief, 10 µL of plasma were added to 30 mL of sialidase solution (10 mU of neuraminidase in 66 mM acetate buffer, pH 5.0) and incubated at 37°C for 2 h. Lipids were extracted with 1.0 mL of chilled chloroform:methanol:ether solution (4:3:1). The apoE isoforms were then separated by 7.2% polyacrylamide gels containing 5 M urea and a 2:1 mixture of 2% ampholytes (pH 4.0–6.0 and pH 5.0–8.0), and then detected by immunoblotting with goat anti-apoE antiserum (Daiichi Pure Chemicals Co., Tokyo, Japan) as the primary antibody. As compared to apoE3/3 of control wild type, the patient had alleles apoE1/2 (Figure 2A). The apoE genotype was determined by restriction fragment length polymorphism (RFLP) analysis as described previously [7,8]. Genomic DNA was amplified by polymerase chain reaction (PCR) using oligonucleotide primers F4 (5' –ACAGAATTCG-CCCCGGCCTGGTACAC-3') and F6 (5' –TAAGCTTGG-CACGGCTGTCCAAGGA-3'). The PCR products were digested with the restriction enzyme HhaI or AccII, electrophoresed on 8% polyacrylamide gel at 200 V for 60–90 min, and then stained with ethidium bromide. As shown in Figure 2B, the 91 and 83 Bp fragments after cleavage by HhaI demonstrated that the patient had genotype {varepsilon}2/2, as compared to the control genotype {varepsilon}3/3. Sequencing of apoE DNA was performed after genomic DNA was extracted from the blood as described previously [7,8]. Genomic DNA was amplified by PCR with primers A (5'-TAGG-TAGCTAGAGCTGGAC-3') and B (5'-AGACTTAGCG-ACAGGGGCAGAATG-3'), and primers E7 (5'-ATCA-AGCTTTCGCCCGCCCCACCCAGCCCTTC-3') and E9 (5'-CGTGAATTCGCATGGCTGCAGGCTTCGGCGTT-C-3'). The PCR-amplified DNA was isolated by electro-phoresis on 0.8% agarose gel and ligated into pT7Blue-T-vector (Novagen, Madison, WI). Twelve clones from each allele were sequenced using T7 DNA polymerase (Sequenase; Amersham International plc, Buckinghamshire, U.K.) according to the dideoxynucleotide chain termination method. DNA sequencing of seven out of twelve separate clones revealed a single nucleotide substitution of arginine for glycine, leading to a novel heterozygous mutation Arg150Gly (Figure 2C). The heterozygous mutation was further confirmed by restriction enzyme digestion with AccII of PCR-amplified DNA fragments (Figure 2D). The patient also had homogenous apoE2 (Arg158Cys) (data not shown). Written informed consent was obtained from the patient prior to commencement of the phenotype and genotype analysis of apoE. ApoE abnormalities were not examined in her family members due to lack of their consent.


Figure 2
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  		Fig. 2. Phenotype and genotype analysis of apoE Okayama in serum from the patient. (A) ApoE phenotype determined by isoelectric focusing polyacrylamide gel electrophoresis (IEF). Lane 1, apoE3/3 (wild type); lane 2, apoE1/2 (patient); lane 3, apoE2/3; lane 4, apoE3/4; lane 5, apoE3/3 (wild type). (B) ApoE genotype examined by restriction fragment length polymorphism (RFLP). PCR-amplified DNA of the apoE gene, including codons 112 and 158, was digested with HhaI and subjected to agarose gel electrophoresis. The 91 and 83 Bp fragments after cleavage by HhaI demonstrated that the patient had genotype {varepsilon}2/2 (lane 1). The 91, 48 and 35 Bp fragments after HhaI digestion showed that control subjects had genotype {varepsilon}3/3 (lanes 2, 3). The lower panel indicates the schematic illustration of apoE2 and apoE3 genes with restriction site of HhaI. (C) Sequence of genomic DNA from normal controls (upper panel) or a patient with LPG (lower panel). The normal apoE allele contains the sequence CGC (arginine) at codon 150, while the mutant apoE Okayama contains GGC (glycine) at the same codon. The non-coding strand has been sequenced. (D) RFLP analysis digested with AccII. The 144 Bp fragment after cleavage by AccII suggested a novel mutation of apoE Okayama in codon 150 (lane 1). The 93 and 51 Bp fragments after cleavage by AccII showed {varepsilon}3/3 alleles in control subjects (lanes 2, 3). The lower panel indicates the schematic illustration of genes for apoE3 and apoE Okayama (Pt) with restriction site of AccII. M, molecular size markers.

 
Clinical course
In the present case, we diagnosed LPG based on renal histopathology and abnormal lipid profiles in the blood. Treatment with pravastatin sodium (10 mg) was initiated. One month after the start of therapy, a significant improvement in the hyperlipidemia was obtained (total cholesterol: 173 mg/dL; triglycerides: 87 mg/dL). However, the degree of proteinuria was increased to 1.0 g/day, suggesting that the statin was not effective against proteinuria. We then combined intensive lipid-lowering therapy with bezafibrate 400 mg and ethyl-icosapentate 1800 mg, and pravastatin sodium was stopped. After the start of this combination therapy, a remarkable decrease in urinary protein excretion was obtained and proteinuria was not detectable by dipstick testing after six weeks. The treatment also improved apolipoprotein profiles (Table 1). The patient remained in clinical remission after one year from the start of intensive combination therapy. A second renal biopsy was performed one year after the initiation of therapy. Four of the nine glomeruli showed small synechiae accompanied by mild mesangial proliferation. The lipoprotein thrombi were no longer observed in the glomerular capillary lumina (Supplementary Figure A–C).

LPG is a rare disease characterized by lipoprotein thrombi in the glomerular capillaries and increased serum apoE levels. It was first described by Saito et al. [1] in 1989, in a patient with proteinuria and dilated glomerular capillaries filled with lipoprotein thrombi. Since then, LPG has been reported in more than 65 patients worldwide, mostly from Japan and east Asian countries [2]. The unique mutation of apoE Okayama, from the southwestern Chugoku region on the main island, indicates that LPG variants have spread throughout the nation.

The pathogenesis of LPG is not fully understood, but it is likely that mutated apoE contributes to the cause of the disease. Ishigaki et al. [12] reported that virus-mediated transduction of apoE Sendai in apoE-deficient hypercholesterolemic mice resulted in the marked intraglomerular lipid deposition that characterizes histologicals alteration in LPG. ApoE Sendai produces severe structural changes in the middle of the {alpha} helix and may alter the three-dimensional conformation of the protein [12]. Hoffman et al. [13] showed that the binding activity of apoE-Sendai to LDL receptors was diminished in a fashion similar to that of apoE2, whereas the heparin binding activity of apoE-Sendai was increased [13]. Many of the apoE mutations occur in the LDL receptor binding site, involving 140–150 amino acids in patients with LPG. Previous studies demonstrated that an apoE2 variant (Gln187Glu), which is not present in the LDL receptor binding site, failed to produce the histological changes in LPG [14]. It is possible that binding of the mutated apoE to the LDL receptor may be abnormal because of an altered three-dimensional structure of mutant protein [8,15,16].

An elevated plasma apoE concentration caused by diminished LDL receptor binding is an important determinant for the development of LPG; however, hyperlipidemia in LPG is often milder than in patients with familial type III hyperlipoproteinemia, or not even recognized in some cases of LPG [9]. Clinical symptoms characteristic of hyperlipidemia, such as xanthoma, corneal arcus, and thickness of the Achilles tendon, are rarely observed in LPG. These findings support the possibility that intrarenal or intraglomerular dysfunction induced by mutant apoE may be involved in the development of LPG. ApoE is synthesized in human kidney tissue, and was shown to regulate proliferation and matrix overproduction of mesangial cells in experimental settings [17]. The interaction between apoE variants and in situ glomerular factors may be an essential determinant for glomerular injury in LPG. In a patient with LPG, the abnormal very low-density lipoprotein (VLDL) had a lesser negative charge as compared with normal VLDL [18]. Since the glomerular basement membrane has a highly anionic charge, the abnormal VLDL may adhere more easily to the glomerular basement membrane and stay within the capillary lumen.

At present, no established therapy is available for patients with LPG. In addition, there are no reports that have investigated the relationship between the variants of apoE and their responsiveness to therapy. Typical treatments for nephrotic syndrome, such as corticosteroids or immunosuppressive therapy, may not adequately reverse the proteinuria in LPG [1]. Indeed, treatment with statins alone did not diminish proteinuria in spite of decreases in serum levels of total cholesterol and triglyceride in our patient. However, subsequent intensive combination therapy with two agents, bezafibrate and ethyl-icosapentate, did dramatically decrease proteinuria and induce clinical remission shortly after the initiation of treatment. In a previous report, administration of bezafibrate decreased plasma apoE and proteinuria in a patient with LPG exhibiting a nephritic syndrome, although the second biopsy after two-year treatment still showed proliferation of mesangial cells, thickening of glomerular basement membrane and effacement of foot processes in podocytes [18]. Ieiri et al. [19] recently reported that the combination of four lipid-lowering agents, fenofibrate, niceritrol, ethyl-icosapentate and probucol, remarkably decreased proteinuria in a patient with LPG exhibiting a nephritic syndrome. Taken together, these findings suggest that early intervention by intensive lipid-lowing therapy including fibrates should decrease proteinuria and prevent progressive renal failure as well as nephritic syndrome in the present type of LPG.

In conclusion, we present a case of LPG with a previously undescribed novel mutation of the apoE gene. Intensive lipid-lowering therapy was very effective for improving proteinuria during the early stage of LPG. Further studies will be necessary to elucidate the relationship between the variants of apoE and their responsiveness to therapy in patients with LPG.



   Supplementary Data
Top
 Introduction
 Case report
 Supplementary Data
Acknowledgements.
 References
 
Supplementary material is available at NDT Journal online.



   Acknowledgements.
Top
 Introduction
 Case report
 Supplementary Data
 Acknowledgements.
References
 
We greatly appreciate the technical assistance of Ms. T. Hashimoto and Ms. Y. Saito in performing histological examination using electron microscopy.

Conflict of interest statement. None declared.



   Notes
 
See http://ndtplus.oxfordjournals.org/



   References
Top
 Introduction
 Case report
 Supplementary Data
 Acknowledgements.
 References
 

  1. Saito T, Sato H, Kudo K, et al. Lipoprotein glomerulopathy: glomerular lipoprotein thrombi in a patient with hyperlipoproteinemia. Am J Kidney Dis (1989) 13:148–153.[Web of Science][Medline]
  2. Sam R, Wu H, Yue L, et al. Lipoprotein glomerulopathy: a new apolipoprotein E mutation with enhanced glomerular binding. Am J Kidney Dis (2006) 47:539–548.[CrossRef][Web of Science][Medline]
  3. Saito T, Matsunaga A, Oikawa S. Impact of lipoprotein glomerulopathy on the relationship between lipids and renal diseases. Am J Kidney Dis (2006) 47:199–211.[Web of Science][Medline]
  4. Weisgraber KH, Innerarity TL, Mahley RW. Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site. J Biol Chem (1982) 257:2518–2521.[Free Full Text]
  5. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science (1988) 240:622–630.[Abstract/Free Full Text]
  6. Corder EH, Saunders AM, Strittmatter WJ, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science (1993) 261:921–923.[Abstract/Free Full Text]
  7. Oikawa S, Matsunaga A, Saito T, et al. Apolipoprotein E Sendai (arginine 145–>proline): a new variant associated with lipoprotein glomerulopathy. J Am Soc Nephrol (1997) 8:820–823.[Abstract]
  8. Matsunaga A, Sasaki J, Komatsu T, et al. A novel apolipoprotein E mutation, E2 (Arg25Cys), in lipoprotein glomerulopathy. Kidney Int (1999) 56:421–427.[CrossRef][Web of Science][Medline]
  9. Konishi K, Saruta T, Kuramochi S, et al. Association of a novel 3-amino acid deletion mutation of apolipoprotein E (Apo E Tokyo) with lipoprotein glomerulopathy. Nephron (1999) 83:214–218.[CrossRef][Web of Science][Medline]
  10. Ando M, Sasaki J, Hua H, Matsunaga A, et al. A novel 18-amino acid deletion in apolipoprotein E associated with lipoprotein glomerulopathy. Kidney Int (1999) 56:1317–1323.[CrossRef][Web of Science][Medline]
  11. Ogawa T, Maruyama K, Hattori H, et al. A new variant of apolipoprotein E (apo E Maebashi) in lipoprotein glomerulopathy. Pediatr Nephrol (2000) 14:149–151.[CrossRef][Web of Science][Medline]
  12. Ishigaki Y, Oikawa S, Suzuki T, et al. Virus-mediated transduction of apolipoprotein E (ApoE)-sendai develops lipoprotein glomerulopathy in ApoE-deficient mice. J Biol Chem (2000) 275:31269–31273.[Abstract/Free Full Text]
  13. Hoffmann M, Scharnagl H, Panagiotou E, et al. Diminished LDL receptor and high heparin binding of apolipoprotein E2 Sendai associated with lipoprotein glomerulopathy. J Am Soc Nephrol (2001) 12:524–530.[Abstract/Free Full Text]
  14. Hayakawa M, Okubo M, Katori H, et al. A patient with apolipoprotein E2 variant (Q187E) without lipoprotein glomerulopathy. Am J Kidney Dis (2002) 39:E15.[Medline]
  15. Dong LM, Parkin S, Trakhanov SD, et al. Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia. Nat Struct Biol (1996) 3:718–722.[CrossRef][Web of Science][Medline]
  16. Feussner G, Albanese M, Valencia A. Three-dimensional structure of the LDL receptor-binding domain of the human apolipoprotein E2 (Arg136–>Cys) variant. Atherosclerosis (1996) 126:177–184.[CrossRef][Web of Science][Medline]
  17. Chen G, Paka L, Kako Y, et al. Regulation of mesangial cell proliferation and matrix expansion. J Biol Chem (2001) 276:49142–49147.[Abstract/Free Full Text]
  18. Arai T, Yamashita S, Yamane M, et al. Disappearance of intraglomerular lipoprotein thrombi and marked improvement of nephrotic syndrome by bezafibrate treatment in a patient with lipoprotein glomerulopathy. Atherosclerosis (2003) 169:293–299.[CrossRef][Web of Science][Medline]
  19. Ieiri N, Hotta O, Taguma Y. Resolution of typical lipoprotein glomerulopathy by intensive lipid-lowering therapy. Am J Kidney Dis (2003) 41:244–249.[CrossRef][Web of Science][Medline]
Received for publication: 31. 5.07
Accepted in revised form: 4. 9.07


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