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Disappearance of intraglomerular lipoprotein thrombi and marked improvement of nephrotic syndrome by bezafibrate treatment in a patient with lipoprotein glomerulopathy
Atherosclerosis 169 (2003) 293 /299 www.elsevier.com/locate/atherosclerosis
Disappearance of intraglomerular lipoprotein thrombi and marked improvement of nephrotic syndrome by bezafibrate treatment in a patient with lipoprotein glomerulopathy Takeshi Arai a, Shizuya Yamashita b,*, Mitsukazu Yamane c, Noriko Manabe b, Toshiyuki Matsuzaki a, Kazuo Kiriyama a, Yoshio Kanayama a, Seiichi Himeno a, Yuji Matsuzawa b a
b
Department of Internal Medicine, Ashiya Municipal Hospital, 39-1 Asahigaoka, Ashiya, Hyogo 659-0012, Japan Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan c Department of Cardiology, Kawasaki Hospital, Kobe, Hyogo 652-0042, Japan Received 21 October 2002; received in revised form 6 May 2003; accepted 9 May 2003
Abstract Lipoprotein glomerulopathy (LPG) is a hereditary disorder characterized by intraglomerular lipoprotein thrombi and increased serum apolipoprotein (apo) E. Patients with LPG usually manifest with nephrotic syndrome, and some progress to renal failure; however, no effective therapeutic regimen has been established for this disease. We experienced a patient with LPG for whom bezafibrate treatment was very effective. This 30-year-old Japanese woman had nephrotic syndrome and type III hyperlipoproteinemia. Renal biopsy showed markedly dilated capillary lumina containing massive lipoprotein thrombi. Plasma apo E concentration was elevated to twice that of normal controls. She was proved to be a heterozygote of apo E2 Kyoto (Arg25Cys). After 2 years treatment with bezafibrate (400 mg/day), her plasma albumin gradually increased from 2.1 to 4.0 mg/dl, and intraglomerular lipoprotein thrombi disappeared almost completely. Bezafibrate decreased plasma apo E and dramatically increased high density lipoprotein (HDL) /cholesterol. The decrease in apo E was observed mainly in the pre-ß-fraction, not in the a fraction. Lipidological analyses of our patient suggest that the origin her lipoprotein thrombi may be mainly from pre-ß-lipoproteins and that HDL might be involved in resolving lipoprotein thrombi. Our case suggests that administration of fibrates such as bezafibrate may be a novel therapeutic strategy for resolving intraglomerular thrombi and improving nephrotic syndrome in patients with LPG. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Apolipoprotein E; Bezafibrate; Type III hyperlipoproteinemia; Kidney failure; Lipoprotein glomerulopathy; Nephrotic syndrome
1. Introduction Lipoprotein glomerulopathy (LPG) is characterized by the presence of intraglomerular lipoprotein thrombi
Abbreviations: apo, apolipoprotein; Chol, cholesterol; HDL, high density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; LPG, lipoprotein glomerulopathy; LPL, lipoprotein lipase; PAGE, polyacrylamide gradient gel electrophoresis; RFLP, Restriction fragment length polymorphism; TG, triglycerides; VLDL, very low density lipoproteins. * Corresponding author. Tel.: /81-6-6879-3732; fax: /81-6-68793739. E-mail address: [email protected] (S. Yamashita).
and an elevation of apolipoprotein (apo) E. Patients with this disease have been reported since 1987 by several investigators [1 /3]. The general findings LPG as presented by Saito et al. [4] are as follows: mild to heavy proteinuria, strikingly dilated glomerular capillaries due to lipoprotein thrombi, hyperlipidemia-like type III hyperlipoproteinemia and elevated apo E, and genetic inheritance. Because several apo E variants have been reported in cases satisfying the above criteria, it has been speculated that these abnormal apo Es may play a pathogenic role in the development of this disease [3,5 / 8]. Type III hyperlipoproteinemia in patients with LPG shows a quite similar lipoprotein profile to that associated with a genetic type III hyperlipoproteinemia
0021-9150/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00194-1
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caused by homozygous apo E2 (R158C) [4,9]. Unlike type III hyperlipoproteinemia, LPG does not appear to cause atherosclerosis [4,10]. In fact, foam cells are rarely found in the glomeruli of patients with LPG. Moreover, lipoprotein thrombi have not been found in organs and tissues other than the kidneys. This finding has led us to speculate that there may be some specific factors involved in the glomeruli besides apo E abnormality. Although the pathogenesis of LPG is still unclear, many therapeutic trials including treatment with steroids, immunosuppressants, hydroxymethyl /glutaryl coenzyme A reductase inhibitors, low density lipoprotein (LDL) apheresis, and renal transplantation [4,11/ 13] have been performed. However, effective therapeutic regimens have not been established yet. We have experienced a patient with LPG in whom proteinuria was markedly improved and intraglomerular thrombi disappeared after bezafibrate administration. Here, we describe a case of LPG, suggest a novel therapeutic strategy using bezafibrate, and discuss the pathophysiological aspects of LPG on the basis of analyses of the lipoprotein changes induced by bezafibrate.
25% polyacrylamide gradient gel [14]. Electrophoresis was performed at 20 mA for 3 h at 4 8C. After electrophoresis, Western blot analysis for apo A-I, B, and E was performed. Immunoreactivity to apo A-I, B, and E (using polyclonal antibodies supplied by Daiichi Pure Chemicals) was identified by goat anti-human polyclonal antibodies using horseradish peroxidase.
2. Methods
3.1. Case report and clinical course
2.1. Plasma lipoprotein analysis
A 30-year-old Japanese woman was admitted to Ashiya Municipal Hospital in May 1998 with a diagnosis of nephrotic syndrome after severe proteinuria was detected during a routine medical checkup in April 1998. She had proteinuria since 1989, and bilateral pretibial edema was noticed for the first time in March 1998. Her first renal biopsy was performed during the May 1998 admission. Light microscopy showed dilated capillary lumina with massive lipoprotein thrombi in every thirteen glomeruli that were examined (Fig. 1A). There were no foam cells or tubulointerstitial lesions. Granular substances and lipid droplets filled the dilated capillary lumina (Fig. 1B). These substances were stained with oil red-O (data not shown). Glomerular basement membranes were not thick but, rather, slightly thin. In addition, effacement of the foot processes was seen (Fig. 1C). Immunohistochemical analysis showed massive and strong immunoreactivity to anti-apo E antibody in the lesions of the lipoprotein thrombi (Fig. 1D) but not to anti-apo B-100 and A-I antibodies (data not shown). Serum total Chol was 252 mg/dl, TG was 274 mg/dl, and HDL /Chol was 42 mg/dl. Plasma apo E concentration was elevated to 10.8 mg/dl (normal is 2.8 / 4.6 mg/dl). The phenotype of her apo E was E4/2 (data not shown). Her brother had already been diagnosed as having LPG with apo E2 Kyoto (Arg25Cys) and was being treated with maintenance hemodialysis despite renal transplantation [6,13]. We performed direct DNA sequencing and found the same mutation, apo E2 Kyoto (Arg25Cys), reported previously by Matsunaga et al. [6]
Venous blood was drawn after an overnight fast. The concentrations of total cholesterol (Chol) and triglycerides (TG) were measured enzymatically. We measured serum HDL /Chol by using commercial kits (L-Type HDL-C, Wako Pure Chemical Industries, Ltd, Osaka, Japan). Serum concentrations of apo A-I, B, and E were measured by immunoturbidimetric assay (Apoauto N ‘Daiichi,’ Daiichi Pure Chemicals, Tokyo, Japan). Very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), LDL, high density lipoproteins (HDL)2, and HDL3 were isolated by sequential preparative ultracentrifugation at densities of 1.006, 1.019, 1.063, 1.125, and 1.210 g/ml, respectively. The lipoprotein profiles were characterized by their electrophoretic mobility in an agarose gel. The lipoprotein profiles were evaluated by staining with Fad Red 7B. The distribution of apolipoproteins in agarose gel electrophoresis was determined by immunoprecipitation using apo A-I, B and E polyclonal antibodies (Daiichi Pure Chemicals). The lipoprotein profiles were also characterized in polyacrylamide disk electrophoresis. 2.2. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis Ultracentrifugally separated plasma at the density 1.21 g/ml (Himac CS 150GX, rotor; S120AT2) was analyzed by nondenaturing gel electrophoresis on 4 /
2.3. Histological analysis Tissue specimens obtained by renal biopsy were processed using routine methods for light microscopy, immunohistochemical analysis, and electron microscopy. Sections (3 mm) were stained with hematoxylin and eosin for light microscopy. Sections were also examined immunohistochemically using mouse anti-human apolipoprotein antibody (A-I and B from ICN Pharmaceuticals Inc., USA, #59430 and #59411, respectively; and E from PROGEN, Germany, #61085).
3. Results
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Fig. 1. Microscopic examinations of glomeruli in the renal specimen obtained at the first biopsy (May 1998) (A /D) and the second biopsy (September 2001) (E, F). Panels A and E show light microscopic photos (hematoxylin and eosin staining, magnification /40). Panels B, C and F are photos from electron microscopy ( /1500). Panel D shows an image from immunofluorescence microscopy with mouse anti-human apo E monoclonal antibody ( /400). The arrows in the photos in panels A and B indicate the lipoprotein thrombi. The arrows with an asterisk in the photos in panels C and F indicate the basement membrane of the glomeruli.
(data not shown). Her brother and father were also heterozygotes. Our patient was being treated with dipyridamole (300 mg/day orally since May 1998) and pravastatin (10 mg/ day since May 1999); however, her proteinuria status gradually deteriorated, and a second admission to our hospital was necessary in August 1999 due to severe
nephrotic syndrome. Physical examination at this time revealed severe bilateral pretibial edema. Her serum albumin had decreased to 2.1 g/dl from 3.4 g/dl. Daily urinary protein excretion reached 24 g/day. Other laboratory examinations showed the following: total protein, 3.7 g/dl; blood urea nitrogen, 16.5 mg/dl; serum creatinine, 0.7 mg/dl; creatinine clearance, 104 ml/min;
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total Chol, 209 mg/dl; TG, 135 mg/dl; HDL /Chol, 41 mg/dl; and apo E, 6.9 mg/dl. To treat her hyperlipoproteinemia, bezafibrate (400 mg/day orally) was started in November 1999 to replace the pravastatin. Over the next 2 years of treatment, her serum albumin gradually increased from 2.1 to 4.0 g/dl, and urinary protein excretion decreased to an average of less than 2 g/day (Fig. 2). Lipid profiles before administration of bezafibrate using the serum obtained on the first admission (June 1998) and after in September 2000 are shown in the Table 1. Serum total Chol and TG decreased from 206 to 190 mg/dl and from 172 to 70 mg/dl, respectively, after bezafibrate administration. HDL /Chol had dramatically increased from 38 to 66 mg/dl. Compared with the lipid profile on the first admission, concentrations of VLDL /Chol and LDL /Chol had decreased after bezafibrate administration; however, those of IDL / Chol were not dramatically changed. VLDL /TG and IDL /TG were decreased from 111.7 to 41.3 mg/dl and from 19.2 to 7.8 mg/dl, respectively. Apo E was decreased from 8.6 to 6.1 mg/dl. Analysis of lipoprotein profiles by polyacrylamide gel disk electrophoresis showed the midband indicating an increase in remnant lipoproteins both before and after bezafibrate administration. The amount of remnant lipoproteins had decreased after administration of bezafibrate but still remained abundant. No deterioration of her nephrotic syndrome was evident by both laboratory and physical examination over the 2-year treatment period. A second renal biopsy was performed in September 2001 after the 2-year bezafibrate treatment. All eight glomeruli which were able to be examined by light microscopy showed complete disappearance of intraglomerular lipoprotein
thrombi (Fig. 1E). Dilated capillary lumina were not observed, whereas mesangial cells had proliferated and glomerular basement membranes became thickened compared with those seen at the first renal biopsy (Fig. 1F). Enlargement of podocytes and effacement of the foot processes were also seen. 3.2. Electrophoretic analyses of lipoproteins To clarify the changes in the patient’s lipoproteins induced by bezafibrate, we performed several electrophoretic analyses. The most manifest change in agarose gel electrophoresis was the marked decrease in pre-blipoproteins and the increase in a-lipoproteins (Fig. 3A). A decrease in apo E concentrations was observed mainly in the pre-b-fraction, not in the a-fraction (Fig. 3B, C). Furthermore, polyacrylamide gradient gel electrophoresis (PAGE) and Western blot analyses indicated that apo E in the HDL fraction was predominantly distributed in the larger HDL fraction (HDL1) and that there was no large quantitative change in the amount of apo E in the HDL1 fraction (Fig. 3C). Furthermore, apo A-I concentration was dramatically increased in the HDL fraction (Fig. 3C).
4. Discussion Here, we have reported a patient with LPG, who was successfully treated with bezafibrate. Laboratory data such as proteinuria, serum albumin, and lipoprotein profiles were markedly improved after bezafibrate treatment. Furthermore, lipoprotein thrombi formerly
Fig. 2. Clinical course of the patient. The upper part shows the admission and treatment time line. The lower graph shows the changes in serum total protein and albumin and daily urinary protein excretion. Renal biopsy was performed twice, in May 1998, and September 2001.
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Table 1 Serum lipid and apolipoprotein levels Before medication (June 1998) Total cholesterol Triglycerides HDL /cholesterol
After medication (September 2001)
206 172 38
190 70 66
VLDL /cholesterola IDL /cholesterol LDL /cholesterol HDL2 /cholesterol HDL3 /cholesterol
36.9 15.3 117.9 14.2 17.4
9.8 13.0 98.8 16.3 42.4
VLDL /TG IDL /TG LDL /TG HDL2 /TG HDL3 /TG
111.7 19.2 27.5 5.9 6.3
41.3 7.8 13.4 2.2 7.8
106 109 8.6
168 74 6.1
Apolipoprotein A-I Apolipoprotein B Apolipoprotein E
Polyacrylamide disk electrophoresisb
HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein. a All values are expressed as mg/dl. b Analysis using polyacrilamide disk electrophoresis before and after administration of bezafibrate.
present in the glomeruli disappeared completely. No effective treatment for LPG has been established yet; however, our case clearly shows that administration of bezafibrate may have potential as a therapy for treatment of patients with LPG. It is well accepted that apo E plays a crucial role in the pathogenesis of LPG because plasma apo E is usually elevated in patients with LPG, and several apo E abnormalities have been identified [3,5/8]. Recently, it has been reported that an adenovirus-mediated transduction of apo E-Sendai resulted in LPG in apo Edeficient mice [15]. However, it has not been clarified how such mutations cause LPG. Apo E mutations related to LPG are not necessarily distributed in the binding site of apo E to the LDL receptor. Indeed, the apo E2 Kyoto (Arg25Cys) mutation, which was detected in our patient, resides in the beginning portion of the ahelix in apo E, even though the LDL receptor-binding activity was reduced to 10% compared with that of normal apo E3 [6]. Matsunaga et al. [6] speculated that Arg25Cys broke the interhelical salt bridge between Arg-25 and Glu-70 in the apo E3 structure, leading to a
change in the tertiary structure. A mutation of the same residue at a different position, apo E2 (Arg145Cys), causes a quite similar lipoprotein profile, whereas apo E2 (Arg145Cys) is not related to LPG [16]. Furthermore, some interaction between mutated apo E and other apolipoproteins may result in production of lipoprotein thrombi because it was reported that apo E formed a homodimer and heterodimer with apo A-II [17,18]. These observations led us to hypothesize that the mechanism of lipoprotein thrombi formation might be related mainly to the three-dimensional conformation changes of apo E, although the hyperlipoproteinemia in patients with LPG caused by reduced binding activity to the LDL receptor may accelerate lipoprotein thrombi formation. However, lipoprotein thrombi have not been found in any other organs or tissues except the glomeruli. Some investigators have speculated the existence of a specific factor in the kidney [4], that has not been identified yet. Remnant lipoproteins existed in our patient in some amounts even after bezafibrate administration, suggesting that our patient could still form lipoprotein thrombi
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Fig. 3. Lipids and lipoprotein analyses. Panel A shows the plasma lipoproteins evaluated by agarose gel electrophoresis. Be.-administration indicates a sample obtained before bezafibrate was administered, and Af.-administration shows a sample obtained during treatment. Panel B shows the distribution of apolipoproteins in agarose gel electrophoresis determined by immunoprecipitation in samples before (upper) and after (lower) bezafibrate administration. C indicates control and P the patient. Panel C shows the lipoprotein profiles of nondenaturing PAGE and Western blot analysis. The plasma was separated ultracentrifugally at a density of 1.210 mg/dl and run on nondenaturing PAGE. Then, Western blot analysis using apo A-I, B, and E antibodies was performed.
again. However, her lipoprotein thrombi disappeared. Her 33-year-old brother, however, requires maintenance hemodialysis despite having the same apo E abnormality [6]. Moreover, healthy carriers of mutant apo E related to LPG have been reported [6,19]. These findings indicate that the balance between formation and removal of lipoprotein thrombi may affect the clinical features. Bezafibrate is one of the fibric acid derivatives, which up-regulates the expression of lipoprotein lipase (LPL) [20] through activation of peroxisome proliferatoractivated receptor a [21]. The increase in LPL synthesis may enhance the hydrolysis of TG in chylomicrons and VLDL on the surface of endothelial cells [22]. Furthermore, bezafibrate inhibits acetyl CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis, leading to a decrease in TG synthesis in the liver [23]. Through these actions, bezafibrate induces a reduction of plasma TG, VLDL /Chol, and VLDL /TG, and an elevation of HDL /Chol [24,25]. Bezafibrate markedly improved the lipoprotein profiles in our subject (Table 1). How then did these changes in plasma lipoprotein levels after bezafibrate treatment result in the disappearance of the intraglomerular thrombi associated with LPG? We speculate the following two mechanisms. First, the supply of lipoproteins to lipoprotein thrombi may have been reduced because of the decrease in VLDL. Secondly, the increased HDL particles might have
removed Chol from the lipoprotein thrombi in the glomeruli. From the distribution of apo E in HDL (Fig. 3), small HDL particles containing apo A-I induced by bezafibrate administration may play an important role in removing lipoprotein thrombi from the glomerulus. Saito et al. [10] reported that in patients with LPG, an inverse correlation was observed between lipid particle size in lipoprotein thrombi and the TG/Chol ratio in plasma and HDL and that LPG patients with smaller lipid particles and a higher TG/Chol ratio were likely to be nephrotic. They concluded that elevated plasma VLDL /TG and HDL /TG levels may make the lipid particle size in lipoprotein thrombi smaller and are likely to lead to heavy proteinuria [4,10]. In our patient, bezafibrate improved the TG/Chol ratio from 1.09 to 0.37 in plasma and from 0.39 to 0.17 in HDL. Thus, bezafibrate may have modified plasma lipoproteins to be less nephrotoxic. In conclusion, lipid-lowering therapy, especially that focused on TG-rich lipoproteins, may be quite important for treatment of patients with LPG. The current study suggests that administration of fibrates such as bezafibrate may be a novel therapeutic strategy to resolve the intraglomerular thrombi and improve the nephrotic syndrome in patients with LPG. Further studies are necessary to establish the efficacy of bezafibrate and other fibrates in treatment of this rare disease.
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Acknowledgements The authors are thankful for the helpful technical support provided by Osamu Miyazaki (Daiichi Pure Chemicals Co.). Seiichi Hirota (Osaka University, Division of Pathology) gave us many important suggestions. This study was supported by a Medical Research Fund from Hyogo Medical Association to T.A. This research was also supported in part by an International HDL Research Awards Program grant to S.Y.
References [1] Faraggiana T, Churg J. Renal lipidoses: a review. Hum Pathol 1987;18:661. [2] 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. [3] Watanabe Y, Ozaki I, Yoshida F, et al. A case of nephrotic syndrome with glomerular lipoprotein deposition with capillary ballooning and mesangiolysis. Nephron 1989;51:265. [4] Saito T, Oikawa S, Sato H, Sasaki J. Lipoprotein glomerulopathy: renal lipidosis induced by novel apolipoprotein E variants. Nephron 1999;83:193. [5] Oikawa S, Matsunaga A, Saito T, et al. Apolipoprotein E Sendai (arginine 1450/proline): a new variant associated with lipoprotein glomerulopathy. J Am Soc Nephrol 1997;8:820. [6] Matsunaga A, Sasaki J, Komatsu T, et al. A novel apolipoprotein E mutation, E2 (Arg25Cys) in lipoprotein glomerulopathy. Kidney Int 1999;56:421. [7] 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. [8] 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. [9] Hazzard WR, Porte D, Jr., Bierman EL. Abnormal lipid composition of very low density lipoproteins in diagnosis of broad-beta disease (type 3 hyperlipoproteinemia). Metabolism 1972;21:1009. [10] Saito T, Oikawa S, Sato H, et al. Lipoprotein glomerulopathy: significance of lipoprotein and ultrastructural features. Kidney Int Suppl 1999;71:S37. [11] Zhang P, Matalon R, Kaplan L, et al. Lipoprotein glomerulopathy: first report in a Chinese male. Am J Kidney Dis 1994;4:942.
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[12] Andrews PA, O’Donnell PJ, Dilly SA, et al. Recurrence of lipoprotein glomerulopathy after renal transplantation. Nephrol Dial Transplant 1997;12:2442. [13] Komatsu T, Kanatsu K, Ochi H, et al. Lipoprotein glomerulopathy with a new apolipoprotein E phenotype. Am J Kidney Dis 1995;25:952. [14] Nichols AV, Krauss RM, Musliner TA. Nondenaturing polyacrylamide gradient gel electrophoresis. Methods Enzymol 1986;128:417. [15] 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. [16] Hsia SH, Connelly PW, Hegele RA. Restriction isotyping of apolipoprotein E (R145C) in type III hyperlipoproteinemia. J Investig Med 1995;43:187. [17] Weisgraber KH, Mahley RW. Apoprotein (E /A-II) complex of human plasma lipoproteins. I. Characterization of this mixed disulfide and its identification in a high density lipoprotein subfraction. J Biol Chem 1978;253:6281. [18] Weisgraber KH, Shinto LH. Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity. J Biol Chem 1991;266:12029. [19] Saito T, Oikawa S, Sato H, Chiba J. Lipoprotein glomerulopathy and its pathogenesis. Contrib Nephrol 1997;120:30. [20] de Man FH, de Beer F, van der Laarse A, et al. The hypolipidemic action of bezafibrate therapy in hypertriglyceridemia is mediated by upregulation of lipoprotein lipase: no effects on VLDL substrate affinity to lipolysis or LDL receptor binding. Atherosclerosis 2000;153:363. [21] Auwerx J, Schoonjans K, Fruchart JC, Staels B. Transcriptional control of triglyceride metabolism: fibrates and fatty acids change the expression of the LPL and apo C-III genes by activating the nuclear receptor PPAR. Atherosclerosis 1996;124(Suppl):S29. [22] Nilsson-Ehle P, Garfinkel AS, Schotz MC. Lipolytic enzymes and plasma lipoprotein metabolism. Annu Rev Biochem 1980;49:667. [23] Stewart JM, Packard CJ, Lorimer AR, et al. Effects of bezafibrate on receptor-mediated and receptor-independent low density lipoprotein catabolism in type II hyperlipoproteinaemic subjects. Atherosclerosis 1982;44:355. [24] Heller F, Harvengt C. Effects of clofibrate, bezafibrate, fenofibrate and probucol on plasma lipolytic enzymes in normolipaemic subjects. Eur J Clin Pharmacol 1983;25:57. [25] Norioka M, Suzuki M, Ryomoto K, et al. Effect of bezafibrate treatment on the altered lipoprotein profiles in hypertriglyceridemic subjects. J Atheroscler Thromb 2000;7:198.
Disappearance of intraglomerular lipoprotein thrombi and marked improvement of nephrotic syndrome by bezafibrate treatment in a patient with lipoprotein glomerulopathy Takeshi Arai a, Shizuya Yamashita b,*, Mitsukazu Yamane c, Noriko Manabe b, Toshiyuki Matsuzaki a, Kazuo Kiriyama a, Yoshio Kanayama a, Seiichi Himeno a, Yuji Matsuzawa b a
b
Department of Internal Medicine, Ashiya Municipal Hospital, 39-1 Asahigaoka, Ashiya, Hyogo 659-0012, Japan Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan c Department of Cardiology, Kawasaki Hospital, Kobe, Hyogo 652-0042, Japan Received 21 October 2002; received in revised form 6 May 2003; accepted 9 May 2003
Abstract Lipoprotein glomerulopathy (LPG) is a hereditary disorder characterized by intraglomerular lipoprotein thrombi and increased serum apolipoprotein (apo) E. Patients with LPG usually manifest with nephrotic syndrome, and some progress to renal failure; however, no effective therapeutic regimen has been established for this disease. We experienced a patient with LPG for whom bezafibrate treatment was very effective. This 30-year-old Japanese woman had nephrotic syndrome and type III hyperlipoproteinemia. Renal biopsy showed markedly dilated capillary lumina containing massive lipoprotein thrombi. Plasma apo E concentration was elevated to twice that of normal controls. She was proved to be a heterozygote of apo E2 Kyoto (Arg25Cys). After 2 years treatment with bezafibrate (400 mg/day), her plasma albumin gradually increased from 2.1 to 4.0 mg/dl, and intraglomerular lipoprotein thrombi disappeared almost completely. Bezafibrate decreased plasma apo E and dramatically increased high density lipoprotein (HDL) /cholesterol. The decrease in apo E was observed mainly in the pre-ß-fraction, not in the a fraction. Lipidological analyses of our patient suggest that the origin her lipoprotein thrombi may be mainly from pre-ß-lipoproteins and that HDL might be involved in resolving lipoprotein thrombi. Our case suggests that administration of fibrates such as bezafibrate may be a novel therapeutic strategy for resolving intraglomerular thrombi and improving nephrotic syndrome in patients with LPG. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Apolipoprotein E; Bezafibrate; Type III hyperlipoproteinemia; Kidney failure; Lipoprotein glomerulopathy; Nephrotic syndrome
1. Introduction Lipoprotein glomerulopathy (LPG) is characterized by the presence of intraglomerular lipoprotein thrombi
Abbreviations: apo, apolipoprotein; Chol, cholesterol; HDL, high density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; LPG, lipoprotein glomerulopathy; LPL, lipoprotein lipase; PAGE, polyacrylamide gradient gel electrophoresis; RFLP, Restriction fragment length polymorphism; TG, triglycerides; VLDL, very low density lipoproteins. * Corresponding author. Tel.: /81-6-6879-3732; fax: /81-6-68793739. E-mail address: [email protected] (S. Yamashita).
and an elevation of apolipoprotein (apo) E. Patients with this disease have been reported since 1987 by several investigators [1 /3]. The general findings LPG as presented by Saito et al. [4] are as follows: mild to heavy proteinuria, strikingly dilated glomerular capillaries due to lipoprotein thrombi, hyperlipidemia-like type III hyperlipoproteinemia and elevated apo E, and genetic inheritance. Because several apo E variants have been reported in cases satisfying the above criteria, it has been speculated that these abnormal apo Es may play a pathogenic role in the development of this disease [3,5 / 8]. Type III hyperlipoproteinemia in patients with LPG shows a quite similar lipoprotein profile to that associated with a genetic type III hyperlipoproteinemia
0021-9150/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00194-1
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T. Arai et al. / Atherosclerosis 169 (2003) 293 /299
caused by homozygous apo E2 (R158C) [4,9]. Unlike type III hyperlipoproteinemia, LPG does not appear to cause atherosclerosis [4,10]. In fact, foam cells are rarely found in the glomeruli of patients with LPG. Moreover, lipoprotein thrombi have not been found in organs and tissues other than the kidneys. This finding has led us to speculate that there may be some specific factors involved in the glomeruli besides apo E abnormality. Although the pathogenesis of LPG is still unclear, many therapeutic trials including treatment with steroids, immunosuppressants, hydroxymethyl /glutaryl coenzyme A reductase inhibitors, low density lipoprotein (LDL) apheresis, and renal transplantation [4,11/ 13] have been performed. However, effective therapeutic regimens have not been established yet. We have experienced a patient with LPG in whom proteinuria was markedly improved and intraglomerular thrombi disappeared after bezafibrate administration. Here, we describe a case of LPG, suggest a novel therapeutic strategy using bezafibrate, and discuss the pathophysiological aspects of LPG on the basis of analyses of the lipoprotein changes induced by bezafibrate.
25% polyacrylamide gradient gel [14]. Electrophoresis was performed at 20 mA for 3 h at 4 8C. After electrophoresis, Western blot analysis for apo A-I, B, and E was performed. Immunoreactivity to apo A-I, B, and E (using polyclonal antibodies supplied by Daiichi Pure Chemicals) was identified by goat anti-human polyclonal antibodies using horseradish peroxidase.
2. Methods
3.1. Case report and clinical course
2.1. Plasma lipoprotein analysis
A 30-year-old Japanese woman was admitted to Ashiya Municipal Hospital in May 1998 with a diagnosis of nephrotic syndrome after severe proteinuria was detected during a routine medical checkup in April 1998. She had proteinuria since 1989, and bilateral pretibial edema was noticed for the first time in March 1998. Her first renal biopsy was performed during the May 1998 admission. Light microscopy showed dilated capillary lumina with massive lipoprotein thrombi in every thirteen glomeruli that were examined (Fig. 1A). There were no foam cells or tubulointerstitial lesions. Granular substances and lipid droplets filled the dilated capillary lumina (Fig. 1B). These substances were stained with oil red-O (data not shown). Glomerular basement membranes were not thick but, rather, slightly thin. In addition, effacement of the foot processes was seen (Fig. 1C). Immunohistochemical analysis showed massive and strong immunoreactivity to anti-apo E antibody in the lesions of the lipoprotein thrombi (Fig. 1D) but not to anti-apo B-100 and A-I antibodies (data not shown). Serum total Chol was 252 mg/dl, TG was 274 mg/dl, and HDL /Chol was 42 mg/dl. Plasma apo E concentration was elevated to 10.8 mg/dl (normal is 2.8 / 4.6 mg/dl). The phenotype of her apo E was E4/2 (data not shown). Her brother had already been diagnosed as having LPG with apo E2 Kyoto (Arg25Cys) and was being treated with maintenance hemodialysis despite renal transplantation [6,13]. We performed direct DNA sequencing and found the same mutation, apo E2 Kyoto (Arg25Cys), reported previously by Matsunaga et al. [6]
Venous blood was drawn after an overnight fast. The concentrations of total cholesterol (Chol) and triglycerides (TG) were measured enzymatically. We measured serum HDL /Chol by using commercial kits (L-Type HDL-C, Wako Pure Chemical Industries, Ltd, Osaka, Japan). Serum concentrations of apo A-I, B, and E were measured by immunoturbidimetric assay (Apoauto N ‘Daiichi,’ Daiichi Pure Chemicals, Tokyo, Japan). Very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), LDL, high density lipoproteins (HDL)2, and HDL3 were isolated by sequential preparative ultracentrifugation at densities of 1.006, 1.019, 1.063, 1.125, and 1.210 g/ml, respectively. The lipoprotein profiles were characterized by their electrophoretic mobility in an agarose gel. The lipoprotein profiles were evaluated by staining with Fad Red 7B. The distribution of apolipoproteins in agarose gel electrophoresis was determined by immunoprecipitation using apo A-I, B and E polyclonal antibodies (Daiichi Pure Chemicals). The lipoprotein profiles were also characterized in polyacrylamide disk electrophoresis. 2.2. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis Ultracentrifugally separated plasma at the density 1.21 g/ml (Himac CS 150GX, rotor; S120AT2) was analyzed by nondenaturing gel electrophoresis on 4 /
2.3. Histological analysis Tissue specimens obtained by renal biopsy were processed using routine methods for light microscopy, immunohistochemical analysis, and electron microscopy. Sections (3 mm) were stained with hematoxylin and eosin for light microscopy. Sections were also examined immunohistochemically using mouse anti-human apolipoprotein antibody (A-I and B from ICN Pharmaceuticals Inc., USA, #59430 and #59411, respectively; and E from PROGEN, Germany, #61085).
3. Results
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Fig. 1. Microscopic examinations of glomeruli in the renal specimen obtained at the first biopsy (May 1998) (A /D) and the second biopsy (September 2001) (E, F). Panels A and E show light microscopic photos (hematoxylin and eosin staining, magnification /40). Panels B, C and F are photos from electron microscopy ( /1500). Panel D shows an image from immunofluorescence microscopy with mouse anti-human apo E monoclonal antibody ( /400). The arrows in the photos in panels A and B indicate the lipoprotein thrombi. The arrows with an asterisk in the photos in panels C and F indicate the basement membrane of the glomeruli.
(data not shown). Her brother and father were also heterozygotes. Our patient was being treated with dipyridamole (300 mg/day orally since May 1998) and pravastatin (10 mg/ day since May 1999); however, her proteinuria status gradually deteriorated, and a second admission to our hospital was necessary in August 1999 due to severe
nephrotic syndrome. Physical examination at this time revealed severe bilateral pretibial edema. Her serum albumin had decreased to 2.1 g/dl from 3.4 g/dl. Daily urinary protein excretion reached 24 g/day. Other laboratory examinations showed the following: total protein, 3.7 g/dl; blood urea nitrogen, 16.5 mg/dl; serum creatinine, 0.7 mg/dl; creatinine clearance, 104 ml/min;
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total Chol, 209 mg/dl; TG, 135 mg/dl; HDL /Chol, 41 mg/dl; and apo E, 6.9 mg/dl. To treat her hyperlipoproteinemia, bezafibrate (400 mg/day orally) was started in November 1999 to replace the pravastatin. Over the next 2 years of treatment, her serum albumin gradually increased from 2.1 to 4.0 g/dl, and urinary protein excretion decreased to an average of less than 2 g/day (Fig. 2). Lipid profiles before administration of bezafibrate using the serum obtained on the first admission (June 1998) and after in September 2000 are shown in the Table 1. Serum total Chol and TG decreased from 206 to 190 mg/dl and from 172 to 70 mg/dl, respectively, after bezafibrate administration. HDL /Chol had dramatically increased from 38 to 66 mg/dl. Compared with the lipid profile on the first admission, concentrations of VLDL /Chol and LDL /Chol had decreased after bezafibrate administration; however, those of IDL / Chol were not dramatically changed. VLDL /TG and IDL /TG were decreased from 111.7 to 41.3 mg/dl and from 19.2 to 7.8 mg/dl, respectively. Apo E was decreased from 8.6 to 6.1 mg/dl. Analysis of lipoprotein profiles by polyacrylamide gel disk electrophoresis showed the midband indicating an increase in remnant lipoproteins both before and after bezafibrate administration. The amount of remnant lipoproteins had decreased after administration of bezafibrate but still remained abundant. No deterioration of her nephrotic syndrome was evident by both laboratory and physical examination over the 2-year treatment period. A second renal biopsy was performed in September 2001 after the 2-year bezafibrate treatment. All eight glomeruli which were able to be examined by light microscopy showed complete disappearance of intraglomerular lipoprotein
thrombi (Fig. 1E). Dilated capillary lumina were not observed, whereas mesangial cells had proliferated and glomerular basement membranes became thickened compared with those seen at the first renal biopsy (Fig. 1F). Enlargement of podocytes and effacement of the foot processes were also seen. 3.2. Electrophoretic analyses of lipoproteins To clarify the changes in the patient’s lipoproteins induced by bezafibrate, we performed several electrophoretic analyses. The most manifest change in agarose gel electrophoresis was the marked decrease in pre-blipoproteins and the increase in a-lipoproteins (Fig. 3A). A decrease in apo E concentrations was observed mainly in the pre-b-fraction, not in the a-fraction (Fig. 3B, C). Furthermore, polyacrylamide gradient gel electrophoresis (PAGE) and Western blot analyses indicated that apo E in the HDL fraction was predominantly distributed in the larger HDL fraction (HDL1) and that there was no large quantitative change in the amount of apo E in the HDL1 fraction (Fig. 3C). Furthermore, apo A-I concentration was dramatically increased in the HDL fraction (Fig. 3C).
4. Discussion Here, we have reported a patient with LPG, who was successfully treated with bezafibrate. Laboratory data such as proteinuria, serum albumin, and lipoprotein profiles were markedly improved after bezafibrate treatment. Furthermore, lipoprotein thrombi formerly
Fig. 2. Clinical course of the patient. The upper part shows the admission and treatment time line. The lower graph shows the changes in serum total protein and albumin and daily urinary protein excretion. Renal biopsy was performed twice, in May 1998, and September 2001.
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Table 1 Serum lipid and apolipoprotein levels Before medication (June 1998) Total cholesterol Triglycerides HDL /cholesterol
After medication (September 2001)
206 172 38
190 70 66
VLDL /cholesterola IDL /cholesterol LDL /cholesterol HDL2 /cholesterol HDL3 /cholesterol
36.9 15.3 117.9 14.2 17.4
9.8 13.0 98.8 16.3 42.4
VLDL /TG IDL /TG LDL /TG HDL2 /TG HDL3 /TG
111.7 19.2 27.5 5.9 6.3
41.3 7.8 13.4 2.2 7.8
106 109 8.6
168 74 6.1
Apolipoprotein A-I Apolipoprotein B Apolipoprotein E
Polyacrylamide disk electrophoresisb
HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein. a All values are expressed as mg/dl. b Analysis using polyacrilamide disk electrophoresis before and after administration of bezafibrate.
present in the glomeruli disappeared completely. No effective treatment for LPG has been established yet; however, our case clearly shows that administration of bezafibrate may have potential as a therapy for treatment of patients with LPG. It is well accepted that apo E plays a crucial role in the pathogenesis of LPG because plasma apo E is usually elevated in patients with LPG, and several apo E abnormalities have been identified [3,5/8]. Recently, it has been reported that an adenovirus-mediated transduction of apo E-Sendai resulted in LPG in apo Edeficient mice [15]. However, it has not been clarified how such mutations cause LPG. Apo E mutations related to LPG are not necessarily distributed in the binding site of apo E to the LDL receptor. Indeed, the apo E2 Kyoto (Arg25Cys) mutation, which was detected in our patient, resides in the beginning portion of the ahelix in apo E, even though the LDL receptor-binding activity was reduced to 10% compared with that of normal apo E3 [6]. Matsunaga et al. [6] speculated that Arg25Cys broke the interhelical salt bridge between Arg-25 and Glu-70 in the apo E3 structure, leading to a
change in the tertiary structure. A mutation of the same residue at a different position, apo E2 (Arg145Cys), causes a quite similar lipoprotein profile, whereas apo E2 (Arg145Cys) is not related to LPG [16]. Furthermore, some interaction between mutated apo E and other apolipoproteins may result in production of lipoprotein thrombi because it was reported that apo E formed a homodimer and heterodimer with apo A-II [17,18]. These observations led us to hypothesize that the mechanism of lipoprotein thrombi formation might be related mainly to the three-dimensional conformation changes of apo E, although the hyperlipoproteinemia in patients with LPG caused by reduced binding activity to the LDL receptor may accelerate lipoprotein thrombi formation. However, lipoprotein thrombi have not been found in any other organs or tissues except the glomeruli. Some investigators have speculated the existence of a specific factor in the kidney [4], that has not been identified yet. Remnant lipoproteins existed in our patient in some amounts even after bezafibrate administration, suggesting that our patient could still form lipoprotein thrombi
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Fig. 3. Lipids and lipoprotein analyses. Panel A shows the plasma lipoproteins evaluated by agarose gel electrophoresis. Be.-administration indicates a sample obtained before bezafibrate was administered, and Af.-administration shows a sample obtained during treatment. Panel B shows the distribution of apolipoproteins in agarose gel electrophoresis determined by immunoprecipitation in samples before (upper) and after (lower) bezafibrate administration. C indicates control and P the patient. Panel C shows the lipoprotein profiles of nondenaturing PAGE and Western blot analysis. The plasma was separated ultracentrifugally at a density of 1.210 mg/dl and run on nondenaturing PAGE. Then, Western blot analysis using apo A-I, B, and E antibodies was performed.
again. However, her lipoprotein thrombi disappeared. Her 33-year-old brother, however, requires maintenance hemodialysis despite having the same apo E abnormality [6]. Moreover, healthy carriers of mutant apo E related to LPG have been reported [6,19]. These findings indicate that the balance between formation and removal of lipoprotein thrombi may affect the clinical features. Bezafibrate is one of the fibric acid derivatives, which up-regulates the expression of lipoprotein lipase (LPL) [20] through activation of peroxisome proliferatoractivated receptor a [21]. The increase in LPL synthesis may enhance the hydrolysis of TG in chylomicrons and VLDL on the surface of endothelial cells [22]. Furthermore, bezafibrate inhibits acetyl CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis, leading to a decrease in TG synthesis in the liver [23]. Through these actions, bezafibrate induces a reduction of plasma TG, VLDL /Chol, and VLDL /TG, and an elevation of HDL /Chol [24,25]. Bezafibrate markedly improved the lipoprotein profiles in our subject (Table 1). How then did these changes in plasma lipoprotein levels after bezafibrate treatment result in the disappearance of the intraglomerular thrombi associated with LPG? We speculate the following two mechanisms. First, the supply of lipoproteins to lipoprotein thrombi may have been reduced because of the decrease in VLDL. Secondly, the increased HDL particles might have
removed Chol from the lipoprotein thrombi in the glomeruli. From the distribution of apo E in HDL (Fig. 3), small HDL particles containing apo A-I induced by bezafibrate administration may play an important role in removing lipoprotein thrombi from the glomerulus. Saito et al. [10] reported that in patients with LPG, an inverse correlation was observed between lipid particle size in lipoprotein thrombi and the TG/Chol ratio in plasma and HDL and that LPG patients with smaller lipid particles and a higher TG/Chol ratio were likely to be nephrotic. They concluded that elevated plasma VLDL /TG and HDL /TG levels may make the lipid particle size in lipoprotein thrombi smaller and are likely to lead to heavy proteinuria [4,10]. In our patient, bezafibrate improved the TG/Chol ratio from 1.09 to 0.37 in plasma and from 0.39 to 0.17 in HDL. Thus, bezafibrate may have modified plasma lipoproteins to be less nephrotoxic. In conclusion, lipid-lowering therapy, especially that focused on TG-rich lipoproteins, may be quite important for treatment of patients with LPG. The current study suggests that administration of fibrates such as bezafibrate may be a novel therapeutic strategy to resolve the intraglomerular thrombi and improve the nephrotic syndrome in patients with LPG. Further studies are necessary to establish the efficacy of bezafibrate and other fibrates in treatment of this rare disease.
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Acknowledgements The authors are thankful for the helpful technical support provided by Osamu Miyazaki (Daiichi Pure Chemicals Co.). Seiichi Hirota (Osaka University, Division of Pathology) gave us many important suggestions. This study was supported by a Medical Research Fund from Hyogo Medical Association to T.A. This research was also supported in part by an International HDL Research Awards Program grant to S.Y.
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