A 79-year-old man was admitted to the Department of Nephrology, Kansai Electric Power Hospital on January 14, 2014, for proteinuria. He had been followed up for hypertension and dyslipidemia in a local clinic since the age of 50. He had experienced several vascular events, such as total occlusion in the common iliac artery at the age of 63 and two histories of cerebral infarction at the ages of 70 and 77. He had 30 years of smoking history but no alcohol habit. He had no family history of kidney disease except for one cousin showing end-stage renal disease of unknown etiology. During his initial admission to our hospital, his urine protein level was 5.4 g/g Cr, and serum albumin was 3.2 g/dL; he was discharged because his condition was stable. Ten months later, edema of his lower limbs worsened, and renal biopsy was performed on his second admission.\nOn the second hospitalization, physical examination showed the following: height of 169 cm, weight of 70 kg, body mass index of 24.5, and blood pressure of 180/88 mmHg. Laboratory findings revealed a nephrotic range of urine protein, 9.15 g/g Cr accompanying microscopic hematuria of dysmorphic 10 – 19 RBC/HPF, and total serum protein and albumin of 4.8 and 1.8 g/dL, respectively. Serum creatinine was 0.95 mg/dL, AST was 29 IU/L, ALT was 14 IU/L, and ALP was 188 IU/L. Serological examination revealed serum IgG, IgA, and IgM of 875, 344, and 52 mg/dL, respectively. Antinuclear and antineutrophil cytoplasmic antibodies were negative.\nLipid analysis revealed elevated total cholesterol (259 mg/dL), triglyceride (376 mg/dL), and LDL cholesterol (167 mg/dL) levels, and a low HDL cholesterol level (45 mg/dL). The serum ApoE level was 13.6 mg/dL (normal range: 2.8 – 4.6 mg/dL). An oral glucose tolerance test showed no abnormality, and his hemoglobin A1c was 5.7%. Although hypertriglyceridemia was marked, systemic abnormalities characteristic of hyperlipidemia, such as corneal opacities, xanthoma, and Achilles tendon hypertrophy, were not found. Moreover, the patient had no family history of hyperlipidemia and diabetes mellitus.\nLight microscopy (LM) revealed that the specimens contained 27 glomeruli, 8 of which showed global sclerosis (29.6%). Most glomeruli showed segmental sclerosis, infiltration of foam cells containing lipid deposits in capillary lumen (a). In periodic methenamine silver (PAM)-stained sections, spike formation and bubbling were detected on the glomerular basement membrane (GBM) (c, d). Mild to severe arteriosclerosis and arteriolosclerosis were also seen. Fibrosis was observed in ~ 20% of the whole interstitial area. Intriguingly, Sudan III staining was strongly positive not only in the mesangial and infiltrating foam cells but also in the focal capillary lumen in the glomerulus (b). Immunofluorescence (IF) studies demonstrated subtle granular staining for IgG along the capillary wall; however, C3 was not detected (data not shown). Electron microscopic (EM) findings analysis showed electron-dense deposits (EDDs) with a variety of densities and distributions. Highly-dense deposits accompanied by spike formation identical to MN were seen in the subepithelial area (a, b). In the subendothelial area, highly dense EDDs were detected but in higher magnification, and microbubbles were contained (a, b). Effacement of foot process was also accompanied with these depositions. In addition, EM findings demonstrated that the capillary lumina was filled with lipid deposits that did not show lamellar formation similar to “lipid thrombi” in LPG.\nPlasma ApoE phenotypes were analyzed by isoelectric focusing in polyacrylamide gel electrophoresis and immunoblotting analysis as previously reported [, ]. The patient’s sample showed the position of ApoE2/2 (a, Lane 1).\nThe APOE genotype was determined by restriction fragment length polymorphism (RFLP) analysis as described previously [, ]. The PCR products were digested with the restriction enzyme HhaI. Genotype ε2/2 in the patient was identified by 91- and 83-bp fragments (b, Lane 1). The ApoE Toyonaka (Ser197Cys) as shown in d was also confirmed by RFLP analysis digested with SacI (c). Genomic DNA was amplified by polymerase chain reaction (PCR) using oligonucleotide primers, sense 5′-CGTGCGGGCCGCCACTGTGAGCT-3′ and antisense 5′-TCGCATGGCTGCAGGCTTCGGCGTTC-3′. The 335-bp fragment after cleavage by SacI suggested normal c.644C of ApoE in codon 197 (Lane 3). The 358- and 335-bp fragments after cleavage by SacI showed a heterozygous novel mutation of ApoE Toyonaka (Lane 2). Direct sequencing of APOE DNA was performed as described previously []. Based on the results, we identified a homozygous polymorphism at codon 158 (e2/2: c.526 C>T: Arg158Cys) (data not shown) and a heterozygous missense mutation (C to G) in exon 4 leading to an amino acid substitution Cys (TGC) for Ser (TCC) at codon 197 (c.644 C>G: p.S215C: Ser197Cys) (d). All these findings were the same as that of a previous case of ApoE2/2 and ApoE Toyonaka showing MN-like glomerular histology [6].\nFor hypertriglyceridemia, fibrate 400 mg/day was started. Obtaining the results of the IF and EM studies, which did not deny immune-related MN like lesions, PSL 10 mg/day and cyclosporine (CyA) 75 mg/day for NS were started. These treatments brought about a substantial improvement of hypertriglyceridemia and complete remission of NS by 300 days after admission. During maintenance therapy with oral PSL 5 mg and CyA 75 mg/day, he had cerebral infarction and aspiration pneumonia. After withdrawal of CyA, his NS relapsed with an increase of proteinuria more than 1 g/day.
A 25-year-old Japanese man was admitted to our hospital because of proteinuria and general edema. Proteinuria and high blood pressure had been detected at a routine medical check-up 3 years earlier. His past history was otherwise unremarkable. Although his mother suffered from nephrolithiasis, there was no family history of renal diseases such as nephrotic syndrome and renal failure. At the first visit to our hospital, his height was 172 cm, weight was 76.2 kg, blood pressure was 170/120 mm Hg, and pulse rate was 91 bpm. Physical examination revealed no particular abnormalities other than pitting edema of the lower extremities. He had neither corneal opacities nor xanthomata. Laboratory findings were as follows: white blood cell count 7,700/μl, (neutrophils 88.0%, lymphocytes 4.0%, eosinophils 4.0%), hemoglobin 11.6 g/dl, platelet count 295,000/μl, total protein 4.0 g/dl, albumin 1.6 g/dl, hemoglobin A1c 5.3%, serum creatinine 1.36 mg/dl, total cholesterol 887 mg/dl, triglycerides 553 mg/dl, and HDL-cholesterol 45 mg/dl. Liver function and total complement were within normal ranges. Serum IgG, IgA, and IgM were 213, 116, and 235 mg/dl, respectively. Anti-nuclear antibody and anti-neutrophil cytoplasmic antibody were negative. The serum apoE level was 14.6 mg/dl (normal range: 2.7-4.3 mg/dl). Urine protein excretion was 12.9 g/g Cr, and urine sediment RBC was 20-29/hpf.\nInitial treatment consisted of high-dose corticosteroid for nephrotic syndrome. However, corticosteroid treatment was withdrawn because the first biopsy suggested LPG. After antihyperlipidemic treatment with atorvastatin, the serum cholesterol and triglyceride levels decreased substantially, but urinary protein excretion continued in the nephrotic range. Thereafter, the use of probucol and bezafibrate brought a significant decrease in urine protein excretion (0.3 g/g Cr). Though serum cholesterol and triglyceride levels were reduced, the serum apoE level did not change with lipid-lowering therapy including bezafibrate (before 14.6 mg/dl, after 12.4 mg/dl).\nFirst Biopsy (January 2013). The light microscopy specimens contained 40 glomeruli, 7 and 2 of which showed global and segmental sclerosis, respectively. Most glomeruli showed not only typical lipoprotein thrombi filled in the dilated capillaries, but also many infiltrating foam cells (fig. ). Diffuse/segmental mesangial cell proliferation and exudative lesions were also observed. Immunohistochemical studies using serial sections demonstrated strong staining for both apoB and apoE within the dilated capillary lumen filled with lipoprotein thrombi (fig. ). In addition, positivity for apoE was also observed at sites other than lipoprotein thrombi (fig. ). Intriguingly, CD68+ foam cells infiltrated in the apoE-positive parts other than lipoprotein thrombi (fig. ). Electron micrographs for the glomerulus showed intra-capillary lipoprotein thrombus-like substances (fig. ) and foamy changes (fig. ).\nSecond Biopsy (September 2013). The light microscopy specimens contained 23 glomeruli. Although lipoprotein thrombi and foam cells were still observed, their number was much lower than on the first biopsy (data not shown). Immunofluorescence examinations showed only non-specific IgM deposition along the capillary wall (data not shown).\nSerum apoE phenotypes were analyzed using isoelectric focusing polyacrylamide gel electrophoresis (IEF), as previously described [,]. As compared to apoE3/3 of control wild type, the patient had alleles apoE1/2 (fig. ).\nThe apoE genotype was determined by restriction fragment length polymorphism (RFLP) analysis as described previously [,]. Genomic DNA was amplified by polymerase chain reaction (PCR) using oligonucleotide primers, sense 5′-ACGCGGGCACGGCTGTCCAA-GGA-3′ and antisense 5′-TCGCGGGCCCCGGCCTGGTACAC-3′. The PCR products were digested with the restriction enzyme HhaI. In addition to the fragments characteristic of genotype ε2 (91-, 78-, and 36-bp fragments), a 30-bp fragment which was not seen in genotype ε2 and an around 60-bp fragment which was not detected in any of the common genotype ε2, ε3, or ε4 were identified (fig. ).\nSequencing of apoE DNA was performed as follows. Three fragments of genomic DNA containing all coding sequence of mature apo E were amplified by polymerase chain reaction with the following primers for apo E gene: 5′-GCTTTCCAAGTGATTAAACCGACT-3′ and 5′-AGAGCTAAAGCCAGGAGTCAG-3′ for exon 3, and 5′-CCTCTTGGGTCTCTCTGGCT-3′ and 5′-CTGCTCCTTCACCTCGTCCA-3′, 5′-GCAGTACCGCGGCGAGGTGCAGG-3′ and 5′-GATCGTGCCAC-TGCACTCTA-3′ for exon 4. The amplified DNA fragments were purified by PCR purification Kit (Qiagen, Germany) and directly sequenced with Genetic Analyzer 3130xl DNA sequencer (Thermo Fisher, USA) by using a BigDye Terminator Cycle Sequencing Kit (Thermo Fisher). A heterozygous deletion of 9 bp was identified in codon 141 to codon 143 of the apoE gene (fig. ). This mutation was consistent with apoE Tokyo/Maebashi mutation in LPG patients, as previously reported [,]. Furthermore, the patient also had heterogeneous apoε2 (Arg158Cys) (fig. ). Because ε2 corresponds to apoE phenotype E2, apoE Tokyo/Maebashi mutation seemed to be expressed as the apoE phenotype E1 by IEF (fig. ). This phenomenon was consistent with a previous report []. These findings indicate that this patient was a compound heterozygote for apoE Tokyo/Maebashi and apoE2. Written informed consent was obtained from the patient before commencement of the phenotype and gene analysis of apoE.