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 However, corticosteroid treatment was withdrawn because the first biopsy suggested LPG.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.
A 20-year-old Japanese female student was admitted to Toyonaka Municipal Hospital in 2006 in order to undergo examinations, including renal biopsy, because she was found to have chance proteinuria and hematuria at the health surveillance in her university. On admission, she was 171 cm tall and weighed 68.1 kg. Her blood pressure was 124/90 mm Hg and her pulse rate was 75/min with no arrhythmia. No abnormal finding was identified by physical examination. Her laboratory findings are described in Table . Significant proteinuria and hematuria with hypoalbuminemia were recognized, but the criteria for nephrotic syndrome were not satisfied. Other values, including lipids and renal functions, were within normal ranges.\nOn renal biopsy, more than 30 glomeruli were contained in LM specimens, some of which showed global sclerotic change. Neither mesangial cell proliferation nor mesangial matrix expansion was marked (Fig. ). In silver methenamine-stained sections, “spike” formation as seen in MN was found in most of the glomeruli (Fig. ). Foam cells were not found in any glomeruli, but in the interstitial area. On IF staining, very weak linear patterns were identified for IgG and C3 in glomeruli, but a granular pattern as seen in MN was not identified (data not shown). EM study showed highly dense deposits in the subepithelial, subendothelial, and mesangial areas, which appeared like microbubbles or microcysts on higher magnification (Fig. ).\nBased on the renal histological findings, in spite of the almost negative results on IF study, MN was diagnosed, and treatment with prednisolone and cyclosporine A was started. The combination of 2 immunosuppressive agents initially seemed effective because of decreased urinary protein. When the dosages were gradually lowered, however, urinary protein was persistent between 0.5 and 2.5 g/gCr. The estimated glomerular filtration rate was slowly reduced but became stable at around 50 mL/min/1.73 m2 after 5 years of the first biopsy. Mild anemia was observed initially, and the hemoglobin level further decreased corresponding with the decline in estimated glomerular filtration rate. After the administration of an erythropoiesis-stimulating agent, however, it was more than 10 g/dL.\nAlthough serum triglycerides and cholesterol were within normal limits (Table ), the plasma ApoE concentration was high (LCAT: 82 nmol/mL; ApoA1: 187 mg/dL; ApoA2: 32.1 mg/dL; ApoB: 52 mg/dL; ApoE: 10.4 mg/dL). Since this finding suggested an ApoE abnormality, the ApoE phenotype, genotype, and ApoE DNA sequence of the patient were examined.\nPlasma ApoE phenotypes were analyzed by isoelectric focusing polyacrylamide gel electrophoresis and immunoblotting analysis, as previously reported [, ]. The patient's sample showed the position of ApoE2/2 (Fig. ).\nThe ApoE genotype was determined by restriction fragment length polymorphism analysis, as described previously [, ]. The polymerase chain reaction (PCR) products were digested with the restriction enzyme HhaI. Genotype ε2/2 in the patient was identified by 91- and 83-bp fragments (Fig. ). The ApoE Toyonaka (Ser197Cys), as shown in Figure , was also confirmed by restriction fragment length polymorphism analysis digested with SacI (Fig. ). Genomic DNA was amplified by 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 (Fig. ). The 358- and 335-bp fragments after cleavage by SacI showed a heterozygous novel mutation of ApoE Toyonaka (Fig. ).\nSequencing of ApoE DNA was performed as described previously []. As a result, in our patient, we identified a homozygous polymorphism at codon 158 (ε2/2: 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) (Fig. ).\nThe first biopsy showed MN-like findings, but immune deposits were not proven there, and immune-genetic MN was doubtful. Meanwhile, the gene examination for ApoE revealed the coexistence of ApoE Toyonaka and ApoE2/2, suggesting the involvement of a new form of ApoE-related disease. To clarify the accurate pathological changes, a second renal biopsy was conducted 9 years after the first one. Although the histological findings by LM and IF were similar to those of the first biopsy, the density of deposits on EM were more obvious, and ApoE was identified by immunohistochemical studies for LM and EM specimens using anti-ApoE antibody (Novus Biologicals, Littleton, CO, USA) (Fig. ) [].\nIn order to determine the glomerular deposits, an analysis by means of liquid chromatography-assisted MS/MS (LC-MS/MS) of glomeruli obtained from both renal biopsies using laser capture microdissection method was performed as previously reported []. In brief, glomeruli in a 10-μm-thick section of formalin-fixed paraffin-embedded tissue stained with hematoxylin were microdissected with laser microdissection (PALM MicroBeam, Carl Zeiss, Oberkochen, Germany). Ten glomeruli were analyzed for each specimen. Microdissected fragments were digested into peptides using trypsin and analyzed on amazon ETD (Bruker Daltonics, Billerica, MA, USA) using LC-MS/MS. Peptides present in raw data files were queried by the use of Mascot and X!Tandem, the results were then assigned peptide and protein probability scores, and they were displayed by the use of Scaffold software (Proteome Software Inc., Portland, OR, USA). As a result, the most abundant protein was ApoE in both samples (Fig. ).