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 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. ).