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. ).
A 58-year-old male was referred to our center by his primary care physician due to nephrotic syndrome. He had massive proteinuria (7.38 g/day) and hypoalbuminemia (1.9 g/dl). He had been treated for diabetes since the age of 50 years and had been started on insulin therapy at the age of 55 years. He had been well-managed (HbA1c, 5.3%), although a pre-proliferative retinopathy and mild renal dysfunction (sCr, 1.38 mg/dl) became evident. His renal biopsy revealed 5 of 22 glomeruli with global sclerosis. The residual glomeruli showed massive foam cell infiltration in the glomerular capillaries and severely widened mesangium (fig. ). These foam cells stained positive for CD68 (not shown). Transmission electron microscope images also revealed foam cell infiltration mainly located in the capillaries containing scattered lipid droplets and lamellar bodies in their cytoplasm (fig. ). The glomerular tufts were enlarged with hyalinosis, mesangiolysis and microaneurysmal formation, which were consistent with diabetic glomerulosclerosis. The interstitium and tubules showed moderate fibrosis and atrophy (50% of the cortical region). The foamy change was seen in some tubular epithelial cells, but no interstitial foamy cells were found. The interlobular arteries showed a moderate degree of fibrous intimal thickening, and the arterioles also showed a moderate degree of subendothelial hyalinosis. Immunofluorescence studies revealed nonspecific linear IgG deposits along the GBM; however, no significant deposit was found by electron microscopy. The patient's lipid profile showed type III HLP (table ). He had an apoE2 homozygote phenotype and the Arg158Cys genotype with no other mutations. Three years after the diagnosis, he progressed to renal failure and was started on hemodialysis.