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. ).
The patient is a 28-year-old Hispanic female with a past medical history of\nhypertension, high serum cholesterol, and gastritis who presented to the emergency\nroom for the evaluation of abdominal pain of 2 days duration. Six months prior, she\nhad similar pain. An upper gastrointestinal (GI) endoscopy at that time showed\npeptic ulcer disease and no other mucosal lesions. The patient had a strong family\nhistory of renal disease and 2 of her siblings required a renal transplant, but she\ndid not know what kidney pathology they had. Her medications included Lisinopril\noral 10 mg tablet daily and Pantoprazole oral 40 mg daily. On presentation at the\nemergency room (ER), her height and weight were 157 cm and 61 kg, respectively, and\nher blood pressure was 206/121 mm Hg. On physical examination, the patient had\nepigastric tenderness. The rest of her physical examination was unremarkable.\nRadiology was ordered to exclude any anatomical abnormality. A computed tomography\nscan showed neither atrophy nor enlargement of the kidneys. She then followed up as\nan outpatient with a nephrologist shortly after her ER visit. Her laboratory work up\nduring that visit revealed the following: nephrotic range proteinuria (9.2 g protein\non 24-hour urine collection; reference range, 0-2.5 g/d), hypoalbuminemia (300\nµmol/L; reference range, 421-662 µmol/L), hypoproteinemia (50 g/L; reference range,\n60-80 g/L), elevated serum creatinine (148.5 µmol/L; reference range, 44-80 μmol/L),\nhigh chloride (110 mmol/L; reference range, 98-106 mmol/L), and low hemoglobin (100\ng/L; reference range, 120-160 g/L). The lipid profile showed an increased total\ncholesterol level (8.3 mmol/L; reference range, <5.2 mmol/L), elevated\ntriglycerides (3.7 mmol/L; reference range, <1.70 mmol/L), elevated low-density\nlipoprotein fraction (5.9 mmol/L; reference range, <2.6 mmol/L), and decreased\nhigh-density lipoprotein fraction (1.1 mmol/L; reference range, ≥1.55 mmol/L). After\ntreating her blood pressure, the patient underwent a US-guided renal biopsy.