Relationships between levels of urinary podocalyxin, number of urinary podocytes, and histologic injury in adult patients with IgA nephropathy.

BACKGROUND AND OBJECTIVES: Podocalyxin (PCX) is present on the apical cell membrane of podocytes and is shed in urine from injured podocytes. Urinary podocalyxin (u-PCX) is associated with severity of active glomerular injury in patients with glomerular diseases. This study examined the relationship between number of urinary podocytes, levels of u-PCX, and glomerular injury in adults with IgA nephropathy (IgAN). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Urine samples voided in the morning on the day of biopsy were obtained from 51 patients with IgAN (18 men and 33 women; mean age, 31 years). All renal biopsy specimens were analyzed histologically. Pathologic variables of IgAN were analyzed per Shigematsu classification, the Oxford classification of IgAN, and the Clinical Guidelines of IgAN in Japan. Levels of u-PCX were measured by sandwich ELISA. RESULTS: Histologic analysis based on Shigematsu classification revealed a significant correlation between levels of u-PCX and severity of acute extracapillary abnormalities (r=0.72; P<0.001), but levels of urinary protein excretion did not correlate with acute glomerular abnormalities. Levels of urinary protein excretion in patients with segmental sclerosis (n=19) were higher than in patients without (n=22) (0.49 [interquartile range (IQR), 0.20-0.88] g/g creatinine versus 0.20 [IQR, 0.10-0.33] g/g creatinine; P<0.01). The number of urinary podocytes in patients with segmental sclerosis was higher than in patients without (1.05 [IQR, 0.41-1.67] per mg creatinine versus 0.28 [IQR, 0.10-0.66] per mg creatinine; P<0.01). CONCLUSIONS: Levels of u-PCX and the number of urinary podocytes are associated with histologic abnormalities in adults with IgAN.

Significance of urinary full-length and ectodomain forms of megalin in patients with type 2 diabetes.

OBJECTIVE: Megalin, an endocytic receptor in proximal tubule cells, is involved in the mechanisms of albuminuria in diabetic nephropathy (DN). To develop efficient novel biomarkers associated with the pathogenesis of DN, we investigated urinary megalin excretion in type 2 diabetes. RESEARCH DESIGN AND METHODS: Sandwich enzyme-linked immunosorbent assay systems were established with monoclonal antibodies against the NH(2) (amino [A]-megalin assay) and COOH (C-megalin assay) termini of megalin to analyze urinary forms of megalin in 68 patients with type 2 diabetes. RESULTS: The A-megalin assay mainly detected a megalin ectodomain form in the soluble urinary fraction, whereas the C-megalin assay identified a full-length form in both soluble and insoluble fractions. Urinary C-megalin levels were significantly high in patients with normoalbuminuria, were elevated in line with increased albuminuria, and showed a better association with estimated glomerular filtration rate (eGFR) (<60 mL/min/1.73 m(2)) than did urinary albumin. In contrast, urinary A-megalin levels were increased in patients with normo- and microalbuminuria but not in those with macroalbuminuria. Urinary C-megalin levels were also positively associated with plasma inorganic phosphate and negatively with hemoglobin levels in those showing no features of bleeding and not taking vitamin D analogs, phosphate binders, or erythropoiesis-stimulating agents. CONCLUSIONS: Urinary full-length megalin excretion as measured by the C-megalin assay is well associated with reduced eGFR and linked to the severity of DN, phosphate dysregulation, and anemia, whereas urinary excretion of megalin ectodomain as measured by the A-megalin assay may be associated with distinctive mechanisms of earlier DN in type 2 diabetes.

Podocyte membrane vesicles in urine originate from tip vesiculation of podocyte microvilli.

Podocyte injury is involved in both the onset and progression of glomerular diseases. Our previous studies revealed that apical cell membranes of podocyte are shed into urine sediment and that urinary podocalyxin is a useful biomarker of podocyte injury. In this study, we examined the origin of urinary podocalyxin. Urine samples and kidney specimens from healthy children (n = 126) and patients with glomerular diseases (n = 77) were analyzed by immunohistologic methods. Immunofluorescence studies demonstrated that urinary podocalyxin was shed as granular structures into both the urine sediment and supernatant. Large amounts of podocalyxin were shed into both the urine sediment (17.2 +/- 3.2 ng/mg creatinine) and the supernatant (172.6 +/- 24.6 ng/mg creatinine) of patients, compared with the small amounts of urinary podocalyxin in healthy controls (sediment, 0.5 +/- 0.1 ng/mg creatinine; supernatant, 24.3 +/- 3.5 ng/mg creatinine). Electron and immunoelectron microscopic examinations showed that podocalyxin-positive vesicles in the sediment (125.6 +/- 8.8 nm) and the supernatant (121.2 +/- 6.4 nm) were similar in size to podocyte microvilli in biopsy specimens (123.6 +/- 8.9 nm), differentiating them from the much smaller urine exosomes (30-80 nm in diameter). Urine podocalyxin-positive vesicles tested negative in immunofluorescence microscopy on both exosomal markers CD24 and CD63. Podocalyxin-positive vesicles also tested negative for cytoskeletal markers, and electron microscopic examination revealed tip vesiculation of microvilli. We conclude that human urinary apical cell membrane vesicles appear to originate not from podocyte exosomes but from tip vesiculation of glomerular podocyte microvilli.