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.
Hara
Masanori
M
Department of Pediatrics, Yoshida Hospital, Niigata 959-0242, Japan. mhara@yoshidea-hosp.jp
Yanagihara
Toshio
T
Hirayama
Yoshiaki
Y
Ogasawara
Shinya
S
Kurosawa
Hiroyuki
H
Sekine
Sakari
S
Kihara
Itaru
I
D000293:Adolescent; D015415:Biomarkers / Q000378:metabolism; D002458:Cell Fractionation; D002648:Child; D002675:Child, Preschool; D022162:Cytoplasmic Vesicles / Q000378:metabolism / Q000648:ultrastructure*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005921:Glomerulonephritis / Q000473:pathology* / Q000652:urine; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D009404:Nephrotic Syndrome / Q000473:pathology* / Q000652:urine; D050199:Podocytes / Q000378:metabolism / Q000473:pathology*; D011507:Proteinuria; D012795:Sialoglycoproteins / Q000378:metabolism*; D016482:Urinalysis / Q000379:methods; D055815:Young Adult
D015415:Biomarkers; D012795:Sialoglycoproteins; C001643:podocalyxin