Tubular epithelium culture from nephronophthisis-affected kidneys: a new approach to molecular disorders of tubular cells.
Abnormalities of tubular membrane structure and composition have been proposed as the primary defect in nephronophthisis (NEF). In order to characterize the protein composition of tubular cells in NEF, in vitro methods were developed to culture and propagate tubular cells obtained from biopsy fragments. Accordingly, microdissected cortical slices (1 x 3 mm) were first digested with collagenase and DNAse and then grown in RPMI medium supplemented with 10% NU serum and conditioned serum deriving from 3T3 cultures. At confluence, cultured cells from NEF showed characteristics which were typical of normal tubules, i.e. presence of cytokeratin and positivity for succinic dehydrogenase and alkaline phosphatase stainings, and presented no morphological alterations compared to cultured cells from normal tubular epithelium. Moreover, no difference was observed for fibronectin, collagen IV and laminin stains. Analysis by two-dimensional electrophoresis of cellular extracts revealed several changes in protein composition of NEF, the main one being the decrease in NEF cells of a polypeptide with a molecular weight of 120 kD and a pI of 4.8; this polypeptide was a constant finding in normal kidneys. These observations demonstrated that human tubular epithelial cells can be successfully cultured from very small biopsy fragments, which represents a new approach to the study of molecular disorders involving tubular cells in inherited disease. Cultured cells from NEF maintain the same morphological, immunological and cytochemical characteristics as normal tubular cells, but present a few alterations in polypeptide composition which may have pathogenetic relevance. A more careful analysis of these alterations is needed to define the molecular disorder(s) involving the tubule in NEF.
Bertelli
R
R
Department of Nephrology, G. Gaslini Institute, Genoa, Italy.
Ginevri
F
F
Candiano
G
G
Ciardi
M R
MR
Tarelli
L T
LT
Meroni
M
M
Sessa
A
A
Ghiggeri
G M
GM
Gusmano
R
R
D001706:Biopsy; D002457:Cell Extracts / Q000032:analysis; D002478:Cells, Cultured; D002648:Child; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007684:Kidney Tubules / Q000737:chemistry / Q000473:pathology*; D008854:Microscopy, Electron; D009395:Nephritis, Interstitial / Q000473:pathology*; D010455:Peptides / Q000032:analysis
D002457:Cell Extracts; D010455:Peptides