title: Spontaneous calcification process in primary renal cells from a medullary sponge kidney patient harbouring a GDNF mutation.
abstract: Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.-27 + 18G>A variant in the GDNF gene encoding glial cell-derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using immunocytochemistry and RT-PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry and RT-PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes; however, Ca₂PO₄ was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes or stromal stem cells and were positive for vimentin, ZO1, αSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin, indicating an osteoblast-like phenotype. In contrast to the control cells, GDNF was down-regulated in the MSK cells. Stable GDNF knockdown was established in the HK2 cell line and was found to promote Ca₂PO₄ deposition when the cells were incubated with calcifying medium by regulating the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which pericyte/stromal-like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down-regulation may have influenced the observed phenomenon.
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Nephrology Division, Department of Medicine DIMED, University of Padua, Padua, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Mezzabotta
authors.forename: Rosalba
authors.initials: R
authors.lastname: Cristofaro
authors.forename: Monica
authors.initials: M
authors.lastname: Ceol
authors.forename: Dorella
authors.initials: D
authors.lastname: Del Prete
authors.forename: Giovanna
authors.initials: G
authors.lastname: Priante
authors.forename: Alessandra
authors.initials: A
authors.lastname: Familiari
authors.forename: Antonia
authors.initials: A
authors.lastname: Fabris
authors.forename: Angela
authors.initials: A
authors.lastname: D'Angelo
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gambaro
authors.forename: Franca
authors.initials: F
authors.lastname: Anglani
mesh_terms: D000199:Actins / Q000378:metabolism; D000368:Aged; D051929:CD146 Antigen / Q000378:metabolism; D002113:Calcification, Physiologic; D002114:Calcinosis; D002460:Cell Line; D002478:Cells, Cultured; D005260:Female; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D007691:Medullary Sponge Kidney / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D009130:Muscle, Smooth / Q000737:chemistry; D009154:Mutation; D015676:Osteonectin / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014746:Vimentin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D000199:Actins; D051929:CD146 Antigen; D051100:Glial Cell Line-Derived Neurotrophic Factor; D015676:Osteonectin; C569042:TJP1 protein, human; D014746:Vimentin; D062826:Zonula Occludens-1 Protein; D053495:Osteopontin
keywords: GDNF; medullary sponge kidney; mesenchymal stromal stem cells; nephrocalcinosis