Generating Conditionally Immortalised Podocyte Cell Lines from Wild-Type Mice

Abstract

Background: Understanding podocyte biology is key to deciphering the pathogenesis of numerous glomerular diseases. However, cultivation of primary podocytes results in de-differentiation with loss of specialised architecture. Human conditionally immortalised podocytes partly overcome this problem, utilising a temperature-sensitive transgene. Conditionally immortalised murine podocytes exist, but are derived from the Immortomouse. Methods: Using retroviral temperature-sensitive SV40 transfection, we created a conditionally immortalised podocyte cell line from wild-type mice. Results: These cells develop characteristic mature podocyte morphology and robustly express slit diaphragm proteins. Functionally, these cells demonstrate comparable responses in motility and glucose uptake to human conditionally immortalised podocytes. Conclusion: Podocyte-specific transgenic mice are extensively used to study glomerular disease and this technique could be used to make podocyte cell lines from any mouse, allowing study at the cellular level. This will help characterise these disease models and add to the laboratory resources used to study podocytopathies and glomerular disease.

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor

“…Studying conditionally immortalised mouse podocytes [ 22 ], we initially demonstrated that, under basal conditions, these cells express key podocyte proteins and components of the insulin signalling cascade, which increase following podocyte differentiation (Fig. 1 ).…”

“…Podocytes were isolated from perfused glomeruli from a male db/db DBA/2J and male wild-type (WT) DBA/2J littermate control mouse at 12 weeks of age. These podocytes were conditionally immortalised with temperature-sensitive SV40 transfection as previously described [ 22 , 23 ].…”

“…Conditionally immortalised human [ 24 ] and mouse [ 22 ] podocytes were maintained in RPMI-1640 containing l -glutamine and NaHCO 3 , supplemented with 10% FBS (Sigma Aldrich, Gillingham, UK). Cells were studied after 12–14 days differentiation at 37°C and were free of Mycoplasma infection.…”

Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress

“…Mouse podocytes immortalised by transduction with a temperature sensitive large T antigen SV40 construct as previously described 45 , 46 , were used as wt cells and as a background for making wt-IR, PTEN kd and PTP1B kd cell lines. The human IR sequence (NM_000208.2) in pLenti-TetCMV(IR)-Rsv(RFP-Bsd) expression vector (Gentarget) was co-transfected into Lenti-X 293 T cells (Clontech) with the packaging vectors pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) as previously reported 47 .…”

Podocytes Produce and Secrete Functional Complement C3 and Complement Factor H

“…Cells were grown on coverslips and fixed in 4% paraformaldehyde (PFA) (Sigma), as previously described (48). Cells were blocked with 3% bovine serum albumin, and permeabilized with 0.05% Triton-X 100 (both Sigma), when needed.…”