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Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes

1998; Elsevier BV; Volume: 54; Linguagem: Inglês

10.1046/j.1523-1755.1998.06733.x

1523-1755

Matthias Kretzler, Bernd Schröppel, Monika Merkle, Stephan M. Huber, Peter Mündel, Michael Horster, Detlef Schlöndorff,

Renal and Vascular Pathologies

Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes. Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms’ tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease. Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes. Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms’ tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease. The glomerular podocyte plays a major role in filtration permselectivity of the glomerulus. Although the mechanisms determining the characteristics of the glomerular filtration unit are only incompletely understood, vascular endothelial growth factor (VEGF) has been implicated1.Simon M. Gröne H.-J. Jöhren O. Kullmer J. Plate K.H. Risau W. Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.Am J Physiol. 1995; 268: F240-F250PubMed Google Scholar,2.Shulman K. Rosen S. Tognazzi K. Manseau E.J. Brown L. Expression of vascular permeability factor (VPF/VEGF) is altered in many glomerular diseases.J Am Soc Nephrol. 1996; 7: 661-666PubMed Google Scholar. VEGF is a conserved, dimeric glycoprotein with structural homology to platelet-derived growth factor. It is a highly selective mitogen in vascular endothelial cells and increases microvascular permeability and nitric oxide-dependent relaxation of small muscular arteries. Chemoattraction of monocytes and expression of plasminogen activator and its inhibitor3.Ikeda E. Achen M.G. Breier G. Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells.J Biol Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar are also enhanced. VEGF is expressed mainly by epithelial cells as a secreted peptide. The corresponding receptors (KDR and flt-1 in human and flk-1 in the mouse) are found on glomerular endothelial cells1.Simon M. Gröne H.-J. Jöhren O. Kullmer J. Plate K.H. Risau W. Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.Am J Physiol. 1995; 268: F240-F250PubMed Google Scholar. In the human kidney, VEGF mRNA and protein localize predominantly to the glomerulus, although lower and less consistent focal tubular expression has also been described. Paracrine secretion with VEGF production in podocytes and consecutive binding to endothelium have been proposed as regulatory mechanisms for normal function of the glomerular filtration barrier1.Simon M. Gröne H.-J. Jöhren O. Kullmer J. Plate K.H. Risau W. Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.Am J Physiol. 1995; 268: F240-F250PubMed Google Scholar,2.Shulman K. Rosen S. Tognazzi K. Manseau E.J. Brown L. Expression of vascular permeability factor (VPF/VEGF) is altered in many glomerular diseases.J Am Soc Nephrol. 1996; 7: 661-666PubMed Google Scholar. The presence of VEGF is crucial for normal renal development that depends on precisely controlled vascularization. Differentiating glomerular epithelia produce VEGF and may attract endothelial cells into the glomeruli1.Simon M. Gröne H.-J. Jöhren O. Kullmer J. Plate K.H. Risau W. Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.Am J Physiol. 1995; 268: F240-F250PubMed Google Scholar. Administration of anti-VEGF antibody during early kidney development in mice leads to formation of abnormal glomerular structures and diminished nephrogenesis as an indirect effect of disturbed blood vessel formation. In VEGF antibody-treated animals, a dual glomerular basement membrane, immature glomerular capillaries with slitlike lumen and amorphous substance surrounded by podocytes occur4.Kitamoto Y. Tokunaga H. Tomita K. Vascular endothelial growth factor is an essential molecule for mouse kidney development: Glomerulogenesis and nephrogenesis.J Clin Invest. 1997; 99: 2351-2357Crossref PubMed Scopus (234) Google Scholar. Various glomerular diseases are characterized by high VEGF expression in glomeruli with preserved architecture, whereas VEGF production is decreased in glomeruli damaged by amyloid deposition, glomerulosclerosis, or compression by crescents. Although up-regulation of VEGF has been postulated as a compensatory response of epithelial cells to reconstruct a glomerulus, limitations of the mRNA detection technique (in situ hybridization) have hindered further analysis of VEGF mRNA expression in glomeruli3.Ikeda E. Achen M.G. Breier G. Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells.J Biol Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar. We have established a method allowing exploration of in vivo mRNA expression of podocytes at the single-cell level5.Schröppel B. Huber S. Horster M. Schlöndorff D. Kretzler M. Analysis of mouse glomerular podocyte mRNA by single cell RT-PCR.Kidney Int. 1998; 53: 119-124Abstract Full Text PDF PubMed Scopus (27) Google Scholar. In this study, we employ this method to analyze the mRNA expression profile of VEGF in podocytes. Single-cell RT-PCR was performed as described5.Schröppel B. Huber S. Horster M. Schlöndorff D. Kretzler M. Analysis of mouse glomerular podocyte mRNA by single cell RT-PCR.Kidney Int. 1998; 53: 119-124Abstract Full Text PDF PubMed Scopus (27) Google Scholar. In brief, microdissected mouse glomeruli were fixed via a holding pipette on a patch-clamp apparatus. Single podocytes were harvested selectively by aspiration of the cell soma into a micropipette at an rate of 10 to 15 podocytes/hour Figure 1. After reverse transcription, PCR was performed using sequence-specific oligonucleotide primers and 40 to 50 amplification cycles5.Schröppel B. Huber S. Horster M. Schlöndorff D. Kretzler M. Analysis of mouse glomerular podocyte mRNA by single cell RT-PCR.Kidney Int. 1998; 53: 119-124Abstract Full Text PDF PubMed Scopus (27) Google Scholar. One third of all samples consisted of negative controls (superfusion medium aspirated next to the glomerulus) to exclude contamination. PCR product authenticity was verified by cycle sequencing of the cloned amplification product. To control for cDNA yield, the housekeeping gene β actin was analyzed in 3-μl aliquots of each sample and was detected in 68% of all cells analyzed. The podocyte nature of the cells was confirmed by detection of mRNA for the putative mouse homologue of glomerular epithelial protein 1 (61% positive, N = 26) and Wilms’ tumor protein 1 (28% positive, N = 32). The endothelial cell marker von Willebrand factor was undetectable (N = 15). Three splice variants generated from a single gene have been found in the human kidney: VEGF121, VEGF165, and VEGF1891.Simon M. Gröne H.-J. Jöhren O. Kullmer J. Plate K.H. Risau W. Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.Am J Physiol. 1995; 268: F240-F250PubMed Google Scholar. The VEGF gene contains eight exons whereby exons 1 to 5 encode a region required for ligand recognition by the KDR receptor and are present in all isoforms. VEGF121 lacks exons 6 and 7; VEGF165 lacks exon 6, and VEGF189 contains all exons Figure 2b6.Poltorak Z.C.T. Sivan R. Kandelis Y. Spira G. Vlodavsky I. Keshet E. Neufeld G. VEGF 145, a secreted VEGF isoform that binds to extracellular matrix.J Biol Chem. 1997; 272: 7151-7158Crossref PubMed Scopus (439) Google Scholar. The major functional difference between the various VEGF isoforms is their ability to bind heparin, encoded for by exon 6. VEGF121, lacking exon 6, does not bind heparin and is freely soluble, whereas VEGF189 is cell and matrix associated, whereas VEGF165 has intermediate heparin-binding characteristics7.Plouet J. Moro F. Bertagnolli S. Coldeboeuf N. Mazarguil H. Clamens S. Bayard F. Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect.J Biol Chem. 1997; 272: 13390-13396Crossref PubMed Scopus (208) Google Scholar. Differences in heparin binding of the various isoforms may affect the diffusion of this paracrine factor from the producing cells to target endothelial cells. The extracellular matrix may serve as a storage for VEGF165 and VEGF189 that can dissociate slowly or be released by the action of proteases7.Plouet J. Moro F. Bertagnolli S. Coldeboeuf N. Mazarguil H. Clamens S. Bayard F. Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect.J Biol Chem. 1997; 272: 13390-13396Crossref PubMed Scopus (208) Google Scholar. Increased protease activity, as occurs in glomerular diseases, could therefore release matrix-stored VEGF and increase glomerular capillary permeability. We used a primer pair combination spanning exons 6 and 7 for amplification Figure 2. Three PCR products, corresponding to the three splice isoforms, were obtained from mouse kidney cortex and isolated glomeruli using standard RT-PCR (Figure 2; technique see8.Kretzler M. Fan G. Rose D. Arend L.J. Briggs J.P. Holzman L.B. Novel mouse embryonic renal marker gene products differentially expressed during kidney development.Am J Physiol. 1996; 271: F770-F777PubMed Google Scholar). VEGF165 was the most abundant isoform, whereas VEGF189 was the least abundant. Single-cell RT-PCR also detected all three isoforms in single, resident glomerular podocytes. For podocyte cell culture, we used a conditionally immortalized cell line9.Mundel P. Reiser J. New aspects of podocyte cell biology.Kidney and Blood Press Res. 1997; 20: 173-176Crossref PubMed Scopus (13) Google Scholar. This transgene mouse podocyte cell line contains the SV40 T antigen under the control of an interferon-γ and thermosensible promoter. The cells are propagated under permissive conditions (interferon-γ, 33°C) in an undifferentiated state (cobblestone epithelium). Withdrawal of interferon-γ and increasing culture temperature to 37°C induce a more differentiated, “arborized” phenotype. For standardization of the cDNA prior to quantification, adenine nucleotide carrier expression was used as a housekeeping gene. As in single podocyte RT-PCR, the VEGF isoforms 121, 165, and 189 were found in cultured podocytes. Interestingly, all isoforms showed marked up-regulation after induction of the arborized phenotype. The techniques described will allow semiquantitative and isoform-specific evaluation of podocyte VEGF mRNA expression. Single podocyte RT-PCR will enable also examination of the role of different splice isoforms during nephrogenesis and in glomerular diseases. The cell culture system of the conditionally immortalized podocyte will provide also a crucial tool for examining the molecular mechanisms controlling VEGF expression in vitro. This study was supported in part by DFG grants to the authors S.H. (grant Ho 485/15-3) and M.K. (grant Kr 1492/2-1). We thank Drs. Harry Holthöfer and Hermann Parvenstädt for helpful discussions and Gabi Fuhrer for expert technical assistance.