Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF
2006; Elsevier BV; Volume: 69; Issue: 9 Linguagem: Inglês
10.1038/sj.ki.5000277
ISSN1523-1755
AutoresSimon C. Satchell, Candida Tasman, Anurag Singh, Lan Ni, Joyce Geelen, Christopher J. von Ruhland, M O'hare, Moin A. Saleem, L.P. van den Heuvel, Peter W. Mathieson,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoGlomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)α, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33°C) and became growth arrested at the non-permissive temperature (37°C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFα upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3′,5′ monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury. Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)α, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33°C) and became growth arrested at the non-permissive temperature (37°C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFα upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3′,5′ monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury. The glomerular microcirculation, unlike other capillary circulations, is highly permeable to water and small solutes, yet, like other capillaries, is relatively impermeable to macromolecules. These properties are essential for filtration function and are a result of the unique three-layer structure of the glomerular capillary wall: the endothelium with its glycocalyx, the glomerular basement membrane, and podocytes. Much interest has recently focused on the podocyte and particularly the slit diaphragm as a possible site of a final barrier to macromolecular passage.1Endlich K. Kriz W. Witzgall R. Update in podocyte biology.Curr Opin Nephrol Hypertens. 2001; 10: 331-340Google Scholar However, models indicate that all three layers have an important contribution and that their combined properties are more than simply the sum of the parts.2Ohlson M. Sorensson J. Haraldsson B. A gel-membrane model of glomerular charge and size selectivity in series.Am J Physiol Renal Physiol. 2001; 280: F396-F405Google Scholar, 3Deen W.M. Lazzara M.J. Myers B.D. Structural determinants of glomerular permeability.Am J Physiol Renal Physiol. 2001; 281: F579-F596Google Scholar Glomerular endothelial cells (GEnC) are highly specialized cells, which form a continuous inner layer of glomerular capillaries. Away from the nucleus the cytoplasm is attenuated to 200 nm and is punctuated by numerous fenestrae. These are circular transcellular pores, 60–80 nm in diameter,4Rostgaard J. Qvortrup K. Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.Microvasc Res. 1997; 53: 1-13Google Scholar which cover 20% of the endothelial surface3Deen W.M. Lazzara M.J. Myers B.D. Structural determinants of glomerular permeability.Am J Physiol Renal Physiol. 2001; 281: F579-F596Google Scholar and are essential for high hydraulic conductivity. Previously, the fenestrations were thought of as empty and therefore providing little barrier to the passage of proteins.5Brenchley P. VEGF/VPF: a modulator of microvascular function with potential roles in glomerular pathophysiology.J Nephrol. 1996; 9: 10-17Google Scholar However, new fixation techniques have allowed the demonstration of a glomerular endothelial glycocalyx of 200–400 nm in thickness, which covers fenestrae and interfenestral domains equally.4Rostgaard J. Qvortrup K. Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.Microvasc Res. 1997; 53: 1-13Google Scholar Although the exact nature of this GEnC glycocalyx is yet to be defined, these observations suggest that the contribution of GEnC to the permeability barrier to proteins may have been underestimated.6Deen W.M. What determines glomerular capillary permeability?.J Clin Invest. 2004; 114: 1412-1414Google Scholar GEnC are also likely to be important in glomerular disease. There is clear evidence of GEnC damage in conditions such as haemolytic uraemic syndrome7van Setten P.A. van Hinsbergh V.W. van der Velden T.J. et al.Effects of TNF alpha on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells.Kidney Int. 1997; 51: 1245-1256Google Scholar and pre-eclampsia.8Maynard S.E. Min J.Y. Merchan J. et al.Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Google Scholar Diseases such as haemolytic uraemic syndrome, vasculitis, and anti-glomerular basement membrane disease involve destruction of glomerular capillaries and experimental models indicate that recovery is dependent on GEnC angiogenesis.9Iruela-Arispe L. Gordon K. Hugo C. et al.Participation of glomerular endothelial cells in the capillary repair of glomerulonephritis.Am J Pathol. 1995; 147: 1715-1727Google Scholar, 10Masuda Y. Shimizu A. Mori T. et al.Vascular endothelial growth factor enhances glomerular capillary repair and accelerates resolution of experimentally induced glomerulonephritis.Am J Pathol. 2001; 159: 599-608Google Scholar, 11Shimizu A. Masuda Y. Mori T. et al.Vascular endothelial growth factor165 resolves glomerular inflammation and accelerates glomerular capillary repair in rat anti-glomerular basement membrane glomerulonephritis.J Am Soc Nephrol. 2004; 15: 2655-2665Google Scholar Systemic conditions such as diabetes, hypertension, and sepsis are associated with microalbuminuria and, although the mechanism of this link is not known, the generalized endothelial dysfunction that accompanies these conditions suggests a contribution from a GEnC lesion.12Deckert T. Feldt-Rasmussen B. Borch-Johnsen K. et al.Albuminuria reflects widespread vascular damage. The Steno hypothesis.Diabetologia. 1989; 32: 219-226Google Scholar, 13Pedrinelli R. Dell'Omo G. Penno G. et al.Non-diabetic microalbuminuria, endothelial dysfunction and cardiovascular disease.Vasc Med. 2001; 6: 257-264Google Scholar In addition to their unique structure and function, GEnC also have a particular embryological origin, arising by a combination of vasculogenesis from mesenchymal precursors (predominantly) and in-growth of existing vessels.14Hyink D.P. Tucker D.C. St John P.L. et al.Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys.Am J Physiol. 1996; 270: F886-F899Google Scholar, 15Woolf A.S. Loughna S. Origin of glomerular capillaries: is the verdict in?.Exp Nephrol. 1998; 6: 17-21Google Scholar Furthermore increasing evidence indicates that EnC from different species, organs, and types of vessels have different properties and behaviours.16Page C. Rose M. Yacoub M. et al.Antigenic heterogeneity of vascular endothelium.Am J Pathol. 1992; 141: 673-683Google Scholar, 17Kelly J.J. Moore T.M. Babal P. et al.Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability.Am J Physiol. 1998; 274: L810-L819Google Scholar, 18Ghitescu L. Robert M. Diversity in unity: the biochemical composition of the endothelial cell surface varies between the vascular beds.Microsc Res Tech. 2002; 57: 381-389Google Scholar All these considerations indicate the importance of the study of human GEnC to enable complete understanding of glomerular filtration, glomerular disease, response to glomerular injury, and the potential for therapeutic manipulations in these contexts. While certain responses are most appropriately examined in vivo, cell culture allows careful analysis of the behaviour of individual cell types and the actions and interactions of particular mediators. Historically, GEnC have been difficult to grow in culture and have therefore been little studied. We have now addressed this problem using conditional immortalization (ci) technology, such as we have successfully applied previously in cultured podocytes.19Saleem M.A. O'Hare M.J. Reiser J. et al.A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression.J Am Soc Nephrol. 2002; 13: 630-638Google Scholar While a number of groups have now successfully cultured rodent or bovine GEnC,20Ballermann B.J. Marsden P.A. Endothelium-derived vasoactive mediators and renal glomerular function.Clin Invest Med. 1991; 14: 508-517Google Scholar, 21Wolf G. Ziyadeh F.N. Zahner G. et al.Angiotensin II is mitogenic for cultured rat glomerular endothelial cells.Hypertension. 1996; 27: 897-905Google Scholar, 22Sorensson J. Bjornson A. Ohlson M. et al.Synthesis of sulfated proteoglycans by bovine glomerular endothelial cells in culture.Am J Physiol Renal Physiol. 2003; 284: F373-F380Google Scholar, 23Rops A.L. van der Vlag J. Jacobs C.W. et al.Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.Kidney Int. 2004; 66: 2193-2201Google Scholar there are few reports convincingly demonstrating isolation of human GEnC.7van Setten P.A. van Hinsbergh V.W. van der Velden T.J. et al.Effects of TNF alpha on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells.Kidney Int. 1997; 51: 1245-1256Google Scholar In this case GEnC were cultured from glomeruli sieved from normal renal cortex. The technique involved partial collagenase digestion of the glomerulus, coating of culture plates with extracellular matrix components, use of medium with EnC growth factors, and purification of GEnC from contaminating cells using an immunomagnetic bead selection technique. Human GEnC, similarly derived from glomeruli decapsulated by sieving, have also become available from a commercial source and we have previously characterized these cells in detail.24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Google Scholar Other described culture methods for ‘glomerular’ EnC, which begin with lysing whole cortex,25Giannini S. Cresci B. Pala L. et al.Human glomerular endothelial cells IGFBPs are regulated by IGF-I and TGF-beta1.Mol Cell Endocrinol. 1999; 154: 123-136Google Scholar, 26Cresci B. Giannini S. Pala L. et al.AT1 and AT2 receptors in human glomerular endothelial cells at different passages.Microvasc Res. 2003; 66: 22-29Google Scholar result in a mixture of EnC, only some of which are glomerular. When successfully isolated, primary culture GEnC form a monolayer of polygonal cells, which express typical EnC-specific markers including von Willebrand factor (vWF), platelet endothelial cell adhesion molecule 1 (PECAM1, CD31), and vascular endothelial (VE)-cadherin (CD144), and growth factor receptors Tie2 and vascular endothelial growth factor receptor 2 (VEGFR2).7van Setten P.A. van Hinsbergh V.W. van der Velden T.J. et al.Effects of TNF alpha on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells.Kidney Int. 1997; 51: 1245-1256Google Scholar, 24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Google Scholar These cells, like other EnC, respond to inflammatory mediators, including tumour necrosis factor (TNF)α, by upregulation of the adhesion molecule E-selectin (CD62E), and they form monolayers in culture whose permeability properties are responsive to cyclic adenosine 3′,5′ monophosphate (cAMP) and thrombin. Human primary culture GEnC express some fenestrations in culture,7van Setten P.A. van Hinsbergh V.W. van der Velden T.J. et al.Effects of TNF alpha on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells.Kidney Int. 1997; 51: 1245-1256Google Scholar while bovine GEnC do not express fenestrations in static culture but can be induced by shear stress.27Ott M.J. Olson J.L. Ballermann B.J. Chronic in vitro flow promotes ultrastructural differentiation of endothelial cells.Endothelium. 1995; 3: 21-30Google Scholar The usefulness of primary culture human GEnC for tissue culture studies is limited by early onset of senescence. Here we describe the ci of human GEnC. We have used a technique restoring functional telomerase activity and introducing the simian virus 40 large tumour antigen (SV40LT), since it has been shown that human EnC require both these elements for successful immortalization.28O’Hare M.J. Bond J. Clarke C. et al.Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.Proc Natl Acad Sci USA. 2001; 98: 646-651Google Scholar A temperature-sensitive (ts) SV40LT construct was used to allow ci, that is, enhanced proliferation at a permissive temperature, while the SV40LT element can be ‘switched off’ by transfer to a non-permissive temperature. At this temperature, cells take on a mature phenotype not seen in cells constitutively expressing SV40LT. This approach has been used successfully in alveolar bone marrow cells,29Salih V. Knowles J.C. O’Hare M.J. et al.Retroviral transduction of alveolar bone cells with a temperature-sensitive SV40 large T antigen.Cell Tissue Res. 2001; 304: 371-376Google Scholar breast microvascular EnC and fibroblasts,28O’Hare M.J. Bond J. Clarke C. et al.Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.Proc Natl Acad Sci USA. 2001; 98: 646-651Google Scholar and in podocytes in our laboratory.19Saleem M.A. O'Hare M.J. Reiser J. et al.A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression.J Am Soc Nephrol. 2002; 13: 630-638Google Scholar Clones of ciGEnC successfully transduced with both SV40LT and the essential catalytic subunit of human telomerase (hTERT) were antibiotic-resistant and proliferated at 33°C with growth arrest occurring on transfer to 37°C. CiGEnC at both 33°C and 37°C retained features of early-passage primary culture GEnC, including small size, homogeneity, and formation of ‘cobblestone’ monolayers up to at least passage 41 (Figure 1 ), while primary culture GEnC became senescent by passage 8 (not shown). CiGEnC at 37°C were used in experiments after at least 5 days at the non-permissive temperature. Scanning electron microscopy of ciGEnC monolayers demonstrated the presence of cytoplasmic pores of 50–400 nm in diameter which were taken to represent fenestrations, although they were slightly larger than the 60–80 nm estimated from ex vivo studies.3Deen W.M. Lazzara M.J. Myers B.D. Structural determinants of glomerular permeability.Am J Physiol Renal Physiol. 2001; 281: F579-F596Google Scholar, 4Rostgaard J. Qvortrup K. Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.Microvasc Res. 1997; 53: 1-13Google Scholar. These were greatly increased in number by VEGF (Figure 2 ). mRNA analysis demonstrated that the overall expression patterns of genes represented on the array used were very similar for primary and ciGEnC at both permissive and non-permissive temperatures, but quite different from the expression pattern for the podocytes used as a control (Figure 3 ). The analysis confirmed the expression of the EnC-specific markers PECAM1, intercellular adhesion molecule 2 (ICAM2, CD102), VEGFR2, and vWF in the three GEnC groups, but not in podocytes (Table 1 ). There were some differences between the three GEnC groups in levels of mRNA expression in these experiments, for example, relatively higher expression of VEGFR2 and vWF in ciGEnC. Interestingly, some genes involved in extracellular matrix production and regulation (plasminogen activator inhibitor-1, thrombospondin, fibronectin) were highly expressed in all GEnC and podocytes (Table 1).Table 1Relative expression of endothelium-specific and matrix-related genes in GEnC, selected from genes analysed by focused gene arrayGene expression level (ratio of gene:GAPDH dot density) for different cell typesGenePrimary GEnCCiGEnC 33°CCiGEnC 37°CPodocytesEndothelium-specific PECAM11.120.930.980.08 ICAM21.041.081.260.02 VEGFR20.270.750.810.01 vWF0.170.480.660.05Matrix-related PAI-11.151.081.151.04 Thrombospondin1.051.111.131.08 Fibronectin1.151.051.221.07ciGENC, conditionally immortalized glomerular endothelial cells; ICAM, intercellular adhesion molecule; PAI, plasminogen activator inhibitor; PECAM, platelet endothelial cell adhesion molecule; VEGFR, vascular endothelial growth factor receptor; vWF, von Willebrand factor. Open table in a new tab ciGENC, conditionally immortalized glomerular endothelial cells; ICAM, intercellular adhesion molecule; PAI, plasminogen activator inhibitor; PECAM, platelet endothelial cell adhesion molecule; VEGFR, vascular endothelial growth factor receptor; vWF, von Willebrand factor. CiGEnC expressed the EnC-specific markers PECAM1, vWF, and VE-cadherin comparable in distribution and level of expression to primary culture GEnC (Figure 4 ). Podocytes were, as expected, negative for these markers. Treatment of ciGEnC with TNFα caused a dose-dependent increase in expression of E-selectin by cell-based fluorescence immunoassay (Figure 5 ), but had no effect on binding of an irrelevant primary antibody (not shown). TNFα (10 ng/ml) caused a 3.2-fold increase in E-selectin expression over 6 h.Figure 5Chart showing the effect of TNFα on e-selectin expression by ciGEnC in a cell-based FIA. CiGEnC at 37°C were incubated with TNFα at various concentrations or control medium for 6 h before fixing and labelling for E-selectin. E-selectin expression is proportional to fluorescence emission. Bars show mean±s.e., n=15, P<0.0001 by analysis of variance.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blotting confirmed that ciGEnC retain the expression of the important EnC-specific molecules PECAM1, ICAM2, VEGFR2, vWF, VE-cadherin, and Tie2 at both 33°C and 37°C (Figure 6 ). These results correspond to and confirm similar gene array results for PECAM1, ICAM2, VEGFR2, and vWF. There were comparable levels of expression in primary and ciGEnC, although as above there appeared to be greater expression of VEGFR2 and vWF in ciGEnC. Western blotting also confirmed expression of the SV40LT antigen in ciGEnC at 33°C and its silencing by transfer of cells to 37°C. Time-course analysis showed SV40LT levels rapidly reducing within 24 h with a greater than 30-fold reduction 5 days after transfer than at 33°C (Figure 7 ). Cells were taken to be quiescent by day 5 and were used in experiments after that time point. CiGEnC monolayers at 33°C reached a mean trans-endothelial electrical resistance (TEER) of 20–25 Ω at 6 days post-seeding, after which the TEER plateaued (data not shown). Monolayers transferred to 37°C on day 6 maintained a similar TEER for a further 7 days. Increasing the effective intracellular concentration of cAMP by use of the ‘cAMP medium’ increased the mean TEER of ciGEnC monolayers at 37°C by 6.3 Ω, while thrombin decreased TEER by 7.6 Ω over 1 h relative to controls (Figure 8 ). We have described for the first time the generation of immortalized human GEnC. Significantly, this immortalization is conditional by use of a temperature-sensitive transgene encoding SV40LT. This allows both an unlimited replication potential and return of cells to a non-proliferative state by transfer to the non-permissive temperature of 37°C. Western blotting confirmed rapid reduction of SV40LT levels after transfer to 37°C. This is consistent with previous observations on other cells, ci in a similar way, which show rapid SV40LT loss on transfer to the non-permissive temperature, leading to growth arrest, while metabolic activity is maintained.30Jat P.S. Sharp P.A. Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.Mol Cell Biol. 1989; 9: 1672-1681Google Scholar Indeed the tsA58 mutant was selected as the source of SV40LT for this property of rapid degradation at the non-permissive temperature.30Jat P.S. Sharp P.A. Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.Mol Cell Biol. 1989; 9: 1672-1681Google Scholar In addition, we have also incorporated a transgene encoding hTERT, shown to be essential for successful immortalization of human EnC in previous studies.28O’Hare M.J. Bond J. Clarke C. et al.Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.Proc Natl Acad Sci USA. 2001; 98: 646-651Google Scholar We have directly compared ciGEnC with primary culture human GEnC, previously characterized in detail,24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Google Scholar and shown that they retain similar morphological appearances at both non-permissive and permissive temperatures. Similar patterns of mRNA expression were seen using a focused gene array including the expression of EnC-specific markers PECAM1, ICAM2, VEGFR2, and vWF. The expression of these markers was validated at the protein level by Western blotting and/or immunofluorescence. GEnC were also shown to express VE-cadherin and Tie2 at comparable levels in primary and ci cells by Western blotting. Further interesting data from the gene array experiments show that GEnC, like podocytes in culture, expressed high levels of mRNA for proteins which are extracellular matrix components (fibronectin, thrombospondin) or involved in regulation of biological reactions occurring in it (plasminogen activator inhibitor-1). This may be important in the production and remodelling of the glomerular basement membrane in vivo. CiGEnC retained EnC behaviours which are important in vivo, and hence will be important in their further use for in vitro studies. Like primary culture GEnC, ciGEnC upregulated E-selectin in response to the inflammatory mediator TNFα and they formed restrictive monolayers in culture. These monolayers were responsive to both cAMP and thrombin as we have previously shown for primary culture GEnC, and comparable to EnC of other types.24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Google Scholar For the first time in cultured GEnC we have shown the upregulation of fenestrations in response to VEGF. The presence of fenestrations in vivo is essential for the high hydraulic conductivity of the glomerular filtration barrier and hence glomerular filtration. The fact that similar structures are expressed in vitro suggests that observations on the barrier-forming properties of these cells will be relevant to pathophysiology of the intact glomerulus. Expression of VEGF receptors and responsiveness to this mediator are particularly noteworthy in this respect as evidence suggests the importance of VEGF in normal glomerular physiology in maintenance of the endothelium as well as in glomerular disease.31Eremina V. Quaggin S.E. The role of VEGF-A in glomerular development and function.Curr Opin Nephrol Hypertens. 2004; 13: 9-15Google Scholar It has long been suggested that VEGF, produced by podocytes, may induce GEnC fenestrations.5Brenchley P. VEGF/VPF: a modulator of microvascular function with potential roles in glomerular pathophysiology.J Nephrol. 1996; 9: 10-17Google Scholar, 32Satchell S.C. Harper S.J. Tooke J.E. et al.Human podocytes express angiopoietin 1, a potential regulator of glomerular vascular endothelial growth factor.J Am Soc Nephrol. 2002; 13: 544-550Google Scholar Although it is yet to be proven that this occurs in vivo, these in vitro observations lend further weight to this hypothesis and are consistent with our previous observations of VEGF reducing the TEER of primary culture GEnC monolayers.24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Google Scholar The ready availability of large numbers of human GEnC now opens the door for detailed in vitro studies. Formation of restrictive monolayers indicates that these cells will be useful for further investigation of GEnC barrier function, including analysis of the role of glycocalyx. This will enable greater understanding of the contribution of GEnC to physiological glomerular permselectivity and to disturbances in disease states. This includes intrinsic glomerular diseases where the exact mechanisms of proteinuria are yet to be elucidated, and also those systemic conditions in which there is a more modest increase in glomerular permeability resulting in microalbuminuria. Study of GEnC in culture may illuminate the important conundrum of why microalbuminuria is a powerful independent risk factor for disease in other vessels and hence for cardiovascular disease.33Hillege H.L. Fidler V. Diercks G.F. et al.Urinary albumin excretion predicts cardiovascular and noncardiovascular mortality in general population.Circulation. 2002; 106: 1777-1782Google Scholar Increasing evidence indicates the importance of interactions between GEnC and podocytes in filtration barrier function in health and disease.24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Google Scholar As we have previously derived ci podocytes, we can now study the two cell types separately and together in further in vitro analyses of factors involved in glomerular permeability. In summary, we have successfully addressed the problem of poor replicative potential of human GEnC in vitro by development of ciGEnC, which represent a unique resource for further study. The importance of studying the relevant cell type in tissue culture studies is clear and hence these cells significantly advance our ability to understand the human renal glomerulus through in vitro investigations. The cells retain the characteristics of primary culture cells, including aspects of their highly specialized in vivo ultrastructure such as fenestration formation. These cells have been developed at a time of rapidly increasing understanding of the ultrastructural and molecular basis of glomerular permselectivity in health and disease through the detailed study of podocytes.34Saleem M.A. Biology of the human podocyte.Nephron Exp Nephrol. 2003; 95: e87-e92Google Scholar In the light of observations indicating that the glomerular endothelium also plays a vital role, a complementary study of these ciGEnC promises to further enhance this progress. Primary culture GEnC derived from isolated human glomeruli were prepared as describ
Referência(s)