Mark J.F. Helbert

Flow cytometric immunodissection of the human nephron in vivo and in vitro.

In the present article, we show that flow cytometric immunodissection of cells immediately following their preparation from a tumor nephrectomy specimen is an accurate way of obtaining pure human primary cultures of proximal convoluted tubule origin, proximal straight tubule origin, distal tubular origin and/or collecting duct origin. By studying the expression of a panel of cell surface markers in these purified cultures, we could identify a number of markers that retain their lineage specificity in vitro. Using these appropriate stable markers, flow cytometry provides a simple yet accurate way of determining cell composition in previously unsorted (mixed type) tubular epithelial cultures in terms of proximal versus distal tubule/collecting duct subpopulations. Both subpopulations in mixed type cultures are shown to retain functional characteristics of their in vivo counterparts (glucose uptake, hormonal stimulation of adenylate cyclase) as well as cell type-specific response patterns (such as inducibility of cell adhesion and histocompatibility molecules), indicating the usefulness of studying cell responses in vitro in a cell-type-dependent way. Finally we illustrate that multi-parameter flow cytometry is a powerful tool for assessing constitutive characteristics of and/or responses by the distinct cell subpopulations present in mixed type cultures.

Tubular Erythropoietin Receptor Expression Mediates Erythropoietin-Induced Renoprotection

Erythropoietin (EPO) has been shown to have tissue protective properties by binding to its receptor (EPOR) which is also expressed on non-haematopoietic cells. The mechanisms underlying this protection have not yet been eluci- dated and the renal cell types mediating these effects remain ill-defined. This study aimed to identify the EPOR expression in human tubular epithelial cells (hTECs) and in rat kidney and to investigate the role of EPOR in EPO-mediated renopro- tection. Male Wistar rats were treated with saline or EPO (3000 U/kg, i.p.) 24h prior to sham-operation or 30 min bilateral renal ischemia. Renal morphology and function, tubular regeneration, apoptosis and expression of EPOR, heme- oxygenase-1 (HO-1) and hepatocyte growth factor (HGF) were analyzed. Primary cultures of human proximal (PTC) and distal/collecting duct (DTC) tubular cells were incubated with EPO (5-50-500 ng/mL) either or not in the presence of so- luble EPOR. Total RNA was extracted and mRNA expression of HO-1 was investigated by quantitative RT-PCR. EPOR mRNA could be demonstrated in hTECs and in cortical tubules of the rat kidney. Furthermore, EPOR protein was expressed at the membrane and as intracellular vesicles in hTECs. In vivo, EPO treatment attenuated histological and func- tional renal damage, decreased both cell necrosis and apoptotic cell death, enhanced tubular regeneration and resulted in an upregulation of HO-1 and HGF mRNA. In vitro, EPO administration resulted in an early upregulation of HO-1 mRNA which was restricted to PTC and inhibited by simultaneous addition of supra-equivalent amounts of soluble EPOR. These data strongly suggest that the EPO-mediated renoprotection results from direct interaction of EPO with EPOR on tubular cells.

Biology of Renal Tubulo-Interstitial Cells

Introduction - biology of renal tubulo-interstitial cells, M.E. De Broe and M.F. Helbert tissue culture of human renal epithelial cells using a defined serum-free growth formulation, D.A. Sens, C.J. Detrisac et al isolation, culture and characterization of human renal proximal tubule and collecting duct cells, A.L. Trifillis flow cytometric immunodissection of the human nephron in vivo and in vitro, M.J.F. Herbert, S. Dauwe and M.E. De Broe renal interstitial fibroblast culture, C. Grupp and G.A. Muller immortalized kidney cells derived from transgenic mice harbouring L-type pyruvate kinase and vimentin promoters, A. Vandewalle modulation of cell differentiation in perfusion culture, W.W. Minuth, P. Steiner et al renal tubular cells cultured from genetically modified animals, G. Friedlander, I. Runembert et al antisense and kidney cell research, M.B. Ganz protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators, C. Zoja, A. Benigni and G. Remuzzi tubular epithelial cells - a critical cell type in the regulation of renal inflammatory processes, C. Van Kooten, M.R. Daha and L.A. van Es cytokine-stimulated nitric oxide production in the human renal proximal tubule and its modulation by natriuretic peptides - a novel immunomodulatory mechanism? P.K. Chatterjee, G.M. Hawksworth and J.S. MacLay decreased degradation of collagen and fibronectin following exposure of proximal cells to glucose, A.O. Phillips, K. Morrisey et al hypoxia-induced changes in extracellular matrix metabolism in renal cells, J.T. Norman, C. Orphanides et al fibrogenic effects of cyclosporin A on the tubulointerstitium - role of cytokines and growth factors, D.W. Johnson, H.J. Saunders et al.