Flow cytometric immunodissection of the human distal tubule and cortical collecting duct system.

BACKGROUND: In recent years, considerable efforts were drawn to isolate human distal tubule (DT) and collecting duct (CD) cells with more or less success. Here, we present a procedure for isolating human DT cells [thick ascending limb (TAL)/distal convoluted tubule (DCT)] and CD system cells (connecting tubule/initial CD) as separate populations within the same kidney specimen, applying monoclonal antibodies in fluorescence-activated cell sorting (FACS) and culturing them. METHODS: We tested antibodies directed against the DT/CD system antigens, epithelial membrane antigen (EMA) and L1-cell adhesion molecule (L1-CAM). Segmental and subsegmental expressions were first assessed by using morphologic and histotopographic criteria, and by comparing sections with adjacent sections stained for expression of well-defined distal subsegment-specific markers. Immunoreactive cells were further characterized by dual immunostaining using cell type-specific markers. As a second step, cells obtained by collagenase digestion of normal renal cortical tissue were flow sorted following labeling with aforementioned antibodies and cultured. RESULTS: EMA expression was found on all cells present in the DT and in the CD system. Its expression was most abundant in TAL and from thereon decreased gradually along the course of the DT and CD system. Flow sorting of all EMA-expressing cells resulted in identification/isolation of DT and CD system cells as a heterogeneous mixture. Flow sorting of only the most strongly EMA-positive cells allowed purification of DT cells only, mainly TAL cells as shown by Tamm-Horsfall protein expression on> 80% of sorted cells. L1-CAM was expressed in only the CD system, and sorting of all L1-CAM-positive cells allowed> 95% purification of CD system cells (connecting tubule/cortical CD). Primary cultures of DT and CD system cells rapidly developed into confluent monolayers, and retained antigenic and functional properties inherent to their segments of origin. CONCLUSION: Our study presents a procedure for isolating and culturing pure populations of human DT cells and CD system cells as separate populations, using antibodies to the best available markers in FACS.

Fibrous intimal thickening at implantation as a risk factor for the outcome of cadaveric renal allografts.

BACKGROUND: During the past decade, the donor age of cadaveric renal allografts steadily increased. Because cerebrovascular injury is the main cause of death in this donor population, an increased prevalence of atherosclerotic lesions in the retrieved grafts could be anticipated. In a prospective study, we investigated the predictive value of morphologic lesions at implantation for the functional and morphologic outcome of cadaveric renal allografts at 1 1/2 years. METHODS: In 50 consecutive adult recipients of a cadaveric renal allograft, under cyclosporine-based regimen, implantation biopsies and subsequent protocol biopsies at 18 months were performed, and morphometrically analyzed for the extent of glomerulosclerosis, interstitial fibrosis, and atherosclerosis. Risk factors were assessed at implantation and during the subsequent observation period of 18 months. Endpoints for this study were: the 24-hr creatinine clearance (normalized for body surface area) and the fractional interstitial volume at 1 1/2 years. RESULTS: In multivariate analysis, fibrous intimal thickening at implantation (FIT) was the main determinant of the functional and morphologic outcome at 1 1/2 years. FIT represented a relative risk of 4.55 for interstitial fibrosis (95% CI=1.855-11.138), and 1.89 for impaired renal function (95% CI=1.185-3.007) at 1 1/2 years. FIT adversely affected fractional interstitial volume at 1 1/2 years (34.3 vs. 27.7%, P=0.004), as well as renal function (54 vs. 68 ml/min/1.73 m2, P=0.028). CONCLUSIONS: Fibrous intimal thickening at implantation is a determinant risk factor for the functional and morphologic outcome of cadaveric renal allografts at 1 1/2 years.

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.

Immunodissection of the human proximal nephron: flow sorting of S1S2S3, S1S2 and S3 proximal tubular cells.

We report on the use of several proximal tubular cell (PTC) surface markets and corresponding antibodies in fluorescence-activated cell sorting (FACS), and their ability to identify and flow sort cells of defined proximal tubular origin (S1S2S3) or of defined proximal subsegmental origin (S1S2 only/S3 only). We tested monoclonal/polyclonal antibodies directed against five different surface peptidases [leucine aminopeptidase (LAP), neutral endopeptidase 24.11 (NEP), dipeptidyl peptidase IV (DPPIV), aminopeptidase A (APA) and gamma-glutamyl transferase (gamma-GT)], the S3 segment-specific marker intestinal type alkaline phosphatase (iAP) and an S1S2 marker (TN20-antigen), originally proposed as a surface marker for interstitial fibroblasts. Segmental (proximal tubular vs. distal tubular) and proximal subsegmental (S1S2 vs. S3) expression of all five surface peptidases and TN20 antigen were first assessed by comparing immunohistochemical staining on normal human kidney tissue with staining for well-known segment-specific differentiation markers (intestinal type alkaline phosphatase, Tamm-Horsfall protein) on adjacent sections. All five peptidases were found to be expressed to a certain degree in all subsegments (S1 S2 and S3) of the proximal nephron, whereas expression was never seen in the more distal parts of the nephron. Flow cytometry was performed on cells obtained following gradient purification of collagenase-digested human renal tissue. Labeling cells for expression of LAP, NEP or DPPIV resulted in high yields of specifically labeled PTC (S1S2S3 origin). Labeling with anti-LAP resulted in the clearest distinction between positive and negative cell subpopulations, and therefore LAP was considered the best PTC marker for use in FACS. iAP histochemical staining on sorted cells showed that flow sorting with monoclonal antibody (moAb) 250 (anti-intestinal type alkaline phosphatase) allowed sorting of S3 cells with > 90% purity. Likewise, moAb TN20 enabled us to obtain a highly purified S1S2 population as confirmed by the absence of iAP on sorted cells.