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

Helbert, Mark; Dauwe, Simonne; Broe, Marc E. De

doi:10.1046/j.1523-1755.2001.059002554.x

Cited by 32 publications

(30 citation statements)

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“…The COPAS technique provides high yield and purity for DCT or CD (20,36,39) but requires the time and expense associated with breeding to fluorescent reporter mice (that may not be of similar background to one's target mouse line), specialized equipment, and technical personnel. The hypotonic lysis method for isolation of the medullary collecting duct is fairly inexpensive, but this method cannot be used for sensitive measurements (e.g., oxygen consumption) (62) and does not capture cortical segments of the ASDN.…”

Section: Discussionmentioning

confidence: 99%

Harvest and primary culture of the murine aldosterone-sensitive distal nephron

Labarca

Nizar

Walczak

et al. 2015

American Journal of Physiology-Renal Physiology

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Labarca M, Nizar JM, Walczak EM, Dong W, Pao AC, Bhalla V. Harvest and primary culture of the murine aldosterone-sensitive distal nephron. Am J Physiol Renal Physiol 308: F1306 -F1315, 2015. First published March 25, 2015 doi:10.1152/ajprenal.00668.2014The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 ⍀ * cm 2 ) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.

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Section: Discussionmentioning

confidence: 99%

Harvest and primary culture of the murine aldosterone-sensitive distal nephron

Labarca

Nizar

Walczak

et al. 2015

American Journal of Physiology-Renal Physiology

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“…Although the resulting cultures contain proximal tubular, distal tubular as well as collecting duct cells, the different cell types can be identified and distinguished from each other by using specific markers for proximal tubular (LAP, leucine aminopeptidase) and distal tubular/collecting duct cells (EMA, epithelial membrane antigen) (13)(14)(15). A similar percentage of proximal and thus endocytosing cells (42 Ϯ 6.5% over 9 kidneys) was present in the cell cultures that were derived from the kidney specimen obtained from different donors.…”

Section: Discussionmentioning

confidence: 99%

“…Since in previous studies, LAP and EMA were found to be specific markers of proximal and distal tubular cells respectively (13)(14)(15), these markers were used to determine whether albumin endocytosis took place into one or both of the tubular cell populations. Microscopic analysis of albumin uptake was combined with immunofluorescent EMA staining (Figure 2 Figure 4 shows that 16 h exposure of the cultures to the 3 statins of interest (simvastatin, pravastatin and rosuvastatin), all resulted in a significant and concentration-dependent inhibition of albumin uptake in proximal tubular cells (P Ͻ 0.02).…”

Section: Albumin Uptake In Proximal Tubular Cellsmentioning

confidence: 99%

“…During the past years, methods for culturing primary human kidney tubular cells have been optimized (13)(14)(15). As well as proximal tubular cells, these cultures also contain distal tubular and collecting duct cells that can be distinguished from each other by means of the specific markers, i.e.…”

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confidence: 99%

“…As well as proximal tubular cells, these cultures also contain distal tubular and collecting duct cells that can be distinguished from each other by means of the specific markers, i.e. leucine aminopeptidase (LAP) for proximal tubular cells and epithelial membrane antigen (EMA) for distal tubular and collecting duct cells (13)(14)(15).…”

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Inhibitors of HMG-CoA Reductase Reduce Receptor-mediated Endocytosis in Human Kidney Proximal Tubular Cells

Verhulst¹,

D’Haese²,

Broe³

2004

Journal of the American Society of Nephrology

123

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Abstract. The proximal tubular cells of the kidney are responsible for reabsorption of proteins from the tubular lumen. In a study using Opossum kidney (OK) cells, receptor-mediated protein endocytosis was reduced by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, which are widely used for therapeutic reduction of plasma cholesterol levels. To explore the possible clinical relevance of the observations in OK cells, protein endocytosis in human kidney tubular cells was investigated in the presence and absence of statins.The uptake of FITC-labeled albumin in these cultures of human kidney tubular cells was investigated by microscopy, flow cytometry and spectrofluorometry. Protein uptake occurred selectively into proximal tubular cells while it was absent in distal tubular/collecting duct cells. Three statins (simvastatin, pravastatin, and rosuvastatin) significantly inhibited the uptake of protein in a concentration-dependent way. This inhibitory effect of statins could be prevented by the co-addition of mevalonate, the product of HMG-CoA reductase. This effect was not the result of a statin-induced cytotoxicity since cell-viability was unaffected. Finally, it was demonstrated that statins strongly inhibited cholesterol synthesis in the human kidney tubular cells.These data suggest that statins have the potential to inhibit albumin uptake by the human proximal nephron as a result of inhibition of HMG-CoA reductase in the proximal tubule cells. Taken into account the data of the accompanying manuscript this inhibitory effect most probably results from a reduced prenylation of some proteins critically involved in endocytosis. It is suggested that these data help to explain the occurrence of proteinuria in some patients treated with high statin doses.Statins, by their ability to inhibit 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting enzyme of the sterol pathway, are potent inhibitors of sterol biosynthesis (1). As a result of the reduction of cellular sterol pools, there is compensatory upregulation of cell-surface receptors for cholesterol-containing low density lipoproteins (LDL), an effect which takes place mainly in the liver (2-4). This mechanism underlies the therapeutic use of the statins to lower plasma cholesterol and particularly the levels of LDL. However, many additional effects of statins on cell function have been described in the literature (5). These appear to be independent of cellular cholesterol homeostasis and are collectively termed "pleiotropic effects". Many of these have been shown to result from the depletion of mevalonate (the HMG-CoA conversion product)-derived intermediates of the sterol pathway, particularly the isoprenoid pyrophosphates such as geranylgeranyl pyrophosphate (GGPP). Isoprenoid pyrophosphates are required by the cells for the post-translational modification of a range of proteins, especially GTP-binding proteins. In the accompanying publication (6), it has been shown that receptormediated endocytosis by opossum kidney (OK) c...

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Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT

Doberstein

Harter

Haberkorn

et al. 2014

Intl Journal of Cancer

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L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers, confers bad prognosis and augments cell motility, invasion and metastasis. Results from xenograft mouse models suggested that L1CAM antibodies might be promising tools for cancer therapy. Here, we generated human L1CAM-transgenic mice to study therapeutic efficacy and putative side effects in a model system. We established three transgenic lines (M2, M3 and F4) expressing the human L1CAM transgene in brain, kidney and colon with decreasing intensity (M2, M3 > F4). The expression pattern was similar to that of L1CAM in humans. No interference of the transgene with the expression of endogenous L1CAM was observed. Immunohistochemical analysis revealed correct expression of the transgene in mouse cortex and collective duct of the kidney. Injection of 125 Ilabeled L1CAM antibodies resulted in specific enrichment in the kidney but not in the brain. The injection of the therapeutic anti-human L1CAM mAb L1-9.3/2a into transgenic mice even at high doses did not cause behavioral changes or other side effects. Similar results were obtained using a mouse specific L1CAM mAb in normal mice. Tumor therapy experiments were performed using syngeneic mouse tumor cells (RET melanoma and Panc02 pancreatic adenocarcinoma) transduced with human L1CAM. MAb L1-9.3/2a efficiently and specifically attenuated local tumor growth in both model systems without apparent side effects. The therapeutic effect was dependent on immune effector mechanisms. Analysis of Panc02-huL1CAM tumors after therapy showed elevated levels of EGF and evidence of immune-induced epithelial-mesenchymal transition. The results suggest that our transgenic mice are valuable tools to study L1CAM-based antibody therapy.The L1 cell adhesion molecule (L1CAM) is a 200-220 kDa transmembrane glycoprotein of the immunoglobulin (Ig) superfamily composed of six Ig-like domains and five fibronectin Type III repeats followed by a transmembrane region and a highly conserved cytoplasmic tail. 1 L1CAM can interact with itself (homophilic) and with heterophilic ligands such as integrins, CD24, neurocan, neuropilin-1 (NRP-1) and other members of the neural cell adhesion family. Cumulative evidence indicates that L1CAM (CD171) is ectopically expressed at high levels in a variety of cancers, including pancreatic, colorectal, ovarian and endometrial cancers, melanoma and neuroblastoma.2-6 Furthermore, high levels of L1CAM were found associated with increased grade of malignancy, 5,6 epithelial-mesenchymal transition (EMT), 6 poor patient prognosis 5,7,8 and worse response to chemotherapy. 4,7These findings prompted extensive research towards development and preclinical testing of L1CAM specific mAbs for selective targeting of cancers. In line with the role of this molecule in cell motility, invasion and tumorigenesis, [9][10][11] L1CAM-specific mAbs were shown to suppress tumor cell outgrowth and metastasis in xenograft models. [12][13][14] The in vivo action of these mAbs was shown to depend on a combination of im...

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

Abstract: 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.

Cited by 32 publications

References 38 publications

Harvest and primary culture of the murine aldosterone-sensitive distal nephron

Harvest and primary culture of the murine aldosterone-sensitive distal nephron

Inhibitors of HMG-CoA Reductase Reduce Receptor-mediated Endocytosis in Human Kidney Proximal Tubular Cells

Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT

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