Glucose Transporters in Human Renal Proximal Tubular Cells Isolated From the Urine of Patients With Non–Insulin-Dependent Diabetes
Article Figures & Tables
Figures
- FIG. 1.
Phase-contrast light micrograph of human primary cells isolated from fresh urine in primary culture after 24 h in culture (A). After, adhering to the collagen after 2–3 days, the cells are adhering to the collagen-I (B). Colonies of cells are formed after a week (C) and formed a confluent monolayer of cuboidal cells with cobblestone-like appearance. D: The cells after passage 2. Original magnification: ×20.
- FIG. 2.
Confocal microscopy of the human exfoliated primary cells (bar = 10 μm). Proximal cell marker: CD13-aminopeptidase-N (green) and distal and loop of Henle cell marker: Tamm-Horsfall protein (red). These markers indicate the presence of distal and proximal cells in the exfoliated primary cell culture.
- FIG. 3.
HEPTEC immunocychemistry characterization prior (A and C) and post (B and D) immuno-magnetic cell separation. TRITC-phalloidin (red) staining actin filaments (A–D) and CD13-aminopeptidase-N (green) enrichment 50% (A) to 90% (B). CD90-FITC (green) a fibroblast-like expression decreasing from ∼10% (C) to <5% post immunomagnetic cell separation (D).
- FIG. 4.
Typical human exfoliated primary cells of two healthy volunteers (V1 and V2). Positive cell counting by confocal microscopy at passage 4 prior (A) to CD13-immunomagnetic cell separation and post separation at passage 6 (B). Five fields on each of two separate coverslips were randomly selected and counted. An average of 30.2 cells per field were counted (range from 19 to 54 cells), and the counts were averaged and converted to percentages.
- FIG. 5.
Glucose transporter mRNA expression in HEPTECs isolated from healthy and type 2 diabetic subjects. A: SGLT2 (n = 4), B: GLUT2 (n = 4), and C: GLUT1 (n = 5). The glucose transporters were analyzed by real-time RT-PCR and normalized to GAPDH. Values are means ± SE (*P < 0.05).
- FIG. 6.
Immunoblot representing SGLT2 and GLUT2 protein expression in HEPTECs isolated from healthy (H) and type 2 diabetic (D) volunteers’ fresh urine (n = 4). The predicted band of 75-kDa molecular weight representing the SGLT2 level of protein expression in HEPTECs isolated from type 2 diabetes increases up to three times compared with healthy volunteers. GLUT2 (55 kDa) level of protein expression in HEPTECs isolated from type 2 diabetes increases up to 10 times compared with healthy volunteers. The level of glucose transporter protein expression is determined from densitometry relative to GAPDH. Values are means ± SE (n = 4; *P < 0.05).
- FIG. 7.
Glucose uptake assay using the AMG uptake, in triplicates, as a tracer to assess HEPTEC glucose transport in healthy and type 2 diabetic subjects (n = 3) as described by Takamotoa et al. (28). AMG uptake HEPTECs isolated from type 2 diabetes increases up to threefold compared with healthy volunteers. AMG cellular accumulation was presented as means ± SE (n = 3; *P < 0.05)
Tables
- TABLE 1
Expression of relevant cellular markers in human exfoliated primary cells and HEPTECs
Cellular marker mRNA* Protein* Cell type Cytokeratin-7 ND +++ High in distal cells and bladder cells and low in proximal cells Aquaporin-2 ND ++ Collecting duct ENaCα +++ +++ Collecting duct and distal cells ENaCβ/γ ++ ND Collecting duct and distal cells PPARγ/α/δ +++ PPARγ only Collecting duct and distal cells/proximal cells NaCl cotransporter +++ ND Proximal cells/connecting tubules Aquaporin-2 ND +++ Collecting duct L1-CAM (cell adhesion molecule) ND +++ Collecting duct Aminopeptidase-N (CD13) ND +++ Proximal cells Sodium-glucose transporters (SGLT2, GLUT1, and GLUT2) +++ +++ Proximal cells Alkaline phosphatase ND +++ Proximal cells Uroplakin III ND +++ Bladder cells Mineralocortcoid receptor +++ ND Connecting tubules/distal cells/collecting duct Expression of cellular markers of human primary exfoliated cells: 1) bladder/urethra (uroplakin III/cytokeratin 7), 2) proximal tubular cells (CD13 aminopepetidase-N/SGLT2, 3) renal distal tubular/collecting duct cells (aldosterone receptor, cytokeratin-7, aquaporin-2, and ENaCα) by PCR and/or immunoblotting. Marker level of presence (+ + +).
- *
* mRNA (Taqman) and proteins were measured and expressed as relative abundance to gapdh. +, weak expression; ++ high expression; +++ very high expression; ND, not been done.
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