Glucagon-like peptide 1 receptor expression in primary porcine proximal tubular cells
Introduction
Glucagon-like peptide 1 (GLP-1) is a hormone secreted from intestinal L-cells after food intake. The peptide is synthesized as a preproglucagon molecule that is subsequently metabolized to GLP-1 [1], [2], [3]. In the following, we refer to the bioactive form GLP-1 7-36 amide, if not stated otherwise. The GLP-1 receptor (GLP-1R) is a class B heptahelical G-protein-coupled receptor with a molecular weight between 62 and 65 kDa [4], [5], [6], [7], [8], [9], [10]. Upon GLP-1 binding, adenylate cyclase is activated and intracellular cAMP is generated [5].
The main effect of GLP-1 is stimulation of glucose-dependent insulin secretion from pancreatic β-cells thereby lowering blood glucose levels [11]. In vivo, GLP-1 is rapidly degraded by dipeptidylpeptidase IV (DPPIV) resulting in a short half-life of about 2 min [12]. Interestingly, DPPIV is highly expressed in renal proximal tubular cells and, therefore, can be used as a specific marker for these cells [13], [14], [15], [16], [17], [18].
Further effects of GLP-1 include induction of satiety and reduction of energy intake, both in healthy volunteers and in patients with type 2 diabetes [14], [15], [19].
Furthermore, in rats and humans, exogenous administration of GLP-1 has a natriuretic effect [15], [20], [21]. In the kidney, about 60–70% of excreted sodium is re-absorbed in the proximal nephron, mainly by a paracellular pathway and by a Na+/H+ exchanger (NHE3; SLC9A3) [22]. NHE3 exists in multimeric complexes with DPPIV at the apical side of the proximal tubular cells [23], [24], but the exact mechanism for this action has not been elucidated.
The aim of the study was therefore 1) to screen human and pig kidney cortex and porcine proximal tubular cells for GLP-1R expression, 2) to characterize the effect of GLP-1 on porcine kidney cells on sodium and glucose re-absorption. Primary porcine proximal tubular cells were used to characterize GLP-1R expression by means of RT-PCR for the detection at mRNA level and immunoassays (Western-blot, immunohisto- and cyto-chemistry) for the detection at protein-level. For sodium transport measurements a fluorescence marker was used and for glucose transport measurements radioactive labelled [3H]-glucose.
Section snippets
Materials
MEM Eagle d-valine with l-glutamine was purchased from Lucerna Chem AG (Luzern, Switzerland), Dulbecco's MEM/Nut Mix F-12 (DMEM/F12), fetal calf serum (FCS) and penicillin/streptomycin from Gibco Life Sciences (Basel, Switzerland), cell flask 75 cm2 from BD (Franklin Lakes, USA), GLP-1 7-36 from Bachem (Bubendorf, Switzerland). P32/98 was a kind gift from Dr. H.-U. Demuth from Probiodrug (Halle/Saale, Germany). All other substances were purchased from Sigma/Fluka in highest quality.
Small
Results
Glucagon like peptide 1 receptor (GLP-1R), dipeptidyl-peptidase IV (DPPIV) and Na+/H+ exchanger isoform 3 (NHE3) mRNA expression was investigated by quantitative RT-PCR (Fig. 1). Glucose had no significant effect on the expression of these three genes (Fig. 1).
GLP-1R expression at the protein level was confirmed in primary porcine proximal tubular cells (Fig. 2, Fig. 3, Fig. 5) by immunocytochemistry, immunohistochemistry and Western blot analysis. In Fig. 4, homogenized kidney cortex slices
Discussion
Renal effects of GLP-1 have been documented in vivo, both in healthy subjects and in obese persons [15]. The findings were supported by animal data in anesthetized rats. When the animals received GLP-1 infusion, a remarkable 13-fold increase of sodium excretion was observed [20]. The authors postulated that the inhibitory effect of GLP-1 on sodium re-absorption was a direct action on the proximal tubules and not due to a change in renal hemodynamics.
In vitro investigations have confirmed the
Acknowledgments
This work was supported by the Swiss National Science Foundation (grant #3200-065588.04/1) and the Senglet foundation, Basel, Switzerland. We express our gratitude to Ursula Behrens for the valuable technical support.
Great thanks go especially to Brigitte Schneider for the excellent technical assistance in immunohistochemistry.
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