Glucagon-like peptide 1 receptor expression in primary porcine proximal tubular cells

https://doi.org/10.1016/j.regpep.2006.12.016Get rights and content

Abstract

Background

GLP-1 is secreted into the circulation after food intake. The main biological effects of GLP-1 include stimulation of glucose dependent insulin secretion and induction of satiety feelings. Recently, it was demonstrated in rats and humans that GLP-1 can stimulate renal excretion of sodium. Based on these data, the existence of a renal GLP-1 receptor (GLP-1R) was postulated. However, the exact localization of the GLP-1R and the mechanism of this GLP-1 action have not yet been investigated.

Methods

Primary porcine proximal tubular cells were isolated from porcine kidneys. Expression of GLP-1R was measured at the mRNA level by quantitative RT-PCR. Protein expression of GLP-1R was verified with immunocytochemistry, immunohistochemistry and Western blot analysis. Functional studies included transport assessments of sodium and glucose using three different GLP-1 concentrations (200 pM, 2 nM and 20 nM), 200 pM exendin-4 (GLP-1 analogue) and an inhibitor of the dipeptidylpeptidase IV (DPPIV) enzyme (P32/98 at 10 μM). Finally, the expression of NHE3, the predominant Na+/H+ exchanger in proximal tubular cells, was also investigated.

Results

GLP-1R, NHE3 and DPPIV were expressed at the mRNA level in porcine proximal tubular kidney cells. GLP-1R expression was confirmed at the protein level. Staining of human and pig kidney cortex revealed that GLP-1R was predominantly expressed in proximal tubular cells. Functional assays demonstrated an inhibition of sodium re-absorption with GLP-1 after 3 h of incubation. Exendin-4 and GLP-1 in combination with P32/98 co-administration had no clear influence on glucose and sodium uptake and transport.

Conclusion

GLP-1R is functionally expressed in porcine proximal tubular kidney cells. Addition of GLP-1 to these cells resulted in a reduced sodium re-absorption. GLP-1 had no effect on glucose re-absorption. We conclude that GLP-1 modulates sodium homeostasis in the kidney most likely through a direct action via its GLP-1R in 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.

References (38)

  • M. Gonzalez-Gronow et al.

    Association of plasminogen with dipeptidyl peptidase IV and Na+/H+ exchanger isoform NHE3 regulates invasion of human 1-LN prostate tumor cells

    J Biol Chem

    (2005)
  • P. Schlatter et al.

    Primary porcine proximal tubular cells as a model for transepithelial drug transport in human kidney

    Eur J Pharm Sci

    (2006)
  • J.L. Dunphy et al.

    Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression

    Mol Cell Endocrinol

    (1998)
  • H. Yoo-Warren et al.

    Regulation of rat glucagon receptor expression

    Biochem Biophys Res Commun

    (1994)
  • Y. Wei et al.

    Tissue-specific expression of the human receptor for glucagon-like peptide-I: brain, heart and pancreatic forms have the same deduced amino acid sequences

    FEBS Lett

    (1995)
  • L.L. Nielsen et al.

    Pharmacology of exenatide (synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes

    Regul Pept

    (2004)
  • R. Goke et al.

    Characterization of the receptor for glucagon-like peptide-1(7-36)amide on plasma membranes from rat insulinoma-derived cells by covalent cross-linking

    J Mol Endocrinol

    (1989)
  • G.G. Holz

    New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells

    Horm Metab Res

    (2004)
  • B. Lankat-Buttgereit et al.

    Detection of the human glucagon-like peptide 1(7-36) amide receptor on insulinoma-derived cell membranes

    Digestion

    (1994)
  • Cited by (0)

    View full text