Characterisation and validation of drug transport and GLP-1R function in primary porcine proximal tubular cells

Overview kidney:
Kidney is, beside the liver, one of the most important organs for the elimination of waste products, toxins, drugs and their metabolites. Due to the anatomy of the kidney, ultra filtrate leaving the glomerulus passes first the proximal tubular cells. Therefore, these cells are exposed to high concentrations of xenobiotics which explains their high metabolic activity. This circumstance results sometimes in proximal tubular nephrotoxicity. In addition, various transmembrane proteins respectively transporters are responsible for the elimination or accumulation of drugs. This complex interplay is also responsible for drug-drug interactions.
Therefore, proximal tubular cells are an interesting tool to study drug transport in the kidney. These cells are mainly localised in kidney cortex which makes it easy to isolate them (Fig. 1.1). Further isolation procedures enables high purity of proximal tubular cells by excluding connective tissue cells, distal tubular cells and other undesirable cells (elucidated in detail in 2.4).
Aim of the thesis
The aim of this thesis was to establish and validate a new in vitro model for drug transport in kidney by using primary porcine proximal tubular cells.
In a first step, primary porcine proximal tubular cells were isolated from pig kidney, which were retrieved freshly at the slaughterhouse. These isolated cells were investigated to verify their origin from proximal tubular cells. In a next step the cells were screened at mRNA, protein and functional level for functional expression of important drug transporters. Finally, it was investigated if this model can be used for screening of drug-drug interactions or for specific drug transporter properties.
Expression of drug transporters
In cultured cells the following drug transporters were expressed (chapter 2.5 and 3.4)
A) at mRNA level:
• abcb1 (pMDR1)
• abcc1 (pMRP1)
• abcc2 (pMRP2)
• slc22a8 (pOAT3)
• slco1a2 (pOATP-A)
• slc15a1 (pPEPT1)
• slc5a2 (pSGLT2)
• slc4a4 (pNBC1)
• slc9a3 (pNHE3)
• abcg2 (pBCRP alias BMDP)
B) at protein level
• slc4a4
• abcb1
• abcc1
• abcc2
• abcg2
C) functionally
• abcb1: transport and uptake of digoxin, inhibition of digoxin transport and higher digoxin uptake with verapamil
• abcc1: uptake of methotrexate, higher methotrexate uptake with indomethacin and MK571
• abcc2: uptake of methotrexate, higher methotrexate uptake with MK571
• slc22a8: transport and uptake of fluorescein, inhibition of fluorescein transport and uptake with estrone sulfate
• slc15a1: transport and uptake of glycylsarcosine, inhibition of glycylsarcosin transport and uptake with benzylpenicillin
• slc5a2: uptake of glucose, inhibition of glucose uptake with phlorizin
• abcg2: uptake of mitoxantrone, higher mitoxantrone uptake with prazosin
Slc22a1 (pOCT1) and slc22a6 (pOAT1) were only expressed in freshly isolated cells but were down-regulated in culture. Freshly isolated proximal tubular cells showed functional activity of slc22a6 as uptake of fluorescein was inhibited with p-aminohippuric acid.
Confirmation of proximal origin
Several transporters are expressed exclusively in proximal tubular cells. This fact was used in order to confirm the origin of the proximal tubular cells with each isolation. Due to expression of slc15a1, slc5a2, slc4a4, slc9a3 and the enzyme dipeptidylpeptidase IV (DPPIV, see below) we confirmed, that our cells are indeed of proximal origin.
BMPD expression:
After validation of the above mentioned drug transporters, we searched for possible inhibitors of BMDP/pBCRP, that might influence elimination of known BCRP substrates such as the chemotherapeutics topo- and irinotecan and mitoxantrone. The tested drugs included various antiepileptic agents, non-steroidal anti-inflammatory drugs, proton pump inhibitor, caffeine, theophylline, thalidomide and dotatoc. Of all investigated drugs, only phenytoin was able to inhibit BMPD significantly in our model.
GLP-1 effect in proximal tubular cells
Glucagon like peptide 1 (GLP-1) is an hormone, secreted after meal ingestion in the intestine. This peptide mediates satiety feelings and most importantly stimulates glucose dependent insulin secretion from pancreatic β-cells, thus lowering plasma glucose levels. Interestingly, GLP-1 also enhances renal sodium secretion in healthy volunteers.
Therefore, we hypothesised that GLP-1 receptor (GLP-1R) is functionally expressed in proximal tubular cells. GLP-1R expression was confirmed at mRNA and protein level (chapter 4.4). More precisely, in kidney cortex the protein expression of GLP-1R seemed to be localised mainly in proximal tubular cells. Furthermore, we could show an inhibitory effect of GLP-1 on sodium re-absorption, indicating functional GLP-1R activity. In contrast to sodium re-absorption the re-absorption of glucose was not affected by GLP1.