The role of transport proteins for the elimination of uremic toxins during chronic kidney disease

慢性肾脏病(CKD)与血浆尿毒症毒素增加有关，并伴有多种临床症状，如动脉高血压。由于透析治疗期间尿毒症毒素的血浆浓度进一步升高，因此加强转运蛋白介导的肾小管毒素分泌对慢性肾脏病患者可能是有益的。到目前为止，人们对近端肾小管上皮细胞转运蛋白调节尿毒症毒素从血液分泌到尿液中的作用知之甚少。因此，研究转运蛋白在尿毒症毒素肾排泄中的作用是本研究项目的重点。丰原和他的同事证明，人类摄取转运体OATP4C1在大鼠肾脏中的重组过表达可以减少肾衰竭背景下的高血压、心脏增大和炎症。此外，OATP4C1过表达降低了尿毒症毒素的血浆水平，如ADMA和琥珀酸胍，这表明该转运蛋白在尿毒症毒素的运输中发挥了作用。此外，几种顶端定位的输出泵，如P-糖蛋白(P-gp)和BCRP，已被确定为尿毒症毒素的可能转运体。为了更深入地了解转运蛋白在尿毒症毒素的肾脏分泌中的作用，将讨论四个子项目。利用已建立的稳定表达OATP4C1的HEK293细胞，尿毒症毒素将被作为OATP4C1介导的转运的调节剂和底物进行研究。此外，利用重组过表达OATP4C1的双转基因MDCKII细胞，结合顶端定位的输出泵如MRP2、MRP4、BCRP或P-gp，可以研究尿毒症毒素的载体转运。对于这两个子项目，几种尿毒症毒素和毒素代谢物将由Masereeuw教授(荷兰奈梅亨大学医学中心)提供。尿毒症毒素对所研究的转运体的调节将通过分析它们在不同浓度的尿毒症毒素处理的小鼠和人的原代近端小管细胞中的表达来解决。Klaus HöCherl教授(弗里德里希-亚历山大-纽伦堡大学药理学系)将为这项分析提供小鼠原代近端小管细胞(PTCs)。最后，将分析OATP4C1及其输出泵MRP2、MRP4、BCRP和P-gp在不同人类慢性肾脏疾病中的表达。在与肾脏病理科的合作下，转运蛋白的表达将在来自不同慢性肾脏疾病组织活检的近端小管细胞样本中进行量化，并通过显微解剖分离出来。这项研究的目标是深入了解转运蛋白在尿毒症毒素肾小管分泌中的作用，以及这些转运蛋白在病理生理条件下的调节，以获得CKD患者可能的药物治疗。

Chronic kidney disease (CKD) is associated with an increase of uremic toxins in plasma accompanied by a variety of clinical symptoms such as arterial hypertension. Because the plasma concentrations of uremic toxins further elevate during dialysis treatment, an enhancement of the transporter-mediated tubular secretion of toxins could be beneficial for patients with CKD. So far, little is known about transporters in proximal tubular cells mediating the secretion of uremic toxins from blood into urine. Therefore, studies on the role of transport proteins for the renal elimination of uremic toxins is in the focus of this research project. Toyohara and coworkers demonstrated that the recombinant overexpression of the human uptake transporter OATP4C1 in rat kidney reduced hypertension, cardiomegaly and inflammation in the setting of renal failure. In addition, OATP4C1 overexpression decreased plasma levels of uremic toxins such as ADMA and guanidine succinate suggesting that this transporter plays a role in the transport of uremic toxins. Furthermore, several apically localized export pumps such as P-glycoprotein (P-gp) and BCRP have been identified as possible transporters of uremic toxins. To gain more insights into the role of transport proteins in the renal secretion of uremic toxins four sub-projects will be addressed. Using established stably-transfected HEK293 cells recombinantly overexpressing OATP4C1, uremic toxins will be investigated as modulators and substrates of OATP4C1-mediated transport. Furthermore, using double-transfected MDCKII cells recombinantly overexpressing OATP4C1 together with an apically localized export pump such as MRP2, MRP4, BCRP or P-gp, the vectorial transport of uremic toxins should be studied. For both sub-projects several uremic toxins and toxin metabolites will be provided by Prof. Dr. Masereeuw (University Medical Center, Nijmegen, The Netherlands). The regulation of the investigated transporters by uremic toxins will be addressed by analyzing their expression in murine and human primary proximal tubule cells treated with different concentrations of uremic toxins. Prof. Dr. Klaus Höcherl (Department of Pharmacology, Friedrich-Alexander-Universität Erlangen-Nürnberg) will provide murine primary proximal tubule cells (PTCs) for this analysis. Finally, the expression of OATP4C1 and of the export pumps MRP2, MRP4, BCRP and P-gp will be analyzed in different human chronic kidney diseases. In a collaboration with the nephropathology department, the expression of the transporters will be quantified in proximal tubule cell samples originating from tissue biopsies of different chronic kidney diseases and isolated by microdissection. The goal of this research project is to get an insight into the role of transport proteins in the tubular secretion of uremic toxins and into the regulation of these transporters under pathophysiological conditions to gain access to a possible pharmacological treatment of patients with CKD.