Engineering and characterization of human renal proximal tubular cells for applications in vitro toxicology and bioartifical kidneys

Abstract

Renal proximal tubular epithelial cells perform a wide variety of kidney-specific functions. Due to their function in glomerular filtrate concentration and drug transport, they are a major target of drug-induced toxicity and hence important for in vitro nephrotoxicology. However, respective approved in vitro models based on renal cells have not been developed yet. One major obstacle is cellular de-differentiation of human primary renal proximal tubular cells (HPTC), which are most interesting for such applications, under in vitro conditions. HPTC are also important for the development of bioartificial kidneys (BAKs) and also in this application cell performance is of critical importance.

In order to establish a reliable source and to characterize cell performance, I established in the laboratory a protocol for isolating HPTC from human kidney samples. The freshly isolated HPTC were characterized using qPCR, immunostaining, immunoblotting and functional assays. In addition, I characterized commercial HPTC. The results showed that both freshly isolated and commercial HPTC displayed many characteristics of HPTC, but showed some changes in gene expression patterns and expressed some markers specific for other parts of the nephron.

I also established a co-culture system between HPTC and human primary endothelial cells. The results showed that HPTC stimulated endothelial cells to secrete a mixture of growth factors, which in turn improved HPTC performance. HPTC showed improved proliferation, marker gene expression and enzyme activity in co-cultures. Also, the long-term maintenance of epithelia formed by HPTC was improved. In order to determine which growth factors were responsible for these effects, qPCR analysis was performed. The results pointed to a central role of transforming growth factor-?1 (TGF-?1) and its antagonist alpha-2-macroglobulin (A2M). The impact of these factors on HPTC was further confirmed by additional experimental approaches involving supplementation with recombinant growth factors. Overall, the results showed that HPTC induced endothelial cells to secrete increased amounts of specific growth factors, which balanced each other functionally and improved cell performance. Together, the results revealed that co-culture systems are useful for analyzing the cross-talk between these cell types which plays an important role in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, is helpful for improving the performance of this cell type in in vitro applications.

The central role of TGF-?1 and its antagonists in regulating HPTC performance was further confirmed by our findings that treatment with bone morphogenetic protein-7 (BMP-7), which is a TGF-?1 antagonist, improved maintenance of epithelia formed by HPTC for extended time periods. In addition, the functional performance of the HPTC was improved. The effects of BMP-7 were strongly concentration-dependent. Following these findings, I generated BMP-7-expressing HPTC by genetic engineering for the development of BMP-7-producing bioartificial kidneys. The hypothesis underlying this work was that HPTC-produced BMP-7 would improve cell performance in the device by autocrine/paracrine signaling. Furthermore, pre-clinical studies revealed beneficial effects of BMP-7 on kidney recovery and hence there is a substantial interest in using BMP-7 for the treatment of kidney disease. Apart from the improvement of cellular functions, a BMP-7-producing BAK would allow the delivery of the growth factor to kidney patients. My results showed that HPTC-produced BMP-7 was bioactive and improved HPTC performance through autocrine signaling. In addition, our results suggested that the amount of BMP-7 produced by HPTC would be sufficient for therapeutic applications.