Introduction

Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na⁺/H⁺ exchange and H₂O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously.

Methods

We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport.

Results

piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport.

Conclusions

Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

中文翻译：

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基因组工程肾上皮细胞增强容量运输功能。

介绍

生物工程植入式人工肾 (IAK) 将需要能够重吸收盐和水的肾上皮细胞。我们使用基因组工程来修饰细胞以改善 Na ⁺ /H ⁺交换和 H ₂ O 重吸收。非病毒piggyBac转座子系统使基因组工程细胞能够同时稳定地过表达一种或多种转基因。

方法

我们生成了表位标记的人钠氢交换剂 3 (NHE3) 和 aquaporin-1 (AQP1) 表达piggyBac转座子载体的 cDNA。通过蛋白质印迹和免疫荧光评估转基因表达。流式细胞术分析用于定量基因组工程克隆文库中的转运蛋白表达。细胞表面生物素化用于评估表面蛋白定位。水泡形成测定用于监测细胞体积运输。

结果

piggyBac能够在培养的肾 (MDCK) 上皮细胞中实现稳定的转座子整合和 cumate 诱导的 NHE3 和/或组成型表达 AQP1 的过表达。使用细胞表面生物素化测定证实 NHE3 和 AQP1 的细胞表面递送。MDCK 克隆库的流式细胞术显示 AQP1 和 NHE3 的不同表达。表达 AQP1 和 cumate-inducible NHE3 的 MDCK 细胞表现出增加的体积运输。

结论

我们的结果表明，肾上皮细胞可以通过基因组工程来增强 IAK 装置所需的体积运输。我们的研究结果为未来研究基因组工程人肾细胞用于肾小管细胞治疗奠定了基础。