Adenosine Triphosphate Protects from Elevated Extracellular Calcium-Induced Damage in Human Proximal Kidney Cells:...

In kidney, extracellular [Ca²⁺] can modulate intracellular [Ca²⁺] to control key cellular processes. Hence, extracellular [Ca²⁺] is normally maintained within narrow range. We tested effect of extracellular ATP on viability of human proximal (HK-2) cells at high calcium. Modulation of intracellular calcium was assessed by imaging cytosolic [Ca²⁺], and expression of calcium-binding proteins (CaBPs). We present an artificial intelligence enabled deep learning model for prediction of injury and protection against extracellular [Ca²⁺] in HK-2 cells.

HK-2 cells were cultured in calcium-free DMEM supplemented with CaCl2. Morphological changes were detected using light microscopy. Cell viability was determined using MTT Assay. Intracellular [Ca²⁺] was detected using fluorescence microscopy. For easy detection of HK-2 cells injury, we performed light microscopy image classification based on Convolutional Neural Network. Expression of CaBPs, p21, and Mcl-1 was measured using real-time PCR.

We show decreased viability of HK-2 cells cultured in elevated calcium levels, which was prevented by adenosine triphosphate (ATP). Exposure of cells to elevated extracellular [Ca²⁺] correlated with increasing fluorescence of intracellular calcium indicator, which was attenuated in presence of ATP. Since features cannot be detected easily by human eyes, we propose a customized deep learning-based CNN model for classification of HK-2 cells injury by extracellular calcium with high accuracy of 98%. Our data demonstrated significant increase in mRNA levels of calmodulin, S100A8, S100A14 and CaBP28k, with elevated extracellular [Ca²⁺]. Expression of these genes was enhanced with ATP.

The results suggest that ATP protects human proximal (HK-2) cells against elevated extracellular calcium levels. We present a CNN model as user friendly tool to study calcium dependent injury in (HK-2) cells. Finally, we show that ATP-mediated protection is correlated with enhanced expression of calcium-binding proteins.