Endocrine Abstracts

Signalling through intracellular pathways is typically monitored by recording the change in abundance of a signalling component of interest in a whole cell population – often using western blotting. However, single cells differ markedly in their response to a given stimuli, even in a clonal cell line. High content imaging enables signalling responses to be quantified in space and time – in single cells – by the semi-automated imaging of many tens of hundreds of single cells under a multitude of different conditions. The variability in response is likely to be due to each cell having a distinct complement of signalling molecules. Furthermore, this inherent cell–cell variability can be exploited to assess the contribution of a particular cell component to a signalling pathway using in silico cell sorting, without the need for disruptive over-expression or knock-down protocols that push protein expression above or below physiological levels. This in silico sorting method will be discussed with respect to expression of the Angiotensin receptor-associated protein (ATRAP) in a human immortalised kidney podocyte cell line. Podocytes are the major constituent cell of the glomerular filtration barrier, but in kidney disease these cells are often injured and lost, resulting in proteinuria. Like other cells in the body, podocytes are prone to receiving damaging signals. ATRAP has been shown to downregulate Angiotensin II signalling, the hormone that regulates blood pressure and is targeted in the treatment of Chronic Kidney Disease to relieve pressure on the kidney tubules. However, further reports indicate that ATRAP interacts with the receptor for TNFα (TNFR1), an inflammatory cytokine. Using immunofluorescence staining followed by in silico cell sorting, I will demonstrate that ATRAP disrupts TNFα survival signalling through c-Jun N-terminal kinase (JNK), Nuclear Factor κ-light-chain-enhancer of activated B cells (NF-κB) and its inhibitor, IκBα, in kidney podocytes.