ABSTRACT BOYD-SHIWARSKI The purpose of this R03 proposal is to develop pilot studies using new research methodology and technology to investigate how dietary potassium (K+) deficiency leads to kidney injury. It is well known that dietary K+ deficiency leads to hypertension, cardiovascular disease, as well as chronic kidney disease. My NIDDK K08 research was funded to study how K+ deficiency alters proteins in the distal convoluted tubule leading to salt-sensitive hypertension. In addition to hypertension, we observed that K+ deficient mice develop kidney injury with a decrease in the glomerular filtration rate (GFR) and an increase in inflammatory markers. It has been proposed that K+ deficiency causes kidney injury through activation of ammoniagenesis in the proximal tubule, yet the link between hypokalemic-induced ammoniagenesis and kidney injury remains obscure. Our preliminary results in K+ deficient mice have shown an increase in ammoniagenesis, an increase in complement activation, an increase in inflammatory markers, and an increase in fibrosis. However, it is unclear whether the kidney injury is due to local effects within proximal tubule cells, or due to an extra-renal immune or endocrine response. To isolate the direct effects of low K+ on the proximal tubule we propose to develop and optimize two cellular model systems. 1) The first is optimizing a 2D permeable support proximal tubule model. This 2D polarized cellular model is the current standard for studying proximal tubule cells in culture systems. 2) The second is developing a tubule- scaffold using 3D bioprinted collagen that can be seeded with proximal tubule cells to recapitulate the 3D environment with tubular constraints, laminar flow, and mechanical forces—factors all known to play a critical role in proximal tubule differentiation and physiology. By developing these two cellular model systems we intend to isolate the direct effects of low K+ on the proximal tubule, including the effects on ammoniagenesis and activation of the de novo immune response. The results from this grant will advance our understanding of how the Western diet, which is deficient in potassium, may increase susceptibility to kidney injury. Additionally, these results will generate publications, facilitate independence, and form the foundation for my R01 application.

PROJECT NARRATIVE BOYD-SHIWARSKI Studies have shown a significant decline in dietary potassium intake over the past several decades and estimate that 98% of the US population is now deficient in dietary potassium—this deficiency has been attributed to the rise in processed food, decreased intake of fruits and vegetables, and the agriculture soil depletion of potassium. There is an urgent and compelling need to study the health consequences of inadequate potassium intake, specifically how it relates to chronic kidney disease. The purpose of this project is to develop new cell culture methodology using innovative 3D bioprinting technology to isolate the pathological effects of low potassium on the kidney’s proximal tubule.