Kidney microphysiological systems (MPS) serve as valuable preclinical instruments in recapitulating physiological conditions and determining underlying interactions involved in renal clearance and osmoregulation. Current kidney MPS models target individual regions of the nephron, such as the glomerulus and proximal tubule, but fail to incorporate multiple filtration and absorption interfaces. In the current study, an in vitro MPS features key filtration and reabsorption properties of the human glomerulus and proximal tubule for seven days of operation. Three human-derived cell types, including the conditionally immortalized human podocytes (CIHP-1), human umbilical vein endothelial cells (HUVECs), and human proximal tubule cells (HK-2), were adapted to serum-free medium prior to being seeded into the three-component MPS (T-Junction splitter, glomerular housing unit, and parallel proximal tubule barrier model), which was optimized using in silico computational modeling. The tri-culture MPS successfully filtered blood serum protein, resorbed glucose, and generated filtrate. This glomerulus and proximal convoluted tubule MPS is a novel system for both human-relevant testing and examining pharmacokinetic interactions.