PON3 is expressed in the aldosterone-sensitive distal nephron, where ENaC plays an essential role in maintaining Na+/K+ homeostasis in the kidney. The aims of our study were to determine the physiological roles of PON3 in renal Na+ and K+ handling. We hypothesize that PON3 functions as a molecular chaperone to regulate ENaC expressionand Na+ reabsorption in the kidney. We have obtained a Pon3 global knockout mouse model and examined the effect of PON3 on ENaC functional expression using several approaches, including biochemistry, immunohistochemistry, electrophysiology, and whole animal metabolic cage studies. Pon3 KO mice have normal kidney histology without evidence of inflammation or injury. At baseline, Pon3 KO mice have a significantly lower blood [K+] and higher blood [Na+] when compared to WT littermates. Amiloride-induced natriuresis was significantly greater in the KO mice, reflecting, in part, an upregulation in ENaC-dependent Na+reabsorption in the absence of the PON3. Immunoblotting of whole kidney lysates indicated that the total abundance of ENaC subunits was not altered in KO mice. Single channel recordings of ENaC in split-open tubules freshly isolated from WT or Pon3 KO mice kidneys demonstrated that open probability ( P O) was similar between the two groups of animals. However, the number of active channels per patch ( N) was significantly higher in Pon3 KO mice, resulting in a higher ENaC activity ( NP O) in the distal nephron segments of Pon3 KO mice. Consistent this notion, we found that ENaC γ subunit surface abundance was increased in mCCD cells when PON3 expression was knocked down by siRNA.Together, our data suggest that Pon3 KO mice have upregulated Na+ reabsorption and K+ secretion, likely a result of increased ENaC functional expression in the absence of PON3. This study was funded by NIH grants R01 DK130901 (to SS), R01 DK111380 (to SS), R01 HL128053 (to TRK and SS), R01 HL147818 (to TRK), and P30 DK079307 (the Pittsburgh Center for Kidney Research). SMM was supported by T32 DK061296, T32 DK007052, and a grant from Relypsa. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.