The diagnosis of acute kidney injury (AKI) traditionally depends on the serum creatinine (Scr) and urine output, which lack sufficient sensitivity and specificity. Using urinary exosomes as a biomarker has unique advantages. To assess whether urinary exosomal Na⁺/H⁺ exchanger isoform 3 (NHE3) protein could serve as a biomarker of AKI, we constructed four AKI rat models: cisplatin (7.5 mg/kg) injected intraperitoneally (IP), furosemide (20 mg/kg, IP) with a low-NaCl (0.03%) diet, a low-NaCl (0.03%) diet with candesartan (1 mg/kg, IP) and bilateral ischemia and reperfusion (I/R) injury for 40 min. Additionally, we assessed six sepsis-associated AKI patients and six healthy volunteers. Urinary exosomes were extracted by ultracentrifugation, and the NHE3 protein abundance was tested by immunoblotting for all the AKI rats and human subjects. The isolated cup-shaped particles with an average diameter of 70 nm and enrichment in CD63 were identified as exosomes. NHE3 abundance was six times higher in exosomes than in the whole urine. In cisplatin-induced AKI rats, urinary exosomal NHE3 was increased on day 2, one day earlier than the increases in Scr and blood urea nitrogen (BUN). In additional rats, urinary exosomal NHE3 decreased along with the decline in Scr after EPO pretreatment. In volume-depletion AKI induced by furosemide injection with a low-NaCl diet, the urinary exosomal NHE3 expression was higher than that in the control. Under a low-NaCl diet with candesartan-related AKI, the urinary exosomal NHE3 was elevated on day 5, earlier than Scr. In I/R-injury AKI, the urinary exosomal NHE3 was also raised compared with that in the control. In humans, the urinary exosomal NHE3 level was also elevated in sepsis-associated AKI patients in comparison with that in the healthy volunteers. The urinary exosomal NHE3 was increased in multiple AKI; it may be used as a diagnostic biomarker of AKI.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)‐induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS‐PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS‐PE than controls on GD14. On GD18, LPS‐PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co‐immunoprecipitated with NKCC2 or NCC in kidney homogenates and co‐immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS‐PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co‐immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS‐induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS‐PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

The diagnosis of acute kidney injury (AKI) traditionally depends on the serum creatinine (SCr) and urine output, which lack sufficient sensitivity and specificity. Urinary exosome as a biomarker has unique advantages. To assessed whether urinary exosome Na+/H+ exchanger isoform 3 (NHE3) protein as a nonivasive diagnostic biomarker of AKI, we constructed 4 AKI rat models: cisplatin (7.5mg/kg) injection intraperitoneally (IP), furosemide (20mg/kg, IP) with low NaCl (0.03%) diet, low NaCl (0.03%) diet with candesartan (1mg/kg, IP) and bilaterally ischemia and reperfusion(I/R) injury for 40 minutes. Besides, we assessed 6 sepsis associated AKI patients and 6 healthy volunteers. Urinary exosomes were extracted by ultra-centrifugation and NHE3 protein abundance was tested by immunoblotting in all AKI rats and human subjects. The isolated cup-shape particles with an average diameter of 70nm and enrichment in CD63 were identified as exosomes. NHE3 abundance was 6 times higher in exosome than in original urine. In cisplatin induced AKI rats, urinary exosome NHE3 was increased at day 2, 1 day earlier than the increases of serum creatinine creatinine (SCr) and blood urea nitrogen (BUN). In additional rats, urinary exosome NHE3 decreased along with the decline of SCr after EPO pretreatment. In volume depletion AKI induced by furosemide injection with low NaCl diet, urinary exosome NHE3 expression was higher than control. In low NaCl diet with candesartan related AKI, urinary exosome NHE3 was elevated at day 5, 2 days earlier than SCr. In I/R injury AKI, urinary exosome NHE3 was also raised compared with control. In humans, the urinary exosome NHE3 level was also elevated in sepsis associated AKI patients in comparison with the healthy volunteers. Then urinary exosome NHE3 protein may be used as a noninvasive diagnostic biomarker of AKI. Impact Statement: The high non-recognition acute kidney injury (AKI) is due to lacking of alarming symptoms or specific early biomarkers. Urinary exosome as a biomarker has unique advantages. In our study, we detected urinary exosome NHE3 protein abundance in 4 different cause of AKI rat model. Urinary exosome NHE3 was increased in all 4 AKI, and even elevated earlier than SCr in some cases. Another novel point was we established a new AKI model of low NaCl diet with candesartan injection, which was common in patients with hypertension or proteinuria clinically. The detailed methods and mechanisms of this new AKI model were presented in another article being submitted. Third, we are not limited to animals, but also selected sepsis associated AKI patients to study. The conclusion that urinary exosome NHE3 may be used as a diagnosis biomarker of AKI has important clinical application value.