PROTECTIVE EFFECT OF P2Y14 RECEPTOR INHIBITION IN ISCHEMIA-INDUCED ACUTE KIDNEY INJURY: TOWARD THE DEVELOPMENT OF A NOVEL THERAPEUTIC STRATEGY

Ischemia-induced acute kidney injury (AKI) affects up to 50% of cardiac surgery (CS) patients, yet early diagnostic biomarkers and targeted therapies remain elusive. We previously showed that P2Y14 receptor inhibition attenuates AKI in male mice. P2Y14 is activated by UDP-sugars. Here, we investigated whether UDP-sugars are predictors of AKI in CS patients and further characterized the beneficial effect of P2Y14 inhibition in a preclinical model of ischemic-AKI in both male and female mice.

1) Plasma and urine samples were collected from 100 CS patients requiring cardio-pulmonary bypass (CPB), prior and up to 5 days after surgery. The primary outcome was AKI within 24 h using modified KDIGO 2024 guidelines. 2) Female and male mice were subjected to bilateral ischemia-reperfusion injury (BIRI), with a clamping time of 35 min and reperfusion for 24 h. SHAM surgeries were performed without clamping. The P2Y14 antagonist PPTN or vehicle were administered using osmotic Alzet minipumps implanted subcutaneously 24 h prior BIRI.

1) CS patients with AKI had elevated urinary UDP-sugars, which correlated with elevated urinary tubular injury and inflammatory markers KIM-1, CXCL1, CCL2 and IL-18, as early as 4 hours post-CPB. Plasma UDP-sugars showed no association with AKI. Immunofluorescence revealed P2Y14 apical localization in collecting duct cells of human ischemic kidneys. 2) Based on plasma creatinine (pCre), female mice showed an apparent AKI protection vs males. However, BIRI induced a significant increase in blood urea nitrogen (BUN) vs SHAM in both female and male mice. Compared with SHAM, BIRI induced in both sexes: i) an increase in the percentage of severely damaged proximal tubules (PTs), characterized by a complete loss of AQP1 polarity; ii) the renal recruitment of Ly6G+ neutrophils, which produced neutrophil extracellular traps (NETs) as evidenced by citrullinated histone H3 labeling; iii) an increase of acyl-CoA synthetase long-chain family member 4 (ACSL4) expression, indicating induction of ferroptosis; iv) increased expression of 12-lipoxygenase (12-LOX) enzyme; v) increased levels of 12-LOX metabolites. Treatment with PPTN significantly reduced all these BIRI-induced effects. Interestingly, a lower dose of PPTN was required in females versus males (5 vs 10 mg/kg/day) to achieve maximal response.

These findings establish UDP-sugars as early predictors of CS-AKI and implicate a mechanistic pathway whereby kidney injury triggers UDP-sugar release, subsequent P2Y14 activation in collecting duct cells, and pro-inflammatory cytokine production. Our preclinical mouse model shows that both females and males can develop ischemia-induced AKI, and that pCre is a poor marker of AKI in females. Finally, attenuation of renal inflammation via P2Y14 inhibition protects against ferroptosis and inflammation, and reduces the extent of kidney dysfunction and damage following ischemia-reperfusion injury in both female and male mice, consolidating its therapeutic efficacy regardless of sex. Our study thus shows that the UDP-sugar/P2Y14 axis comprises both a biomarker and a therapeutic target for identifying and preventing ischemia-induced AKI.