Fig 2: (A) Staining of whole kidney sections showed the presence of GLUT1, (pro)renin receptor (PRR), and OXGR1 (red staining). L indicates lumen. Nuclei are stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). (B) Inner medullary collecting duct (IMCD) cells were grown until reach confluence and assessed for the presence of NKCC to rule out the presence of cortical tissues cells. Western blot analysis showed the presence of aquaporin (AQP)-2 in homogenates of total kidney tissue and IMCD cells. (C) NKCC was absent in IMCD. Functional IMCD cells were assessed by the activation of the vasopressin V2 with a V2 receptor agonist (DDAVP, 10−8 M). (D) After 45 min, AQP-2 immunofluorescence was mostly detected in plasma membrane. Coimmunostaining demonstrated the coexistence of OXGR1 and PRR, which also co-localizes with AE-1, a marker of intercalated cell. (E) OXGR1 did not co-localizes with AQP-2, a marker of principal cell.

Fig 3: Working hypothesis representing the effects of high glucose on the activation of tricarboxylic acid (TCA) cycle and accumulation of αKG leading to OXGR1 activation. Elevated plasma glucose levels and increased expression of GLUT1 boost glycolysis and TCA cycle, causing αKG accumulation and secretion. Prorenin (and renin) released into the lumen by the principal cell, binds to (pro)renin receptor (PRR), consequently increasing renin activity and activating enzymatic activity of prorenin. Angiotensinogen in the urine (secreted by proximal tubules or coming from blood filtrate) is cleaved by activated prorenin or renin. Since ACE activity is present along the nephron, this intratubular RAS activation ends with AngII intratubular formation and angiotensin type 1 receptor (ATR1)-dependent stimulation of sodium reabsorption through epithelial sodium channels (ENaC).

Fig 4: Effect of high glucose (HG) conditions (25 mM) on GLUT1 and (pro)renin receptor (PRR) expression in cultured inner medullary collecting duct (IMCD) cells subjected or not to OXGR1 pharmacological blockade. (A) PRR messenger RNA (mRNA) and (B) protein levels were augmented by 24-h HG treatment while pretreatment with 10−7 M of montelukast (ML) blunted the induction of PRR expression. Augmentation of GLUT1 at (C) mRNA and (D) protein levels was not altered by the pretreatment with ML. *p < 0.05 vs. control group, n = 5. Reblotting of blot (B) is shown in Panel (D), and same representative β-actin loading control was used.