Organic anion transporter OAT3 enhances the glucosuric effect of the SGLT2 inhibitor empagliflozin

The sodium-glucose cotransporter SGLT2 inhibitor empagliflozin (plasma protein binding ~88%) may reach its target in the brush border of the early proximal tubule by glomerular filtration and tubular secretion. Here we determined whether empagliflozin is secreted by renal tubules in mice and whether genetic knockout of the basolateral organic anion transporter 3 ( Oat3−/−) affects its tubular secretion or glucosuric effect. Renal clearance studies in wild-type (WT) mice showed that tubular secretion accounted for 50–70% of empagliflozin urinary excretion. Immunostaining indicated that SGLT2 and OAT3 localization partially overlapped in proximal tubule S1 and S2 segments. Glucosuria in metabolic cage studies was reduced in Oat3−/− vs. WT mice for acute empagliflozin doses of 1, 3, and 10 mg/kg, whereas 30 mg/kg induced similar maximal glucosuria in both genotypes. Chronic application of empagliflozin (~25 mg·kg−1 ·day−1) in Oat3−/− mice was associated with lower urinary glucose-to-creatinine ratios despite maintaining slightly higher blood glucose levels than WT. On a whole kidney level, renal secretion of empagliflozin was largely unchanged in Oat3−/− mice. However, the absence of OAT3 attenuated the influence of empagliflozin on fractional glucose excretion; higher levels of plasma or filtered empagliflozin were needed to induce similar increases in fractional renal glucose excretion. We conclude that empagliflozin is excreted into the urine to similar extent by glomerular filtration and tubular secretion. The latter can occur largely independent of OAT3. However, OAT3 increases the glucosuric effect of empagliflozin, which may relate to the partial overlap of its localization with SGLT2 and thus OAT3-mediated tubular secretion of empagliflozin in the early proximal tubule.

N-acetyl-beta-glucosaminidase urine activity as a marker of early proximal tubule damage and a predictor of the long-term function of the transplanted kidneys.

Ischaemia-reperfusion injury (IRI) is a factor leading to the damages of the transplanted kidney, what affects mainly the proximal tubules. Early monitoring of tubule damage can be an efficient tool to predict the allograft dysfunction. Present in proximal tubules, N-acetyl-beta-glucosaminidase (NAG) is a lysosomal enzyme whose excretion rises as a result of IRI or acute rejection. The aim of this study was to monitor the NAG urine activity to evaluate the early proximal tubule damage, and to try to predict the long-term function of the transplanted kidney. The study enrolled 87 Caucasian renal transplant recipients (61.7% males, 38.3% females, mean age 45.56±14.34 years). Urine samples were collected for NAG and creatinine analysis on the 1st day after transplantation, and then in the 3rd and 12th month. Protocol biopsies were performed in the 3rd and 12th month. A significant positive correlation between NAG urine activity in the 3rd month after transplantation and creatinine concentration on the 14th (p=0.004) and 30th day (p=0.05), in the 3rd month (p=0.009) and after the 1st (p=0.005) and 2nd year (p=0.003) was observed. A statistically significantly higher urinary NAG activity in samples collected in the first 3 days and in the 3rd month after transplantation among patients with DGF (p=0.006 and p=0.03 respectively) was found. There was a significant positive correlation between NAG urine activity in the 3rd month and the grade of tubular atrophy in specimens collected in the 3rd (p=0.03) and 12th (p=0.04) month. Monitoring of NAG urine activity is useful in the evaluation of early proximal tubule damage and predicting the long-term function of the transplanted kidneys.

Fluid flow in the juxtaglomerular interstitium visualized in vivo

Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 ± 3.5% of that in the AA plasma ( n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5–10 s) and a slow (one every 45–50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2–5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 ± 4% ( n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function).