FP001Expression Pattern of Renal Tubular Transporters in Urinary Exosomes from Patients with Acute and Chronic Hypokalemia

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INTRODUCTION: Urinary exosomes contain membrane and cytosolic proteins from each renal epithelial cell type and has been used as an index of renal tubular transporter expression in primary aldosteronism or renal tubular disorders. Urinary exosome analysis of renal sodium (Na+) and potassium (K+) associated transporters in hypokalemia patients has not been studied. However, whether analysis of proteins in urine faithfully reflects their changes in the kidney tissue remains unclear. Our purpose to evaluate the expression of renal Na+ and K+ associated transporters in patients with acute and chronic hypokalemia. METHODS: Nanoparticle tracking analysis (NTA) were used for quantification of urinary exosomes in healthy controls (n=7). We also have collected timely spot urine from thirty-one patients with hypokalemia. Urinary exosomes were further isolated by ultracentrifugation method. Membrane transporters proteins abundance including NaCl cotransporter (NCC), phospharylated NaCl cotransporter (pNCC), Na+-hydrogen exchanger 3 (NHE3), Na/K/2Cl cotransporter (NKCC2), epithelial Na+ channel β (ENaCβ), and renal outer medullary K1 channel (ROMK) were analyzed by immunoblotting. RESULTS: Quantification of urine exosomes in healthy controls revealed a range of particle sizes with mean concentration was 8×10 11/ml, and urinary exosomes particles number correlated strongly with urine creatinine. In Gitelman syndrome (GS, n=11) patients, immunoblotting, NCC and pNCC abundance significantly decreased corresponding to NCC mutation compared to healthy control. Na+ associated transporters abundance of NHE3 and ENaCβ significantly increased while K+ associated transporters abundance of ROMK significantly increased. Expression pattern of transporters in urinary exosomes was consistent with that in kidney immunofluorescence staining. In thyrotoxic periodic paralysis patients (TPP, n=9), there were no significant change of transporter abundance in acute hypokalemia phase compared to recovery phase. Patients with Sjogren syndrome with distal renal tubular acidosis (distal RTA, n=4), aldosterone producing adenoma (APA, n=3), and gastrointestinal disorders (n=4) exhibited similar renal transporter expression that abundance of NHE3 and ENaCβ significantly increased while abundance of ROMK significantly increased. Of note, there were significant increased NCC abundance in APA patients and increased NKCC2 abundance in distal RTA patients compared to healthy control. CONCLUSIONS: Urine exosomics could be used to evaluate the renal Na+ and K+ associated transporters expression in hypokalemia. Acute hypokalemia could not affect expression in Na+ or K+ associated transporters. Chronic hypokalemia could activate both upstream NHE3 and downstream ENaCβ with concomitant increased expression of ROMK in response to flow.

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