Urinary Extracellular Vesicles for Renal Tubular Transporters Expression in Patients With Gitelman Syndrome

Abstract

Background: The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na⁺) or potassium (K⁺) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3. Methods: uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na⁺-hydrogen exchanger 3 (NHE3), Na⁺/K⁺/2Cl^- cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na⁺ channel β (ENaCβ), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca²⁺-sensitive K⁺ channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled. Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCβ, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCβ, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues. Conclusion: uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.

Keywords: Gitelman syndrome; hypokalemia; renal tubular disease; renal tubular transporters; urinary extracellular vesicles (exosomes).

Figure 1

Characterization of urinary extracellular vesicles (uEVs) from healthy controls. (A) Screen shot from 1:2,000 diluted urine sample reveals a range of particle sizes by nanoparticle tracking analysis (NTA). (B) Concentration and size distribution of uEVs (0–150 nm diameter) by NTA were shown. The concentration is expressed as number of particles per ml. (C) uEVs particles were correlated strongly with urine creatinine (r² for 0.81, P < 0.0001). (D) Transmission electron microscopy of uEVs was shown (scale bar 100 nm). (E) uEVs markers (AQP2, TSG101, NSE, and CD9) were assessed by immunoblotting. (F) Expression pattern of renal transporters including NHE3, NKCC2, NCC, p-NCC, ENaCβ, pendrin, ROMK, and Maxi-K from healthy controls was similar.

Figure 2

Renal Na⁺ and K⁺ associated transporters expression from urinary extracellular vesicles in patients with GS (n = 10) compared with healthy controls. (A) Immunoblotting of renal transporters (NHE3, NKCC2, NCC, p-NCC, ENaCβ, pendrin, ROMK, Maxi-K, and CD9). (B) Quantification of immunoblotting of NHE3, NKCC2, NCC, p-NCC, ENaCβ, pendrin, ROMK, Maxi-K, and CD9. Error bars, standard deviation. *P < 0.05, **P < 0.01.

Figure 3

Immunofluorescence of biopsied kidney tissues from another 3 representative GS patients (GS 1, GS 2, and GS 3) compared with the control kidney tissue. Renal transporters including NHE3, NKCC2, NCC, p-NCC, ENaCβ, pendrin, ROMK, Maxi-K were stained with green. AQP2 was stained with red for localization. Scale bar, 50 μm.

Figure 4

Renal transporters expression from urinary extracellular vesicles (uEVs) and immunofluorescence of biopsied kidney tissues from bulimic patients. (A) Immunoblotting of renal transporters (NHE3, NKCC2, NCC, p-NCC, ENaCβ, pendrin, ROMK, Maxi-K, and CD9) from uEVs in bulimic patients (n = 3) compared with healthy control. (B) Immunofluorescence of NHE3, NKCC2, NCC, p-NCC (green, right) and ENaCβ, pendrin, ROMK, Maxi-K (green, left) from one representative bulimia patient compared with the control. AQP2 was stained with red for localization. Scale bar, 50 μm.