Impaired Na+-K+-ATPase signaling in renal proximal tubule contributes to hyperuricemia-induced renal tubular injury

Abstract

Hyperuricemia contributes to renal inflammation. We aimed to investigate the role of Na⁺-K⁺-ATPase (NKA) in hyperuricemia-induced renal tubular injury. Human primary proximal tubular epithelial cells (PTECs) were incubated with uric acid (UA) at increasing doses or for increasing lengths of time. PTECs were then stimulated by pre-incubation with an NKA α1 expression vector or small interfering RNA before UA (100 μg ml^-1, 48 h) stimulation. Hyperuricemic rats were induced by gastric oxonic acid and treated with febuxostat (Feb). ATP levels, the activity of NKA and expression of its α1 subunit, Src, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and interleukin 1β (IL-1β) were measured both in vitro and in vivo. Beginning at concentrations of 100 μg ml^-1, UA started to dose-dependently reduce NKA activity. UA at a concentration of 100 μg ml^-1 time-dependently affected the NKA activity, with the maximal increased NKA activity at 24 h, but the activity started to decrease after 48 h. This inhibitory effect of UA on NKA activity at 48 h was in addition to a decrease in NKA α1 expression in the cell membrane, but an increase in lysosomes. This process also involved the subsequent activation of Src kinase and NLRP3, promoting IL-1β processing. In hyperuricemic rats, renal cortex NKA activity and its α1 expression were upregulated at the 7th week and both decreased at the 10th week, accompanied with increased renal cortex expression of Src, NLRP3 and IL-1β. The UA levels were reduced and renal tubular injuries in hyperuricemic rats were alleviated in the Feb group. Our data suggested that the impairment of NKA and its consequent regulation of Src, NLRP3 and IL-1β in the renal proximal tubule contributed to hyperuricemia-induced renal tubular injury.

Figure 1

UA dose- and time-dependently reduced intracellular ATP levels altered NKA activity and its α1 subunit cellular expression in human PTECs. PTECs were incubated with different concentrations of UA (25 to 200 μg ml⁻¹) for 48 h or different time courses (15 min to 48 h of 100 μg ml⁻¹ UA). UA dose- (A) and time-dependently (B) reduced intracellular ATP levels. UA 25 μg ml⁻¹ exhibited a tendency to increase NKA activity, but UA concentrations of 100 μg ml⁻¹ started to dose-dependently reduce the NKA activity (C). UA100 μg ml⁻¹ time-dependently affected the NKA activity, with the maximal increased NKA activity at 24 h that started to decrease until 48 h (D). Immunofluorescence showed that the expression of the α1 subunit of NKA (green) was linearly expressed on the cell surface, but when the cells were incubated with 100 μg ml⁻¹ UA for 48 h, NKA α1 membrane expression was reduced and scattered in the cytoplasm (E). Immunoblotting of cell surface proteins confirmed the changed cell membrane expression of the α1 subunit. Twenty-four hours of incubation of 100 μg ml⁻¹ UA increased the α1 subunit, but 48 h of incubation of UA reduced the α1 subunit expression on the cell surface of PTECs (F). *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs UA 24 h.

Figure 2

NKA α1 expression vector and siRNA altered its activity and subcellular expression. To overexpress or inhibit NKA α1 expression, the NKA α1 expression vector or siRNA was added to PTECs for 30 h before 48 h of UA 100 μg ml⁻¹ stimulation. The NKA α1 expression vector enhanced its activity and cell membrane expression, whereas its siRNA reduced its activity (A, B). We used Lyso-Tracker Red to label the lysosomes of cells (red) and then fixed the cells before proceeding to immunohistochemistry staining of NKA α1 (green). Confocal microscopy immunofluorescence showed that, under normal circumstances, NKA α1 was linearly expressed along the cell membrane (C–c). After adding UA, NKA α1 translocated into the cell plasma and were scattered into the lysosomes (C–f). The NKA α1expression vector increased its cell membrane expression and decreased its lysosome expression (C–i), whereas NKA α1 siRNA decreased its cell membrane expression and increased its lysosome expression (C–l). *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs UA, ##P<0.01 vs UA.

Figure 3

NKA α1 alleviated UA induced ROS production and autophagy in cultured human PTECs. The NKA α1 expression vector or siRNA was added to cells for 30 h to overexpress or inhibit NKA α1 before 48 h UA 100 μg ml⁻¹ stimulation. UA significantly increased ROS production (a, b), LDH release (c), early apoptosis (d, e) and induced autophagy, as indicated by reduced p62 (g) and an increased LC3II/LC3I ratio (h), beclin-1 (i) and LAMP2 (j) when compared with the control. The NKA α1 expression vector significantly alleviated ROS production (a, b) had a tendency to reduce early apoptosis (d, e), but had no effect on late apoptosis (d, f). The NKA α1 expression vector increased p62 expression (g) and reduced LAMP2 expression (j). The NKA α1 expression vector exerted a tendency to reduce the LC3II/LC3I ratio (h). *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs UA, ##P<0.01 vs UA.

Figure 4

NKA α1 alleviated UA induced mitochondrial dysfunction in cultured human PTECs. NKA α1 expression vector or siRNA was added to cells for 30 h to overexpress or inhibit NKA α1 before 48 h UA 100 μg ml⁻¹ stimulation. UA significantly reduced mitochondrial membrane potential (a, b), increased UCP2 expression (c), reduced mtDNA copies (d), and complex I (e) and V (I) activities. UA did not change complex II (f), III (g) and IV (h) activities. The NKA α1 expression vector alleviated mitochondrial dysfunction by increasing the mitochondrial membrane potential (a, b), reducing UCP2 expression (c), increasing mtDNA copies (d), as well as complex I (e) and V (i) activities. The NKA α1 expression vector exert no effect on complex II (f), III (g) and IV (h) activities. *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs UA, ##P<0.01 vs UA.

Figure 5

NKAα1 altered urate transporter expression in cultured human PTECs. NKA α1 expression vector or siRNA was added to cells for 30 h to overexpress or inhibit NKA α1 before 48 h of UA 100 μg ml⁻¹ stimulation. Except OAT3 (m), UA significantly increased the expressions of SLC5A8 (a), SLC5A12 (b), URAT1 (c), OAT10 (d), OAT4 (e), GLUT9 (f), MRP4 (g), ABCG2 (h), NPT1 (i), NPT4 (j), OAT2 (k), OAT1 (l) and SLC13A3 (n). NKA α1 expression vector significantly increased UARAT1 (c) and reduced MRP4 (g) and NPT4 (j) expressions, whereas it had no effect on other urate transporters. NKA α1 siRNA reduced SLC5A8 (a), UARAT1 (c) and OAT4 (e) expressions, wheresa it had no effect on other urate transporters. *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs UA, ##P<0.01 vs UA.

Figure 6

Src activation and NLRP3-IL-1β signaling in UA activated PTECs and hyperuricemic rats. UA (100 μg ml⁻¹, 48 h) stimulation significantly activated Src (indicated as phosphor-Src (pSrc)/Src, a), NLRP3 (b) and IL-1β (c), and reduced intracellular ATP (d). The NKA α1 expression vector or siRNA was added to PTECs for 30 h before 48 h UA 100 μg ml⁻¹ stimulation. NKA α1 siRNA significantly increased intracellular ATP level; however, it did not alleviate Src, NLRP3 and IL-1β activation induced by UA, whereas NKA α1 expression vector had no effect on intracellular ATP level, but significantly alleviated Src, NLRP3 and IL-1β activation induced by UA. (a–d) *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs UA, ##P<0.01 vs UA. Renal cortex expressions of Src, NLRP3 and IL-1β were detected by western blotting. Src (e), NLRP3 (f) and IL-1β (g) in OA-treated rats started to increase at week 7 and significantly higher than that of the control group at week 10. The expression of Src, NLRP3 and IL-1β were lower in the febuxostat group. Renal cortex ATP levels (h) were not different among Cont 10w, OA10w and Feb 10w groups. (e, f) *P<0.05 vs Cont, **P<0.01 vs Cont, #P<0.05 vs OA 10w.

Figure 7

Hyperuricemicrats developed microalbuminuria and slight mesangial expansion. OA gastric administration for 7 weeks showed a tendency of elevated UA levels and significantly increased serum UA level at the 10th week in SD rats (A). Urinary UA were not different among Cont 10w, OA 10w and Feb 10w groups (B). OA started to increase urinary albumin/creatinine ratio (ACR) at the 4th week and maintained this effect till the 10th week (C). Feb treatment showed a tendency of reducing urinary ACR in hyperuricemic rats (D). OA induced a slight increase in glomerular size and mesangial expansion at the 7th week and the changes were more significant at the 10th week (E). Feb treatment reduced serum UA levels and renal mesangial expansion in OA-administered rats.*P<0.05 vs Cont, **P<0.01 vs Cont, ##P<0.01 vs OA 10w.

Figure 8

Impairment of NKA in hyperuricemic rats. The activity of NKA in the renal cortex of rats was higher than in the control group at the 7th week, but was lower than that in the normal group at the 10th week. Feb treatment increased NKA activity compared with the OA group (A). The renal cortex expression of the NKA α1 subunit was correlated with its activity and the NKA α1 subunit expression showed an increased tendency in the OA versus the control group at the 7th week, but it was lower in the OA group than in the control group at the 10th week. Feb treatment increased NKA α1 subunit expression in OA administered rats (B). Immunohistochemistry showed that NKA α1subunit expression was expressed on the basolateral side of the proximal tubular epithelial cells and was higher in the OA 7w group than in the Cont 7w group, but NKA α1 subunit expression was lower in the OA 10w group than in the Cont 10w group (C). *P<0.05 vs Cont.