NQO1 Deficiency Aggravates Renal Injury by Dysregulating Vps34/ATG14L Complex during Autophagy Initiation in Diabetic Nephropathy

Abstract

Diabetic nephropathy (DN) is one of the causes of end-stage renal failure, featuring renal fibrosis. However, autophagy, a vital process for intracellular homeostasis, can counteract renal fibrosis. Moreover, NAD(P)H: quinone dehydrogenase 1 (NQO1) modulates the ratios of reduced/oxidized nicotinamide nucleotides, exerting a cytoprotective function. Here, to examine the role of NQO1 genes in DN progression, the levels of autophagy-related proteins and pro-fibrotic markers were assessed in silencing or overexpression of NQO1 in human proximal tubular cells (HK2), and C57BL/6 (wild-type) and Nqo1 knockout (KO) mice injected to streptozotocin (50 mg/kg). NQO1 deficiency impaired the autophagy process by suppressing basal expression of ClassⅢ PI 3-kinase (Vps34) and autophagy-related (ATG)14L and inducing the expressions of transforming growth factor beta (TGF-β1), Smad3, and matrix metallopeptidase9 (MMP9) in high-glucose (HG) -treated HK2 cells. Meanwhile, NQO1 overexpression increased the expression of Vps34 and ATG14L, while, reducing TGF-β1, Smad3 and MMP9 expression. In vivo, the expression of Vps34 and ATG14L were suppressed in Nqo1 KO mice indicating aggravated glomerular changes and interstitial fibrosis. Therefore, NQO1 deficiency dysregulated autophagy initiation in HK2 cells, with consequent worsened renal cell damage under HG condition. Moreover, STZ-treated Nqo1 KO mice showed that NQO1 deficiency aggravated renal fibrosis by dysregulating autophagy.

Keywords: NQO1; autophagy; diabetic nephropathy; renal injury.

Figure 1

Increased autophagy related protein and kidney damage accompanied by enhanced expression of NQO1 in high glucose treated HK2 cells. Cells were harvested 24 h after high glucose (10 mM, 25 mM) treatment, and total cell lysates were subjected to Western blotting. Band intensities of NQO1, NRF2 and KIM1 (a), were normalized to those of β-actin (b). Values are expressed as means ± SEM of triplicate experiments. ** p < 0.01, compared with control cells.

Figure 2

Autophagy inhibition increased renal fibrosis-related proteins in HG-treated HK2 cells. Cells were harvested 24 after high glucose (25 mM). Cell was treated with rapamycin (100 nM) for 24 h or 3-MA (5 mM) for 3 h before harvesting. Total cell lysates were subjected to western blotting. Conversion ratio of LC3Ⅱ/Ⅰ (b). The band intensities of p62, TGF-β1 were normalized to those of β-actin (a,c). Values are expressed as means ± SEM of triplicate experiments. ^# p < 0.01, compared with control cells, *** p < 0.001 and * p < 0.01, compared with HG cells. Cell nuclei were visualized by 6-diamino-2 phenylindole (DAPI; Blue) and the expression of collagen (red) and fibronectin (green) were visualized by Zeiss LSM 880 with Airyscan confocal microscopy at 400× magnification (scale bar = 20 μm) (d).

Figure 3

NQO1 silencing aggravated kidney damages and dysregulated autophagy-related proteins in HG-treated HK2 cells. After siControl or siNQO1 transfection, cells were incubated with vehicle of HG (25 mM) for 24 h. Total cell lysates were analyzed using western blot. Conversion ratio of LC3Ⅱ/Ⅰ (d). The expression intensities of kidney damages (a) and autophagy-related proteins (c) were normalized to those of β-actin (b,e). Values are expressed as means ± SEM of triplicate experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with wild-type. Cell nuclei were visualized by 6-diamino-2 phenylindole (DAPI; Blue) and the expressions of ATG14L, Vps34, LC3-Ⅱ, and p62 were visualized by AlexaFlour594 conjugate (Red) at 400× magnification (scale bar = 10 μm) (f).

Figure 4

NQO1 overexpression induces the autophagy and reduces kidney damages in HG-treated HK2 cells. After transfection with NQO1 overexpression plasmids (Strep-tag pEXPR-IBA 105-NQO1), all cells were incubated for 24 h. Total cell lysates were analyzed using western blot. The expression intensities of kidney damages (a) and autophagy-related proteins (c) were normalized to those of β-actin (b,d,e). Values are expressed as means ± SEM of triplicate experiments, * p < 0.05, *** p < 0.001 compared with wild-type.

Figure 5

Increased renal damage in Nqo1 KO mice in STZ-induced DN. C57BL/6 and Nqo1 KO mice were sacrificed 12 week after STZ treatment. (n = 5/group) Blood glucose level (mg/dl) (a), serum HbA1c levels (b). Histopathology determined via hematoxylin and eosin (H&E), period acid-Schiff stain (PAS) and Sirius red stain at 400× magnification (scale bar = 30μm) (c). The % area of fibrosis was quantified (d). The expressions of kidney damages related proteins (e) were normalized to those of β-actin (f). Values are expressed as means ± SEM of triplicate experiments. ** p < 0.01, *** p < 0.001 compared with wild-type. ^††† p < 0.001 compared with wild type control. ^‡‡‡ p < 0.001 compared with Nqo1 KO control.

Figure 6

NQO1 dysregulated autophagy initiation related proteins in STZ-induced DN. C57BL/6 and Nqo1 KO mice were sacrificed 12 week after STZ treatment. (n = 5/group) Homogenized tissues were analyzed using western blot. The expression intensities of autophagy-related proteins (a) were normalized to those of β-actin (b,c). Values are expressed as means ± SEM of triplicate experiments, *** p < 0.001 compared with wild-type.

Figure 7

Schematic representation of NQO1, ATG14L, Vps34, autophagy, and renal fibrosis in diabetic nephropathy. In diabetic nephropathy, under the stress, NQO1 is induced. In the absence of NQO1, autophagy initiation was dysregulated, resulting suppressing autophagy. In turn, ensuring of NQO1 pathway and autophagy flux reduces renal fibrosis.