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Nox4 is predominantly localized to mitochondria in mouse podocytes. A : mitochondria in podocytes labeled with mitochondria-targeted cyan fluorescence protein (mitoCFP; left ), rabbit isotype control IgG ( middle ), and the merged image ( right ). C : podocytes infected with mitoCFP adenovirus ( left ), labeled with Nox4 antibody ( middle ), and the merged image ( right ). E : podocytes stained with endogenous mitochondrial protein mitochondrial complex IV subunit I (Cox1; left ), Nox4 ( middle ), and the merged image ( right ). G : mouse podocytes transfected with D1ER plasmid ( left ), Nox4 ( middle ), and the merged image ( right ). B , D , F , and H : relative fluorescence intensity plots along the line in the merged images in A , C , E , and G , respectively. I : the Nox4 protein level was checked in mitochondrial and cytosolic fractions using Western blots. Antibodies against complex I and  -actin were used to check the purity of the mitochondrial and cytosolic fractions, respectively. J : Western blots showing the increase in the mitochondrial Nox4 protein level by TGF-  1 stimulation.
Source publication
Injury to podocyte leads to the onset of chronic renal diseases characterized by proteinuria. Elevated TGF-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through...
Contexts in source publication
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... colocalization, the relative intensities of the two fluorescent signals were plotted along a line drawn on the merged images. Colocalization of the two proteins was determined to be where the peaks of the two different intensities overlapped. mitoCFP was visualized in differentiated podocytes by an adenovirus-mediated overex- pression system (Fig. 3, A and C). Immunostaining of Nox4 using rabbit polyclonal antibody clearly colocalized with mitoCFP, as indicated by the presence of multiple yellow puncta in the merged image (Fig. 3, C and D). Endogenous mitochon- drial protein Cox1 labeled with mouse monoclonal Cox1 an- tibody colocalized with Nox4 (Fig. 3, E and F). In the presence ...
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... be where the peaks of the two different intensities overlapped. mitoCFP was visualized in differentiated podocytes by an adenovirus-mediated overex- pression system (Fig. 3, A and C). Immunostaining of Nox4 using rabbit polyclonal antibody clearly colocalized with mitoCFP, as indicated by the presence of multiple yellow puncta in the merged image (Fig. 3, C and D). Endogenous mitochon- drial protein Cox1 labeled with mouse monoclonal Cox1 an- tibody colocalized with Nox4 (Fig. 3, E and F). In the presence of rabbit isotype control IgG antibody, however, no signal was detected, indicating the specificity of Nox4 antibody (Fig. 3, A and B). We also considered the possibility that Nox4 might be ...
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... overex- pression system (Fig. 3, A and C). Immunostaining of Nox4 using rabbit polyclonal antibody clearly colocalized with mitoCFP, as indicated by the presence of multiple yellow puncta in the merged image (Fig. 3, C and D). Endogenous mitochon- drial protein Cox1 labeled with mouse monoclonal Cox1 an- tibody colocalized with Nox4 (Fig. 3, E and F). In the presence of rabbit isotype control IgG antibody, however, no signal was detected, indicating the specificity of Nox4 antibody (Fig. 3, A and B). We also considered the possibility that Nox4 might be localized in the ER of podocytes. To address that question, the ER was labeled by transfection of D1ER fluorescent protein, which ...
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... by the presence of multiple yellow puncta in the merged image (Fig. 3, C and D). Endogenous mitochon- drial protein Cox1 labeled with mouse monoclonal Cox1 an- tibody colocalized with Nox4 (Fig. 3, E and F). In the presence of rabbit isotype control IgG antibody, however, no signal was detected, indicating the specificity of Nox4 antibody (Fig. 3, A and B). We also considered the possibility that Nox4 might be localized in the ER of podocytes. To address that question, the ER was labeled by transfection of D1ER fluorescent protein, which is specifically targeted to the luminal side of the ER. As shown in Fig. 3, G and H, D1ER fluorescence did not colo- calize with Nox4, implying ...
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... however, no signal was detected, indicating the specificity of Nox4 antibody (Fig. 3, A and B). We also considered the possibility that Nox4 might be localized in the ER of podocytes. To address that question, the ER was labeled by transfection of D1ER fluorescent protein, which is specifically targeted to the luminal side of the ER. As shown in Fig. 3, G and H, D1ER fluorescence did not colo- calize with Nox4, implying that Nox4 was primarily localized to mitochondria, not the ER. In a different approach, podocytes were fractionated into mitochondrial and cytosolic fractions, and Nox4 protein levels were checked using Western blot analysis. Antibodies against complex I and -actin ...
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... mitochondrial and cytosolic fractions, and Nox4 protein levels were checked using Western blot analysis. Antibodies against complex I and -actin were used to check the purity of the mitochondrial and cytosolic fractions, respectively. Compared with the cytosolic fraction, the maxi- mum amount of Nox4 was detected in the mitochondrial fraction (Fig. 3I). The protein level of Nox4 in the mitochon- drial fraction was increased by TGF-1 (64.5%, n 3; Fig. 3J), which was consistent with changes of the total Nox4 protein ...
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... Antibodies against complex I and -actin were used to check the purity of the mitochondrial and cytosolic fractions, respectively. Compared with the cytosolic fraction, the maxi- mum amount of Nox4 was detected in the mitochondrial fraction (Fig. 3I). The protein level of Nox4 in the mitochon- drial fraction was increased by TGF-1 (64.5%, n 3; Fig. 3J), which was consistent with changes of the total Nox4 protein ...
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... adenovirus targeted to mitochondria and the endogenous mitochondrial marker pro- tein Cox1 of the mitochondrial electron transport chain com- plex IV, showed that Nox4 is localized to mitochondria. Our Western blot data also supported similar findings that revealed the presence of the majority of Nox4 protein in the mitochon- drial fraction (Fig. 3F). Mitochondrial Nox4 in podocyte is fully functional as TGF-1 treatment increased a substantial degree of Nox activity in the mitochondrial fraction, which was almost completely suppressed by a Nox inhibitor (Fig. 4B). It has been known that mitochondrial ROS precipitate in collapse of the mitochondrial transmembrane potential preceding ...
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Citations
... We, therefore, used TGF-β1 for all of the current in vitro experiments. Consistent with previous reports [26,27], TGF-β1 induced significant mitochondrial ROS production in a dose-dependent manner compared to control cells (Fig. 3a). Both preand post-treatment of cells with amifostine significantly reduced mitochondrial ROS levels (Fig. 3b). ...
Background
Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disease characterized by irreversible scarring of the lung parenchyma. Despite various interventions aimed at mitigating several different molecular aspects of the disease, only two drugs with limited clinical efficacy have so far been approved for IPF therapy.
Objective
We investigated the therapeutic efficacy of amifostine, a detoxifying drug clinically used for radiation-caused cytotoxicity, in bleomycin-induced murine pulmonary fibrosis.
Methods
C57BL6/J mice were intratracheally instilled with 3 U/kg of bleomycin. Three doses of amifostine (WR-2721, 200 mg/kg) were administered intraperitoneally on days 1, 3, and 5 after the bleomycin challenge. Bronchoalveolar lavage fluid (BALF) was collected on day 7 and day 21 for the assessment of lung inflammation, metabolites, and fibrotic injury. Human fibroblasts were treated in vitro with transforming growth factor beta 1 (TGF-β1), followed by amifostine (WR-1065, 1–4 µg/mL) treatment. The effects of TGF-β1 and amifostine on the mitochondrial production of reactive oxygen species (ROS) were assessed by live cell imaging of MitoSOX. Cellular metabolism was assessed by the extracellular acidification rate (ECAR), the oxygen consumption rate (OCR), and the concentrations of various energy-related metabolites as measured by mass spectrum (MS). Western blot analysis was performed to investigate the effect of amifostine on sirtuin 1 (SIRT1) and adenosine monophosphate activated kinase (AMPK).
Results
Three doses of amifostine significantly attenuated lung inflammation and pulmonary fibrosis. Pretreatment and post-treatment of human fibroblast cells with amifostine blocked TGF-β1-induced mitochondrial ROS production and mitochondrial dysfunction in human fibroblast cells. Further, treatment of fibroblasts with TGF-β1 shifted energy metabolism away from mitochondrial oxidative phosphorylation (OXPHOS) and towards glycolysis, as observed by an altered metabolite profile including a decreased ratio of NAD + /NADH and increased lactate concentration. Treatment with amifostine significantly restored energy metabolism and activated SIRT1, which in turn activated AMPK. The activation of AMPK was required to mediate the effects of amifostine on mitochondrial homeostasis and pulmonary fibrosis. This study provides evidence that repurposing of the clinically used drug amifostine may have therapeutic applications for IPF treatment.
Conclusion
Amifostine inhibits bleomycin-induced pulmonary fibrosis by restoring mitochondrial function and cellular metabolism.
... This organelle, when damaged, leads to the release of pro-apoptotic factors, including cytochrome c, which in turn, can activate caspase-9 and initiate the intrinsic pathway of apoptosis. 21,39,40 Consequently, in this study the increase in NOX4 expression found in BD adolescent rats leads to an increase in caspase-9 expression. ...
Binge drinking (BD) is an especially pro‐oxidant pattern of alcohol consumption, particularly widespread in the adolescent population. In the kidneys, it affects the glomerular filtration rate (GFR), leading to high blood pressure. BD exposure also disrupts folic acid (FA) homeostasis and its antioxidant properties. The aim of this study is to test a FA supplementation as an effective therapy against the oxidative, nitrosative, and apoptotic damage as well as the renal function alteration occurred after BD in adolescence. Four groups of adolescent rats were used: control, BD (exposed to intraperitoneal alcohol), control FA‐supplemented group and BD FA‐supplemented group. Dietary FA content in control groups was 2 ppm, and 8 ppm in supplemented groups. BD provoked an oxidative imbalance in the kidneys by dysregulating antioxidant enzymes and increasing the enzyme NADPH oxidase 4 (NOX4), which led to an increase in caspase‐9. BD also altered the renal nitrosative status affecting the expression of the three nitric oxide (NO) synthase (NOS) isoforms, leading to a decrease in NO levels. Functionally, BD produced a hydric‐electrolytic imbalance, a low GFR and an increase in blood pressure. FA supplementation to BD adolescent rats improved the oxidative, nitrosative, and apoptotic balance, recovering the hydric‐electrolytic equilibrium and blood pressure. However, neither NO levels nor GFR were recovered, showing in this study for the first time that NO availability in the kidneys plays a crucial role in GFR regulation that the antioxidant effects of FA cannot repair.
... [68][69][70][71] Previous studies have reported that under high-glucose conditions, the TGF-β signaling pathway is active and can be involved in podocyte apoptosis by mediating the Smad signaling pathway. Das et al. 72 found that TGF-β1 selectively upregulated the transcription of Nox4 mRNA by Smad2/3, leading to elevated mitochondrial Nox4 protein levels, oxidative stress, mitochondrial dysfunction, and podocyte apoptosis. Another study also found that TGF-β1 increased the translation of Nox4 through activation of the Smad-ERK1/2-mTORC1 pathway, increasing reactive oxygen species (ROS) production and mitochondrial dysfunction, leading to apoptosis in foot cells. ...
Podocytes, the key component of the glomerular filtration barrier (GFB), are gradually lost during the progression of diabetic kidney disease (DKD), severely compromising kidney functionality. The molecular mechanisms regulating the survival of podocytes in DKD are incompletely understood. Here, we show that membrane‐associated guanylate kinase inverted 2 (MAGI2) is specifically expressed in renal podocytes, and promotes podocyte survival in DKD. We found that MAGI2 expression was downregulated in podocytes cultured with high‐glucose in vitro, and in kidneys of db/db mice as well as DKD patients. Conversely, we found enforced expression of MAGI2 via AAV transduction protected podocytes from apoptosis, with concomitant improvement of renal functions. Mechanistically, we found that MAGI2 deficiency induced by high glucose levels activates TGF‐β signaling to decrease the expression of anti‐apoptotic proteins. These results indicate that MAGI2 protects podocytes from cell death, and can be harnessed therapeutically to improve renal function in diabetic kidney disease.
... Within podocytes, mtROS are mostly formed by the mitochondrial respiration chain and NOXs (Galvan et al., 2017;Gao et al., 2020;Yu et al., 2014). In the case of DN, mitochondrial NOX4 in podocytes is the main inducer of mitochondrial dysfunction and podocyte apoptosis (Das et al., 2014). ...
... which generates moreROS (Abe et al., 2013). The TGF-β1 induces proteinuric glomerular diseases including DN by the TGF-β receptor/ Smad2/3 pathway that upregulates mitochondrial NADPH oxidase 4 (NOX4)(Das et al., 2014). Mitochondrial dysfunction mediated by Smad3/NOX4 axis enhances ROS products and activates intrinsic apoptosis (cytochrome c and caspases 3 and 9) in podocytes (Yu et al.from some substrates and regulate apoptosis, cell cycle, and immune responses(Mathis et al., 2015). ...
Podocytes are terminally differentiated kidney cells acting as the main gatekeepers of the glomerular filtration barrier; hence, inhibiting proteinuria. Podocytopathies are classified as kidney diseases caused by podocyte damage. Different genetic and environmental risk factors can cause podocyte damage and death. Recent evidence shows that mitochondrial dysfunction also contributes to podocyte damage. Understanding alterations in mitochondrial metabolism and function in podocytopathies and whether altered mitochondrial homeostasis/dynamics is a cause or effect of podocyte damage are issues that need in-depth studies. This review highlights the roles of mitochondria and their bioenergetics in podocytes. Then, factors/signalings that regulate mitochondria in podocytes are discussed. After that, the role of mitochondrial dysfunction is reviewed in podocyte injury and the development of different podocytopathies. Finally, the mitochondrial therapeutic targets are considered.
... NaB was further able to counteract the increases in PKC observed when these cells were treated using TPA, which is a PKC agonist. The NOX4 oxidase functions downstream of PKC to generate H 2 O 2 directly without the need for additional activation steps, unlike other NOX family Excess oxidase activity can drive ROS production [60], altered mitochondrial membrane potential, reduced SOD activity, higher MDA levels, and the overall disruption of normal free radical scavenging and mitochondrial dysfunction [61]. NF-κB serves as a key transcription factor that regulates inflammatory activity, immune response induction, and apoptosis in the context of organismal stress responses [62]. ...
Background
Elevated levels of oxidative stress as a consequence of estrogen deficiency serve as a key driver of the onset of osteoporosis (OP). In addition to increasing the risk of bone fractures, OP can reduce the bone volume proximal to titanium nails implanted to treat these osteoporotic fractures, thereby contributing to titanium nail loosening. Sodium butyrate (NaB) is a short-chain fatty acid produced by members of the gut microbiota that exhibits robust antioxidant and anti-inflammatory properties.
Methods
OP fracture model rats parameters including bone mineral density (BMD), new bone formation, and the number of bonelets around the implanted nail were analyzed via micro-CT scans, H&E staining, and Masson’s staining. The protective effects of NaB on such osseointegration and the underlying mechanisms were further studied in vitro using MC3T3-E1 cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce oxidative stress. Techniques including Western immunoblotting, electron microscopy, flow cytometry, alkaline phosphatase (ALP) staining, and osteoblast mineralization assays were employed to probe behaviors such as reactive oxygen species production, mineralization activity, ALP activity, protein expression, and the ability of cells to attach to and survive on titanium plates.
Results
NaB treatment was found to enhance ALP activity, mineralization capacity, and Coll-I, BMP2, and OCN expression levels in CCCP-treated MC3T3-E1 cells, while also suppressing PKC and NF-κB expression and enhancing Nrf2 and HO-1 expression in these cells. NaB further suppressed intracellular ROS production and malondialdehyde levels within the cytosol while enhancing superoxide dismutase activity and lowering the apoptotic death rate. In line with these results, in vivo work revealed an increase in BMD in NaB-treated rats that was associated with enhanced bone formation surrounding titanium nails.
Conclusion
These findings indicate that NaB may represent a valuable compound that can be postoperatively administered to aid in treating OP fractures through the enhancement of titanium nail osseointegration.
... Liang et al. 40 identified that salvianolate could prevent glucose-induced oxidative injury of podocytes through modulation of NOX4 activity in DN mice. Das et al. 41 suggested that TGF-b1 activates NOX4 in mouse podocyte through Smad2/3 pathways; knockdown of either Smad2 or Smad3 attenuates NOX4-induced podocyte apoptosis in DN. Furthermore, activation of mammalian target of rapamycin (mTOR) 1 initiates NOX4-dependent podocyte injury in diabetic renal injury. ...
Introduction
Podocyte apoptosis is a common mechanism driving progression in Alport syndrome(AS). The aim of this study was to investigate the mechanism of podocyte apoptosis caused by COL4A3 mutations.
Methods
We recruited autosomal dominant AS(ADAS) patients. Patients with minimal change disease (MCD) were recruited as controls. Microarray analysis was carried out on isolated glomeruli from the patients and validated. Then, corresponding mutant human podocytes(p.C1616Y) and 129 mice(p.C1615Y, the murine homolog to the human p.C1616Y) were constructed. The highest differentially expressed genes (DEGs) from microarray analysis were validated in transgenic mice and podocytes before and after admnistration of MMP-2 inhibitor(SB-3CT), and NOX4 inhibitor(GKT137831). We further validated NOX4/MMP-2/apoptosis pathway by real-time PCR, immunohistochemistry and western blot in renal tissue from the ADAS patients.
Results
Using microarray analysis, we observed that DEGs including NOX4/H2O2, MMP-2 and podocyte apoptosis-related genes were significantly upregulated. These genes were validated by real-time PCR, histologic analysis and western blot in corresponding mutant human podocyte (p.C1616Y) and/or mice models (p.C1615Y). Moreover, we found podocyte apoptosis was abrogated and MMP-2 expression was downregulated both in vivo and in vitro by NOX4 inhibition, urinary albumin-to-creatinine ratio, 24 hour proteinuria and renal pathologic lesion was attenuated by NOX4 inhibition in vivo; Furthermore, podocyte apoptosis was attenuated while NOX4 expression remained the same by inhibition of MMP-2 both in vivo and in vitro.
Conclusion
These results indicate that NOX4 might induce podocyte apoptosis through the regulation of MMP-2 in patients with COL4A3 mutations. Our findings provided new insights into the mechanism of ADAS.
... 6 The level of transforming growth factor-β1 (TGF-β1) in kidney tissue is elevated in diabetic nephropathy and other chronic kidney diseases. 7 TGF-β-driven fibrosis is mediated by Smaddependent or Smad-independent pathways. 8 Original Article deposition and prevent tubulointerstitial fibrosis. ...
Objective:
To observe the efficacy of Danggui Buxue decoction (, DBD) on diabetic nephropathy-induced renal fibrosis in rats, and to study the possible mechanism.
Methods:
Sixty male Goto Kakizaki (GK) rats were randomly assigned to the model group, gliquidone group, astragaloside IV group, and high-, medium- and low-doses DBD groups. After 8 weeks, changes in body weight, blood glucose, serum creatinine, serum urea nitrogen, and total cholesterol were observed. Changes in transforming growth factor-β1 (TGF-β1), Smad3, and Smad5 pathways and the expression of the fibrosis-related proteins collagen IV (col IV), α-smooth muscle actin (α-SMA), and vimentin were assessed. The degree of renal fibrosis was observed by immunohistochemistry and Mason staining. The expression of interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor (TNF-α), and C-reactive protein (CRP) in the kidneys was assessed using enzyme linked immunosorbent assay.
Results:
Our experiments showed that DBD effectively reduced blood glucose, blood urea nitrogen, and creatinine levels after 8 weeks of administration, improved renal function in diabetic rats, alleviated renal fibrosis, and reduced the renal tissue levels of IL-6, IL-10, TNF-α, and CRP. Furthermore, DBD decreased the expression of TGF-β1, Smad3, col IV, α-SMA, and vimentin in renal tissues and increased the expression of Smad5.
Conclusions:
DBD ameliorates diabetic renal interstitial fibrosis by modulating the TGF-β1/Smads pathway.
... The above evidence strongly suggests that the Smad3/ezrin/PKA/Nox4 pathway may be one of the potential molecular pathways of podocyte apoptosis caused by high glucose, and the molecules are inter-regulative. It has also been demonstrated that Nox4 is involved in TGF-β1-triggered podocyte apoptosis in mice, accompanied by increased ROS production and abnormal activation of caspase-3, while knockdown of Smad2 or Smad3 could block the TGF-β1-induced upregulation in Nox4 expression, ROS production, and abnormal activation of caspase-3 [67]. This study further suggests that the TGF-β receptor-Smad2/3 pathway may serve as a novel pathway for TGF-β1-induced podocyte Nox4 upregulation and podocyte apoptosis. ...
Diabetic vascular complications can affect both microvascular and macrovascular. Diabetic microvascular complications, such as diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and diabetic cardiomyopathy, are believed to be caused by oxidative stress. The Nox family of NADPH oxidases is a significant source of reactive oxygen species and plays a crucial role in regulating redox signaling, particularly in response to high glucose and diabetes mellitus. This review aims to provide an overview of the current knowledge about the role of Nox4 and its regulatory mechanisms in diabetic microangiopathies. Especially, the latest novel advances in the upregulation of Nox4 that aggravate various cell types within diabetic kidney disease will be highlighted. Interestingly, this review also presents the mechanisms by which Nox4 regulates diabetic microangiopathy from novel perspectives such as epigenetics. Besides, we emphasize Nox4 as a therapeutic target for treating microvascular complications of diabetes and summarize drugs, inhibitors, and dietary components targeting Nox4 as important therapeutic measures in preventing and treating diabetic microangiopathy. Additionally, this review also sums up the evidence related to Nox4 and diabetic macroangiopathy.
... TGF-β1 can also induce a fibrotic response through indirect mechanisms (non-Smad-based). On the other hand, TGF-β1 can induce the apoptosis of endothelial cells and podocytes [27], promoting glomerular and interstitial fibrosis. In addition, TGF-β1 is a potent inducer of a mesenchymal gene expression program that induces the transition of epithelial cells, endothelial cells, and intrinsic renal fibroblasts into α-smooth muscle actin (SMA)-expressing myofibroblasts [28]. ...
Arterial hypertension (AH) is a global challenge that greatly impacts cardiovascular morbidity and mortality worldwide. AH is a major risk factor for the development and progression of kidney disease. Several antihypertensive treatment options are already available to counteract the progression of kidney disease. Despite the implementation of the clinical use of renin–angiotensin aldosterone system (RAAS) inhibitors, gliflozins, endothelin receptor antagonists, and their combination, the kidney damage associated with AH is far from being resolved. Fortunately, recent studies on the molecular mechanisms of AH-induced kidney damage have identified novel potential therapeutic targets. Several pathophysiologic pathways have been shown to play a key role in AH-induced kidney damage, including inappropriate tissue activation of the RAAS and immunity system, leading to oxidative stress and inflammation. Moreover, the intracellular effects of increased uric acid and cell phenotype transition showed their link with changes in kidney structure in the early phase of AH. Emerging therapies targeting novel disease mechanisms could provide powerful approaches for hypertensive nephropathy management in the future. In this review, we would like to focus on the interactions of pathways linking the molecular consequences of AH to kidney damage, suggesting how old and new therapies could aim to protect the kidney.
... Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is a constitutive enzyme expressed in the kidney and primarily contributes to the growth in reactive oxygen species (ROS) [6], and exerts a pivotal part in the modulation of oxidative stress and downstream signals [7][8][9][10]. Growing evidence showed that NOX4 plays a role in the pathogenesis of various kidney diseases, such as diabetic nephropathy [11], hypertensive nephropathy [12], obstructive nephropathy [13], as well as AKI induced by ischemia/reperfusion injury and cisplatin [14][15][16][17][18]. Recent research suggested NOX4 was regulated by SH3YL1 in generating ROS in LPS-induced AKI model [19]. Our previous study demonstrated that over-expression of NOX4 in renal tubular epithelial cells (RTECs) in LPS-induced AKI was ameliorated after Maresin 1 administration [20], while the therapeutic potential and regulatory mechanism of targeting NOX4 in SAKI remain largely unknown. ...
Rationale: Sepsis is a severe clinical syndrome featured through organ dysfunction due to infection, while the accompanying acute kidney injury (AKI) is linked to significant incidence of morbidity as well as mortality. Recently, emerging evidence has revealed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is implicated in various renal diseases, while its role and modulation in septic acute kidney injury (S-AKI) remains largely unknown.
Methods: In vivo, S-AKI in wild-type and renal tubular epithelial cell (RTEC)-specific NOX4 knockout mice was induced by lipopolysaccharides (LPS) injection or cecal ligation and puncture (CLP). In vitro, TCMK-1 (mouse kidney tubular epithelium cell line) cells were treated with LPS. Serum and supernatant biochemical, mitochondrial dysfunctional, inflammatory and apoptotic parameters were measured and compared across groups. The activation of reactive oxygen species (ROS) and NF-κB signaling was also assessed.
Results: NOX4 was predominantly upregulated in RTECs of S-AKI mouse model induced by LPS/CLP and cultured TCMK-1 cells exposed to LPS. RTEC-specific deletion of NOX4 or pharmacological inhibition of NOX4 by GKT137831 both alleviated LPS/CLP-injured renal function and pathology in mice. Furthermore, NOX4 inhibition alleviated mitochondrial dysfunction supported by ultrastructural damage, reduction of ATP production and mitochondrial dynamics imbalance, together with inflammation and apoptosis in kidney injured by LPS/CLP and TCMK-1 cells injured by LPS, while NOX4 overexpression aggravated the above-mentioned indices in TCMK-1 cells with LPS stimulation. Mechanism-wise, the raised NOX4 in RTECs may induce ROS and NF-κB signaling activation in S-AKI.
Conclusions: Collectively, genetic or pharmacological inhibition of NOX4 protects from S-AKI by reducing generation of ROS and activation of NF-κB signal, which suppress mitochondrial dysfunction, inflammation together with apoptosis. NOX4 may act as a novel target for the S-AKI therapy.
