Tubular von Hippel-Lindau Knockout Protects against Rhabdomyolysis-Induced AKI

Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA–based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.No specific therapy is currently available for human AKI, a clinical entity of increasing incidence and high morbidity and mortality.^1–4 Rhabdomyolysis, one of the leading causes of AKI, develops after trauma, drug toxicity, infections, burns, and physical exertion.^5–8 The animal model using an intramuscular glycerol injection with consequent myoglobinuria is closely related to the human syndrome of rhabdomyolysis.⁹ Experimental data demonstrate renal vasoconstriction,^9–15 tubular hypoxia,^15,16 normal or even reduced intratubular pressure,^9–11 as well as large variation in single nephron GFR.^10,11 Intratubular myoglobin casts, a histologic hallmark, seem not to cause tubular obstruction,^9–11 but rather scavenge nitric oxide^17,18 and generate reactive oxygen species¹⁹ followed by vasoconstriction.The traditional discrimination between ischemic and toxic forms of AKI has been challenged because an increasing amount of evidence suggests that renal hypoxia is a common denominator in AKI of different etiologies.²⁰ Pimonidazole adducts, which accumulate in tissues at oxygen tensions <10 mmHg,²¹ have been demonstrated in various AKI forms.^16,22–24 During AKI, hypoxia-inducible factors (HIFs), which are mainly regulated by oxygen-dependent proteolysis, were found to be upregulated in different renal tubular segments.^{16,20,22,24,25} HIFs are heterodimers of a constitutive β subunit, HIF-β (ARNT), and one of three oxygen-dependent α-subunits, HIF-1α, HIF-2α, and HIF-3α. The α-β dimers bind to hypoxia-response elements (HREs) in the promoter-enhancer region of HIF target genes.^26–28 Although the 5′-RCGTG-3′ (R = A or G) core HRE appears >1 million times in the entire genome²⁹ and in >4000 promoter regions of validated genes,³⁰ a recent study demonstrated HIF binding in roughly 350 genes.³¹ Multiple HIF-based biologic effects are known, and it is widely accepted that a broad panel of these promote cellular survival in a hostile and oxygen-deprived environment.^27–29 In all types of AKI tested thus far, HIF activation along the nephron correlates with tubular survival, and the cells most vulnerable to injury exhibit no or only very limited HIF activity.²⁰ This observation led to the concept of insufficient HIF-based hypoxic adaptation in AKI. Consequently, maneuvers of preconditional HIF activation are utilized to ameliorate AKI. Indeed, many of these attempts are successful but the majority are conducted in ischemia-reperfusion injury.²⁰ It is largely unclear whether HIF can rescue kidneys exposed to AKI forms other than ischemia-reperfusion injury, and it is unclear which HIF target genes are involved in AKI protection if so. In many tumors, constitutive HIF activation promotes anaerobic ATP production, a process known as the Warburg effect.³²von Hippel-Lindau protein (VHL) is a ubiquitin ligase engaged in the stepwise HIF-α degradation process, which constantly occurs during normoxia.³³ Inducible Pax8-rtTA–based knockout of VHL (VHL-KO) achieves strong, selective, and persistent upregulation of HIF in all nephron segments.³⁴ In this study, we use this transgenic technique in conjunction with rhabdomyolysis in mice to address two issues: (1) Does HIF activation through VHL-KO protect from rhabdomyolysis-induced AKI? (2) If so, what are the biologic mechanisms and HIF target genes that are responsible for renal protection against acute injury? We demonstrate that indeed VHL-KO mice are largely protected against rhabdomyolysis-induced AKI, and provide evidence for a metabolic shift toward anaerobic ATP generation as the central protective mechanism.     

Renalase Prevents AKI Independent of Amine Oxidase Activity

AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide–induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20–amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI.AKI is a common clinical condition affecting up to 20% of hospitalized patients and is frequently associated with sepsis, surgery, and certain drugs. Epidemiologic data indicate a positive association between the severity of AKI and in-hospital and long-term mortality.^1,2 Unfortunately, the development of effective therapy for AKI has been hampered by (1) an inherent delay in diagnosis, a consequence of relying on serum creatinine, which only increases 48–72 hours after the original insult, and (2) the paucity of validated targets of therapy. There is an urgent need to identify novel therapeutic modalities.Renalase is a novel secretory flavoprotein with amine oxidase activity.^3–5 In vitro, renalase metabolizes epinephrine, norepinephrine, and dopamine and also possesses significant intrinsic nicotinamide adenine dinucleotide (NADH) oxidase activity.^6,7 Other investigators have questioned the amine oxidase activity of renalase.^8,9 We had proposed that, in contrast to the classic amine oxidases, renalase reacts with oxygen to generate superoxide anions and hydrogen peroxide, with subsequent oxidation of catecholamines to their respective aminochromes. Recent results indicate that renalase functions as an oxidase/anomerase, using molecular oxygen to convert α-NAD(P)H to β-NAD⁺, with hydrogen peroxide as reaction byproduct.¹⁰ Because it was also shown to bind epinephrine, the authors pointed out that the hydrogen peroxide (H₂O₂) generated from the anomerase reaction will drive the oxidation of epinephrine to adrenochrome, albeit at a slower rate. Single-nucleotide polymorphisms present in the gene are associated with hypertension, cardiac disease, and diabetes.^6,11–14 The administration of renalase in wild-type (WT) mice lowers plasma catecholamines and systemic BP. In contrast, the deficiency of renalase in renalase knockout (KO) mice raises catecholamine levels and BP.¹⁵ Renalase also modulates the severity of renal ischemia and reperfusion injury.¹⁶ In WT mice, the administration of recombinant human renalase before induction of renal ischemia significantly blunts the severity of renal injury, with less renal tubular necrosis, inflammation, and apoptosis. In contrast, the lack of renalase in renalase KO mice exacerbates the renal damage after similar ischemic injury.AKI is characterized by an elevation in plasma catecholamine levels. It has been postulated that, in addition to causing hypertension, excess catecholamines in AKI may produce an inflammatory response, aggravating tissue damage and contributing to multiorgan dysfunction.¹⁷ Interestingly, renalase levels in the blood and kidneys of WT mice are reduced following acute renal ischemia. Because the renalase in blood is secreted from the kidneys and is thought to metabolize circulating catecholamines, the excess catecholamines in AKI may be a direct consequence of the concurrent renalase deficiency. Notably, the administration of renalase to WT mice before induction of ischemia, which greatly attenuates ischemic renal injury, dampens the rise in blood catecholamine levels. On the basis of these findings, it is inviting to speculate that the renal protective effect in ischemic injury of renalase and its hemodynamic effect stem from its amine oxidase activity.The role of amine oxidase activity of renalase in mediating its hemodynamic effect has been questioned. First, measurement of the amine oxidase activity of renalase relies on the production of H₂O₂ as an indirect measure of oxidase activity. Because the measured rate of H₂O₂ synthesis is low, the putative oxidase activity of renalase has been deemed unlikely to have physiologic significance.⁹ Second, the recombinant renalase synthesized in Escherichia coli with a histidine-tag possesses no detectable oxidase activity, and yet it markedly lowers BP when injected into rats.^8,18In this study, we sought to clarify the role of amine oxidase activity in the renal protective effects of renalase and to explore an additional mechanism of action of renalase that is independent of its oxidase activity.     

Remifentanil Preconditioning Protects against Ischemic Injury in the Intact Rat Heart

Background: Opioid receptors mediate cardiac ischemic preconditioning. Remifentanil is a new, potent ultra-short-acting phenylpiperidine opioid used in high doses for anesthesia. The authors hypothesize that pretreatment with this drug confers cardioprotection.

Methods: Male Sprague-Dawley rats were anesthetized and the chest was opened. All animals were subjected to 30 min of occlusion of the left coronary artery and 2 h of reperfusion. Before the 30-min occlusion, rats received either preconditioning by ischemia (ischemic preconditioning, 5-min occlusion, 5-min reperfusion x 3) or pretreatment with remifentanil, performed with the same regime (3 x 5-min infusions) using 0.2, 0.6, 2, 6, or 20 [mu]g[middle dot]kg-1[middle dot]min-1 intravenously. The experiment was repeated with naltrindole (a selective [DELTA]-opioid receptor antagonist, 5 mg/kg), nor-binaltorphimine (a selective [kappa]-OR antagonist, 5 mg/kg), or CTOP (a selective [mu]-opioid receptor antagonist, 1 mg/kg) administered before remifentanil-induced preconditioning or ischemic preconditioning, respectively. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining.

Results: There was a dose-related reduction in infarct size/area at risk after treatment with remifentanil that was similar to that seen with ischemic preconditioning. This effect was prevented or significantly attenuated by coadministration of a [mu], [kappa], or [DELTA]-opioid antagonist. The infarct-sparing effect of ischemic preconditioning was abolished by blockade of [kappa]-opioid receptors or [DELTA]-opioid receptors but not by [mu]-opioid receptors.     

Oxypurinol Protects Normothermic Ischemic Hearts

Rabbit hearts were mounted on a Langendorff apparatus and after measurement of baseline hemodynamic function exposed to 30 minutes normothermic arrest. Hearts were reperfused at 37 degrees C with buffer solution containing oxypurinol in different concentration: group II (0.01 mM), group III (0.1 mM), group IV (1 mM). Group I did not receive active drug and served as control. Each group consisted of eight hearts. After reperfusion, hemodynamic function was again measured and compared to baseline. Groups III and IV showed significantly less deterioration (p less than 0.05) than the control, while group II was better than the control, but in general differences were nonsignificant. We conclude that oxypurinol ameliorates ischemic cardiac damage following normothermic cardiac arrest. The beneficial effect of oxypurinol is most likely due to the drug's scavenging effect of oxygen-free radicals.     

缺血后处理对老年大鼠再灌注心肌的保护作用及其与P-Akt的相关性

目的 探讨缺血后处理对老年大鼠离体心脏缺血-再灌注损伤的影响及其与P-Akt的相关性。 方法21～23个月龄 （老年鼠） 健康SD大鼠30只,雌雄不拘,体重450～500 g,分为空白对照组（N组）、缺血-再灌注组（IR组）和缺血后处理组 （Post组） 3组,每组10只,观测心脏冠状动脉流量、心肌梗死范围、磷酸化的蛋白激酶B （P-Akt）表达、心肌和线粒体的改变。 结果 Post组较IR组冠状动脉流量明显增加 〔（6.4±1.2） ml/min vs. （3.1±1.2） ml/min,P＜0. 01〕,心肌梗死范围明显减少（35.0%±2.0% vs. 55.7%±3.6%）,P-Akt的表达明显增强,心肌纤维和线粒体的完整程度明显较好。 结论 缺血后处理对老年大鼠离体心脏具有显著的保护作用,这在一定程度与P-Akt激活有关。     

Delayed Ischemic Preconditioning Protects Against Liver Ischemia-Reperfusion Injury In Vivo

Objectives

Ischemic preconditioning (IP) affords resistance to liver ischemia-reperfusion (IR) injury, providing an early phase of protection. Development of delayed IP against IR injury was assessed using partial IR in rat liver.

Methods

The IP manuver (10 minutes of ischemia and up to 72 hours of reperfusion) was induced before 1 hour of ischemia and 20 hours of reperfusion. At the end of the reperfusion period, blood and liver samples were analyzed for serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), haptoglobin and tumor necrosis factor-α (TNF-α) levels, hepatic histology, protein carbonyl and glutathione (GSH) contents as well as nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) DNA binding.

Results

The IP manuver significantly increased protein carbonyl/GSH ratios (275%), serum ALT (42%), and AST (58%); these changes normalized after 12 hours. Serum AST, ALT, and LDH levels were significantly increased by IR (4-, 5.6-, and 7.0-fold, respectively), with significant changes in liver histology, protein carbonyl/GSH ratio (481% enhancement), and serum TNF-α (6.1-fold increase). Delayed IP in IR animals reduced serum AST (66%), ALT (57%), and LDH (90%) and liver GSH depletion (89%), with normalization of protein carbonyl content, serum TNF-α levels, and liver histology. Enhanced AP-1/NF-κB DNA binding ratios and diminished haptoglobin expression induced by IR were normalized by IP.

Conclusion

These data support that delayed IP suppresses IR-induced liver injury, oxidative stress, and TNF-α response, which coincide with recovery of IR-altered signaling functions represented by normal AP-1/NF-κB DNA binding ratios and acute phase responses.     

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP^−/−, and TxNIP^+/− mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP^−/− mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP^−/− mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms’ tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP^−/− mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O₂⁻ generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.     

Fetuin-A Protects against Atherosclerotic Calcification in CKD

Reduced serum levels of the calcification inhibitor fetuin-A associate with increased cardiovascular mortality in dialysis patients. Fetuin-A–deficient mice display calcification of various tissues but notably not of the vasculature. This absence of vascular calcification may result from the protection of an intact endothelium, which becomes severely compromised in the setting of atherosclerosis. To test this hypothesis, we generated fetuin-A/apolipoprotein E (ApoE)-deficient mice and compared them with ApoE-deficient and wild-type mice with regard to atheroma formation and extraosseous calcification. We assigned mice to three treatment groups for 9 wk: (1) Standard diet, (2) high-phosphate diet, or (3) unilateral nephrectomy (causing chronic kidney disease [CKD]) plus high-phosphate diet. Serum urea, phosphate, and parathyroid hormone levels were similar in all genotypes after the interventions. Fetuin-A deficiency did not affect the extent of aortic lipid deposition, neointima formation, and coronary sclerosis observed with ApoE deficiency, but the combination of fetuin-A deficiency, hyperphosphatemia, and CKD led to a 15-fold increase in vascular calcification in this model of atherosclerosis. Fetuin-A deficiency almost exclusively promoted intimal rather than medial calcification of atheromatous lesions. High-phosphate diet and CKD also led to an increase in valvular calcification and aorta-associated apoptosis, with wild-type mice having the least, ApoE-deficient mice intermediate, and fetuin-A/ApoE-deficient mice the most. In addition, the combination of fetuin-A deficiency, high-phosphate diet, and CKD in ApoE-deficient mice greatly enhanced myocardial calcification, whereas the absence of fetuin-A did not affect the incidence of renal calcification. In conclusion, fetuin-A inhibits pathologic calcification in both the soft tissue and vasculature, even in the setting of atherosclerosis.Hemodialysis (HD) patients experience a cardiovascular mortality of up to 20% per year, and vascular calcification is a strong independent risk factor of cardiovascular death.^1,2 Pathologic calcification is driven both by an elevated serum calcium phosphate product and by differentiation of vascular or mesenchymal cells into osteoblast-like cells, becoming mineralization competent.Serum is a metastable solution with respect to calcium phosphate precipitation. Once started, calcification proceeds rapidly in the presence of calcifiable templates such as collagen, elastin, and cell debris.^3–5 Fetuin-A accounts for approximately 50% of the capacity of serum to inhibit the spontaneous apatite formation from solutions supersaturated in calcium and phosphate.⁶ The inhibition is achieved by rapid formation of soluble colloidal fetuin-A calcium phosphate complexes, termed calciprotein particles (CPPs).^7–9We previously showed that HD and calciphylaxis patients have depressed fetuin-A serum levels accompanied by a reduced capacity of their serum to inhibit calcium phosphate precipitation.⁵ In cross-sectional studies in HD patients, fetuin-A deficiency was identified as an inflammation-related predictor of cardiovascular and all-cause mortality, respectively.^10,11 In patients without chronic kidney disease (CKD), fetuin-A levels correlated inversely with advanced coronary calcification.^12,13 Fetuin-A–deficient (Ahsg−/−) mice maintained on the DBA/2 background exhibit a fully penetrating phenotype with extensive soft tissue calcification, whereas C57BL/6 Ahsg−/− mice represent “borderline calcifying” mice whereby rapid calcification can be induced by additional metabolic challenges or induction of CKD.^5,14Calcification of the aorta or larger vessels is conspicuously absent in Ahsg−/− mice; therefore, the role for fetuin-A as an inhibitor of vascular calcification was uncertain.^15,16 Absent vascular calcification in Ahsg−/− mice may be related to the protective mechanisms by an intact endothelium, which is severely compromised in humans with atherosclerosis and thus may serve as a nidus for subsequent calcification. To test this hypothesis, we created Ahsg−/−/apolipoprotein E double-deficient (ApoE−/−) mice maintained on the C57BL/6 genetic background to dissect the contribution of fetuin-A, CKD, and an elevated calcium-phosphorus product (Ca × P) to the development of atheroma formation and vascular calcification in an established murine model of atherosclerosis.     

Extracellular Superoxide Dismutase Protects against Proteinuric Kidney Disease

Extracellular superoxide dismutase (EC-SOD), also known as SOD3, is an antioxidant expressed at high levels in normal adult kidneys. Because oxidative stress contributes to a variety of kidney injuries, we hypothesized that EC-SOD may be protective in CKD progression. To study this hypothesis, we used a murine model of ADR nephropathy characterized by albuminuria and renal dysfunction. We found that levels of EC-SOD diminished throughout the course of disease progression and were associated with increased levels of NADPH oxidase and oxidative stress markers. EC-SOD null mice were sensitized to ADR injury, as evidenced by increases in albuminuria, serum creatinine, histologic damage, and oxidative stress. The absence of EC-SOD led to increased levels of NADPH oxidase and an increase in β-catenin signaling, which has been shown to be pathologic in a variety of kidney injuries. Exposure of EC-SOD null mice to either chronic angiotensin II infusion or to daily albumin injections also caused increased proteinuria. In contrast, EC-SOD null mice subjected to nonproteinuric CKD induced by unilateral ureteral obstruction exhibited no differences compared with wild-type mice. Finally, we also found a decrease in EC-SOD in human CKD biopsy samples, similar to our findings in mice. Therefore, we conclude that EC-SOD is protective in CKDs characterized by proteinuria.     

Programmed anti-inflammatory macrophages protect against AKI and promote repair through trophic actions

Because pharmacological interventions for the treatment of acute kidney injury (AKI) have remained ineffective, cell-based therapies have been developed. Marrow stromal cells have been found to be safe and potentially effective in study subjects. Jung et al. demonstrate that macrophages made to overexpress anti-inflammatory interleukin-10 protect rats with AKI through iron-mediated upregulation of lipocalin-2 and its receptors, eliciting both anti-inflammatory and proliferative responses. These data further advance our understanding of cell-based therapies for AKI.     

Renalase deficiency aggravates ischemic myocardial damage

Chronic kidney disease (CKD) leads to an 18-fold increase in cardiovascular complications not fully explained by traditional risk factors. Levels of renalase, a recently discovered oxidase that metabolizes catecholamines, are decreased in CKD. Here we show that renalase deficiency in a mouse knockout model causes increased plasma catecholamine levels and hypertension. Plasma blood urea nitrogen, creatinine, and aldosterone were unaffected. However, knockout mice had normal systolic function and mild ventricular hypertrophy but tolerated cardiac ischemia poorly and developed myocardial necrosis threefold more severe than that found in wild-type mice. Treatment with recombinant renalase completely rescued the cardiac phenotype. To gain insight into the mechanisms mediating this cardioprotective effect, we tested if gene deletion affected nitrate and glutathione metabolism, but found no differences between hearts of knockout and wild-type mice. The ratio of oxidized (NAD) to reduced (NADH) nicotinamide adenine dinucleotide in cardiac tissue, however, was significantly decreased in the hearts of renalase knockout mice, as was plasma NADH oxidase activity. In vitro studies confirmed that renalase metabolizes NADH and catecholamines. Thus, renalase plays an important role in cardiovascular pathology and its replacement may reduce cardiac complications in renalase-deficient states such as CKD.     

NADPH-Oxidase 4 Protects against Kidney Fibrosis during Chronic Renal Injury

NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD_cl1) cell line increased TGF-β1–induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.CKD is a worldwide health problem defined as an alteration of kidney function usually estimated by GFR decline or histologic damage to the kidney.¹ CKD is associated with high mortality and morbidity as well as tremendous costs associated with renal replacement when patients reach ESRD.Interstitial fibrosis and glomerulosclerosis are hallmarks of CKD. Tubulointerstitial fibrosis is better correlated with loss of kidney function than glomerulosclerosis independently of primary injury.² Tubulointerstitial fibrosis arises from numerous, confounding events. These include tubular atrophy, via necrosis or apoptosis, and local hypoxia, due to the loss of peritubular capillaries, which are early events.^3–5 Tubular cells are both targets of tubulointerstitial fibrosis and important players in its progression via secretion of chemotactic factors and cytokines.Oxidative stress has been associated with aging and CKD progression.^6,7 Increased levels of kidney reactive oxygen species (ROS), such as angiotensin II–induced and diabetic nephropathies, are found in CKD.^8–13 However, low levels of ROS also play important roles in oxygen sensing, neoangiogenesis, and cell survival pathways, which may play protective roles against kidney fibrosis.^14,15 NADPH oxidases (NOXs) are enzymes that produce ROS as primary products.¹⁶ The NOX family is composed of seven members (NOX1–5 and DUOX 1 and 2). Among these, NOX1, NOX2, and NOX4 are expressed in both mice and human kidney, whereas NOX5 is only expressed in human kidney. NOX4 is highly expressed in the tubular cell compartment, where its physiologic role is unknown.¹⁷ This isoform is mainly regulated by its controlled abundance.¹⁸ The role of NOX4 in kidney injury is not yet elucidated. Increased renal NOX4 expression has been shown in diabetic nephropathy and its silencing has been demonstrated to be protective.^8,19 However, NOX4 may participate to oxygen sensing and exhibit anti-inflammatory properties via regulation of the NRF2 pathway in the heart.^15,20To determine the role of NOX4 and NOX2 in kidney fibrosis progression, we performed unilateral ureteral obstruction (UUO) on wild-type (WT), and NOX4 knockout (KO) mice as well as on NOX2 and NOX4/NOX2 KO mice. Indeed, UUO is a well described model of renal tubular stress leading to kidney fibrosis.^21,22 We demonstrate that deletion of NOX4 is associated with increased kidney fibrosis in obstructed kidneys. This effect was associated with enhanced tubular cell apoptosis as well as defective hypoxia-inducible factor-1α (HIF-1α) oxygen sensing and NRF2 antioxidant pathways. NOX4 is therefore crucial for kidney tubular cell survival under stressed conditions.     

Drugs and AKI

Acute kidney injury is a common complication in hospitalized patients and is associated with substantially increased morbidity and mortality. Frequently, it is caused by impaired renal perfusion, ischemia and reperfusion injury, sepsis or urinary tract obstruction, but often its etiology is multifactorial. In this context, the contribution of nephrotoxic medications to the development of AKI plays an important role. This review begins with an attempt to evaluate the importance of drug-related acute kidney injury in general. Then, a selected list of 7 classes of drugs or compounds, namely aminoglycosides, aristolochic acid, cytostatic drugs, nonsteroidal anti-inflammatory drugs, osmotic agents, radiocontrast, and phosphate salts are discussed in depth, including their epidemiology, pathophysiology, clinical features and treatment. While not attempting to be exhaustive, this review attempts to provide an overview with additional in-depth information on certain classes of drugs that are either of general importance or have recently emerged in the literature.