Background and Aims Massive intravascular hemolysis is a common condition of several pathologies. It is associated with acute kidney injury (AKI) and progressive impairment of renal function. In this context, free hemoglobin (Hb) can exert harmful effects by accumulating in the kidney, where induces oxidative stress and it becomes cytotoxic. NADPH oxidase 4 (Nox4) is the principal source of reactive oxygen species (ROS) in the kidney. Nox4 is mostly expressed in proximal tubular cells with lower levels in glomerulus. The role of Nox4 in renal damage is not clear, with studies reporting beneficial or deleterious actions depending of the environmental conditions. For that reason we aimed to investigate the role of Nox4 in massive intravascular hemolysis-associated AKI. Method To study the role of Nox4 in AKI caused by massive intravascular hemolysis, we performed an experimental model of intravascular hemolysis by intraperitoneal injection of phenylhydrazine (200 mg/kg) in wild type (Nox4+/+) and Nox4 knockout mice (Nox4-/-). Mice were sacrificed 24 and 72 hours after intravascular hemolysis induction. We collected serum, urine and tissues sample. We analyzed renal function, oxidative stress, cell death and inflammation in these samples. In other experiments, wild type mice were treated with GKT137831 (10mg/kg/day), a potent Nox4 and Nox1 inhibitor, and mice were sacrificed 72h after induction of hemolysis. We also performed in vitro experiments in murine tubular epithelial cells (MCT) and murine podocytes cells to investigate the regulation of Nox4 in Hb-stimulated cells treated or not with GKT137831. Results Induction of intravascular hemolysis in Nox4+/+ mice increased creatinine and BUN levels and enhanced the expression of tubular injury markers, such as NGAL. These pathological effects were reduced in Nox4 knockout mice. Then, we analyzed oxidative stress in our experimental model thought determination of HO-1, ferritin, GSH and lipid peroxidation levels. All of these oxidative markers were reduced in Nox4-/- mice with intravascular hemolysis as compared with Nox4+/+ mice. We also observed that inflammatory markers such as IL-6, cell death and podocytes injury markers were reduced in Nox4-/- mice than in wild type mice, specially 72 hours after phenylhydrazine injection. In line with these results, GKT137831 administration ameliorated intravascular hemolysis-associated renal function impairment. Moreover, oxidative stress, tubular injury markers and podocyte injury were reduced in hemolytic mice treated with GKT137831. GKT137831 also reduced Hb- and heme-mediated oxidative stress in MCT and podocytes. Conclusion Our results show the important role of Nox4 in renal injury associated to massive intravascular hemolysis. Moreover, the inhibition of Nox4 may be a potential therapeutic target to prevent renal damage associated to Hb accumulation. These findings provide new insights into novel aspects of Hb-toxicity and may have important pathogenic and therapeutic implications for intravascular hemolysis related diseases

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Background and Aims Hematuria is a common finding in patients with IgA nephropathy (IgAN), occurring mainly after upper respiratory tract infections. Hematuria can lead to acute kidney injury and chronic loss of renal function in IgAN. However, the mechanisms involved in egression of erythrocytes from the glomerular capillaries into the urinary space are unknown. To answer this question, we developed an infection with Streptococcus pneumoniae (SP) in a humanized experimental IgAN model (α1KICD89tg mice) that resembles the pathological and clinical findings of disease (IgA1 and soluble CD89 mesangial deposits, complement activation, proteinuria and hematuria). Method α1KICD89tg mice (12 weeks old) received an intranasal instillation of SP (107 bacteria). Blood, urine and renal samples were obtained during 1 month after induction of respiratory infection. The presence of SP in lungs from these mice was confirmed by microbiological analysis. Hematuria was quantified in the urinary sediment and renal function was determined by biochemical analysis. Renal histological characteristics were evaluated by hematoxylin/eosin, masson's trichrome and PAS staining. IgA glomerular deposits, activation of complement system and infiltration of proinflammatory cells was examined by immunohistochemistry or immunofluorescence. Circulating leukocyte populations were studied on a hemocytometer. Renal inflammatory cytokines, metalloproteases, as well as markers of tubular and glomerular damage were determined in kidneys by RT-PCR and western-blot. To further validate the role of neutrophils in this pathological setting, we selective depleted these cells through a single injection of anti-Ly6G mAb (200 µg/kg i.p). Results SP-intranasal instillation in α1KICD89tg mice increased hematuria, microalbuminuria and proteinuria, peaking at 48h after induction of the respiratory infection. SP instillation caused disruption of the glomerular basement membrane, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin, as well as higher glomerular accumulation of IgA and proteins of complement system (C3, MBL). Hematuria intensity was positively correlated with the presence of interstitial F4/80+ macrophages, matrix metalloproteinase 9 (MMP-9), inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, CCL-2, CCL5 and CX3CL1/CX3CR1) as well as p65 NF-κB activation. Hematuria was negatively correlated with anti-inflammatory IL-10 mRNA expression, Factor H levels and collagen IV content. Notably, SP infection induced expression of the tubular injury markers N-GAL and KIM-1. Increased peripheral neutrophils levels were observed in the SP-infected α1KICD89tg mice. Mechanistically, anti-Ly6G-mediated neutrophil depletion reduced SP-mediated hematuria, proteinuria and albuminuria, prevented loss of synaptopodin and nephrin, decreased renal inflammation and MMP-9 expression in α1KICD89tg mice Conclusion In a humanized mouse model of IgAN, hematuria bouts following respiratory tract infections are caused by a neutrophil-mediated alteration of the glomerular filtration barrier (podocyte damage, complement deposits and loss of Collagen IV). These findings may help to unveil novel potential therapeutic approaches to combat one of the key elements in the progression of IgAN and related conditions.

Background and Aims Rhabdomyolysis is characterized by the breakdown of the skeletal muscle and the subsequent myoglobin (Mb) release into the bloodstream. A common complication of this syndrome is acute renal injury (AKI). Once filtered by the kidney, Mb causes oxidative stress, inflammation and tubular cell death. There is no specific treatment for rhabdomyolysis-AKI, so it is crucial a better understanding of this syndrome to identify new therapeutic targets. Klotho is an anti-aging protein mostly expressed by the kidney. In addition to its functions in the regulation of mineral metabolism, Klotho protects from AKI-harmful effects. However, no previous studies analyzed the role of Klotho in rhabdomyolysis. Method We performed a pre-clinical model of rhabdomyolysis in C57BL/6J mice (male, 12 weeks old, n=30) by intramuscular injection of 10 ml/kg of 50% glycerol (≥99.5% m/v). Mice were sacrificed 3 and 6 hours or 1, 3, 7 and 30 days after glycerol administration. To evaluate to beneficial effect of Klotho in rhabdomyolysis, C57BL/6J mice were injected intraperitoneally with 0.1 mg/kg recombinant mouse Klotho (1819-KL, R&D Systems), or vehicle (PBS) 30 minutes before and 1, 3 and 5 days after glycerol injection. Blood, urine and renal samples were collected to analyze renal function, Klotho/FGF23 levels, oxidative stress, inflammation, fibrosis and cell death, all of them pathological processes affecting Klotho expression. In addition, we carried out studies in murine tubular cells (MCTs) to study the molecular mechanisms involved in Klotho regulation. Results Our results indicate that rhabdomyolysis induces an early decrease in Klotho renal mRNA and protein expression as well as Klotho serum levels. Klotho levels decreased in line with augmentation of creatinine concentration, kidney inflammation (CCL2 and IL-6 mRNA expression) and tubular injury marker NGAL. Moreover, patients with rhabdomyolysis-AKI also showed lower plasma Klotho levels and increased FGF23 plasma concentration than age-matched healthy individuals. Renal klotho protein expression remained reduced one month after rhabdomyolysis-induction, in line with long term renal fibrosis and pro-inflammatory macrophage accumulation (F4/80+ cells). Exogenous recombinant Klotho administration ameliorated renal function and reduced rhabdomyolysis-mediated tubular cell death oxidative stress (4-HNE staining) and tubular injury 24h after glycerol injection. In the same line, Klotho administration during AKI development reduced long term renal fibrosis and macrophage infiltration one month later. Antioxidant therapies with N-acetylcysteine (NAC) and sulforaphane, a potent Nuclear factor erythroid-2-related factor 2 (Nrf2) inducer, reduced Mb-mediated Klotho decrease in cultured tubular cells. Inhibition of TNF-α and IL-6 with infliximab and tocilizumab, respectively, also reverted Mb-mediated Klotho decrease. Inhibition of the inflammatory NFkB and p38 pathways also prevented Mb-mediated Klotho reduction. Conclusion Our findings are the first to demonstrate decreased renal and soluble Klotho levels not only in the early phases of rhabdomyolysis-induced AKI, but also when renal function was recovered, indicating that long-term consequences of AKI, such as inflammation and fibrosis, are also involved in Klotho downregulation. In addition, our results also indicate that Klotho administration may be a potential strategy to decrease rhabdomyolysis- long term negative effects.