Evolution of hypoxia and hypoxia-inducible factor asparaginyl hydroxylase regulation in chronic kidney disease.

BACKGROUND: The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). METHODS: We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. RESULTS: In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. CONCLUSIONS: The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

Potential benefits and harms of NADPH oxidase type 4 in the kidneys and cardiovascular system.

The main function of NADPH oxidases is to catalyse the formation of reactive oxygen species (ROS). NADPH oxidase 4 (NOX4) is expressed at high levels in kidney tubular cells, and at lower levels in endothelial cells, cardiomyocytes and other cell types under physiological conditions. NOX4 is constitutively active producing hydrogen peroxide (H2O2) as the prevalent ROS detected, whereas other NOX isoforms present in the renal and cardiovascular systems (i.e. NOX1, NOX2 and NOX5) generate superoxide radical anions as main products. Pharmacological inhibition of NOX4 has received enormous attention for its potential therapeutic benefit in fibrotic disease and nephropathologies. Ongoing clinical trials are testing this approach in humans. Diabetes elevates NOX4 expression in podocytes and mesangial cells, which was shown to damage glomeruli leading to podocyte loss, mesangial cell hypertrophy and matrix accumulation. Consequently, NOX4 represents an interesting therapeutic target in diabetic nephropathy. On the contrary, experiments using NOX4-deficient mice have shown that NOX4 is cytoprotective in tubular cells, cardiomyocytes, endothelial cells and vascular smooth muscle cells, and has a metabolism-regulating role when these cells are subjected to injury. Mice with systemic NOX4 deletion are more susceptible to acute and chronic tubular injury, heart failure and atherosclerosis. Overall, the current literature suggests a detrimental role of increased NOX4 expression in mesangial cells and podocytes during diabetic nephropathy, but a cytoprotective role of this enzyme in other cellular types where it is expressed endogenously. We review here the recent evidence on the role of NOX4 in the kidneys and cardiovascular system. With the emergence of pharmacological NOX4 inhibitors in clinical trials, caution should be taken in identifying potential side effects in patients prone to acute kidney injury and cardiovascular disease.

[Renal urinary shear stress: a novel actor in nephropathies]. / Quand l'urine cisaille les cellules rénales - Un nouveau rôle pour le shear stress dans les néphropathies.

The role of fluid shear stress is well established in vascular pathophysiology. However, urinary shear stress now also appears as a key mechanism in the regulation of renal function. In addition, there is a growing body of evidence showing that modified urinary shear stress is involved in the development of nephropathies. Therefore we review here the state-of-the-art on the pathophysiological roles of urinary shear stress.

Renal tubular fluid shear stress facilitates monocyte activation toward inflammatory macrophages.

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. Recently, we reported that renal tubular FSS promotes endothelial cell activation and subsequent adhesion of human monocytes, thereby suggesting that changes in urinary FSS can induce the development of inflammation (Miravète M, Klein J, Besse-Patin A, Gonzalez J, Pecher C, Bascands JL, Mercier-Bonin M, Schanstra JP, Buffin-Meyer B, BBRC 407: 813-817, 2011). Here, we evaluated the influence of tubular FSS on monocytes as they play an important role in the progression of inflammation in nephropathies. Human renal tubular cells (HK-2) were exposed to FSS 0.01 Pa for 30 min or 5 h. Treatment of human THP-1 monocytes with the resulting conditioned medium (FSS-CM) modified the expression of macrophage differentiation markers, suggesting differentiation toward the inflammatory M1-type macrophage. The effect was confirmed in freshly isolated human monocytes. In contrast to endothelial cells, the activation of monocytes by FSS-CM did not require TNF-α. Cytokine array analysis of FSS-CM showed that FSS modified secretion of cytokines by HK-2 cells, particularly by increasing secretion of TGF-ß and by decreasing secretion of C-C chemokine ligand 2 (CCL2). Neutralization of TGF-ß or CCL2 supplementation attenuated the effect of FSS-CM on macrophage differentiation. Finally, FSS-injured HK-2 cells expressed and secreted early biomarkers of tubular damage such as kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. In conclusion, changes in urinary FSS should now also be considered as potential insults for tubular cells that initiate/perpetuate interstitial inflammation.

Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution.

BACKGROUND: NADPH oxidase 4 (NOX4) catalyzes the formation of hydrogen peroxide (H2O2). NOX4 is highly expressed in the kidney, but its role in renal injury is unclear and may depend on its specific tissue localization. METHODS: We performed immunostaining with a specific anti-NOX4 antibody and measured NOX4 mRNA expression in human renal biopsies encompassing diverse renal diseases. We generated transgenic mice specifically overexpressing mouse Nox4 in renal tubular cells and subjected the animals to the unilateral ureteral obstruction (UUO) model of fibrosis. RESULTS: In normal human kidney, NOX4 protein expression was at its highest on the basolateral side of proximal tubular cells. NOX4 expression increased in mesangial cells and podocytes in proliferative diabetic nephropathy. In tubular cells, NOX4 protein expression decreased in all types of chronic renal disease studied. This finding was substantiated by decreased NOX4 mRNA expression in the tubulo-interstitial compartment in a repository of 175 human renal biopsies. Overexpression of tubular NOX4 in mice resulted in enhanced renal production of H2O2, increased NRF2 protein expression and decreased glomerular filtration, likely via stimulation of the tubulo-glomerular feedback. Tubular NOX4 overexpression had no obvious impact on kidney morphology, apoptosis, or fibrosis at baseline. Under acute and chronic tubular injury induced by UUO, overexpression of NOX4 in tubular cells did not modify the course of the disease. CONCLUSIONS: NOX4 expression was decreased in tubular cells in all types of CKD tested. Tubular NOX4 overexpression did not induce injury in the kidney, and neither modified microvascularization, nor kidney structural lesions in fibrosis.

NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury.

NADPH oxidase 4 (NOX4) is highly expressed in kidney proximal tubular cells. NOX4 constitutively produces hydrogen peroxide, which may regulate important pro-survival pathways. Renal ischemia reperfusion injury (IRI) is a classical model mimicking human ischemic acute tubular necrosis. We hypothesized that NOX4 plays a protective role in kidney IRI. In wild type (WT) animals subjected to IRI, NOX4 protein expression increased after 24 hours. NOX4 KO (knock-out) and WT littermates mice were subjected to IRI. NOX4 KO mice displayed decreased renal function and more severe tubular apoptosis, decreased Bcl-2 expression and higher histologic damage scores compared to WT. Activation of NRF2 was decreased in NOX4 KO mice in response to IRI. This was related to decreased KEAP1 oxidation leading to decreased NRF2 stabilization. This resulted in decreased glutathione levels. In vitro silencing of NOX4 in cells showed an enhanced propensity to apoptosis, with reduced expression of NRF2, glutathione content and Bcl-2 expression, similar to cells derived from NOX4 KO mice. Overexpression of a constitutively active form of NRF2 (caNRF2) in NOX4 depleted cells rescued most of this phenotype in cultured cells, implying that NRF2 regulation by ROS issued from NOX4 may play an important role in its anti-apoptotic property.

Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, ß-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and ß-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and ß-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

Long term metabolic syndrome induced by a high fat high fructose diet leads to minimal renal injury in C57BL/6 mice.

Metabolic syndrome can induce chronic kidney disease in humans. Genetically engineered mice on a C57BL/6 background are highly used for mechanistic studies. Although it has been shown that metabolic syndrome induces cardiovascular lesions in C57BL/6 mice, in depth renal phenotyping has never been performed. Therefore in this study we characterized renal function and injury in C57BL/6 mice with long-term metabolic syndrome induced by a high fat and fructose diet (HFFD). C57BL/6 mice received an 8 months HFFD diet enriched with fat (45% energy from fat) and drinking water enriched with fructose (30%). Body weight, food/water consumption, energy intake, fat/lean mass ratio, plasma glucose, HDL, LDL, triglycerides and cholesterol levels were monitored. At 3, 6 and 8 months, renal function was determined by inulin clearance and measure of albuminuria. At sacrifice, kidneys and liver were collected. Metabolic syndrome in C57BL/6 mice fed a HFFD was observed as early 4 weeks with development of type 2 diabetes at 8 weeks after initiation of diet. However, detailed analysis of kidney structure and function showed only minimal renal injury after 8 months of HFFD. HFFD induced moderate glomerular hyperfiltration (436,4 µL/min vs 289,8 µL/min; p-value=0.0418) together with a 2-fold increase in albuminuria only after 8 months of HFFD. This was accompanied by a 2-fold increase in renal inflammation (p-value=0.0217) but without renal fibrosis or mesangial matrix expansion. In addition, electron microscopy did not show alterations in glomeruli such as basal membrane thickening and foot process effacement. Finally, comparison of the urinary peptidome of these mice with the urinary peptidome from humans with diabetic nephropathy also suggested absence of diabetic nephropathy in this model. This study provides evidence that the HFFD C57BL/6 model is not the optimal model to study the effects of metabolic syndrome on the development of diabetic kidney disease.