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.

Decreased Renal Gluconeogenesis Is a Hallmark of Chronic Kidney Disease.

INTRODUCTION: CKD is associated with alterations of tubular function. Renal gluconeogenesis is responsible for 40% of systemic gluconeogenesis during fasting, but how and why CKD affects this process and the repercussions of such regulation are unknown. METHODS: We used data on the renal gluconeogenic pathway from more than 200 renal biopsies performed on CKD patients and from 43 kidney allograft patients, and studied three mouse models, of proteinuric CKD (POD-ATTAC), of ischemic CKD, and of unilateral urinary tract obstruction. We analyzed a cohort of patients who benefitted from renal catheterization and a retrospective cohort of patients hospitalized in the intensive care unit. RESULTS: Renal biopsies of CKD and kidney allograft patients revealed a stage-dependent decrease in the renal gluconeogenic pathway. Two animal models of CKD and one model of kidney fibrosis confirm gluconeogenic downregulation in injured proximal tubule cells. This shift resulted in an alteration of renal glucose production and lactate clearance during an exogenous lactate load. The isolated perfused kidney technique in animal models and renal venous catheterization in CKD patients confirmed decreased renal glucose production and lactate clearance. In CKD patients hospitalized in the intensive care unit, systemic alterations of glucose and lactate levels were more prevalent and associated with increased mortality and a worse renal prognosis at follow-up. Decreased expression of the gluconeogenesis pathway and its regulators predicted faster histologic progression of kidney disease in kidney allograft biopsies. CONCLUSION: Renal gluconeogenic function is impaired in CKD. Altered renal gluconeogenesis leads to systemic metabolic changes with a decrease in glucose and increase in lactate level, and is associated with a worse renal prognosis.

Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress.

Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.

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.

Beneficial Effects of Elderly Tailored Mediterranean Diet on the Proteasomal Proteolysis.

Aging is a multifactorial process characterized by the accumulation of proteins undergoing oxidative modifications, either due to enhanced levels of oxidative stress or due to their decreased clearance; both facts are related to the establishment of chronic inflammatory processes. These processes are directly associated with functional and structural modifications of a key cellular component, namely the proteasome. In this study, levels of oxidized proteins, along with proteasome and immunoproteasome composition and activity on a selected group of 120 elderly volunteers were analyzed before and after the administration of a specific dietary protocol, based on an elderly tailored Mediterranean diet (the "NU-AGE diet"). A significant negative correlation between levels of oxidized/carbonylated proteins and proteasome function was confirmed, both before and after intervention. Furthermore, it was demonstrated that subgroups of non-frail subjects and women receive a greater benefit after the intervention, concerning specifically the proteasome content and activity. These data highlight the putative beneficial effects of Mediterranean diet on the major cellular proteolytic mechanism, the proteasome, in elderly people.

Anti-ageing properties of Khelma Longevity™: treatment of human fibroblasts increases proteasome levels and decreases the levels of oxidized proteins.

We have determined the putative anti-ageing properties of Khelma Longevity™, a formula based on various natural compounds from the Mediterranean area. Human primary fibroblast cultures were treated with a wide range of concentrations of Khelma Longevity™ for 1 day or 3 consecutive days. Following these treatments, two major and complementary biomarkers of ageing were measured, namely, the proteasome and the amount of oxidized proteins. It was observed that 24h of treatment with Khelma Longevity™ resulted in a maximum increase of about 41% of the total protein levels of 20S proteasome. Levels of oxidized proteins were reduced by almost 6.5-fold following longer treatments. Specifically we have observed a maximum decrease of protein carbonyls to 84.7% in comparison with nontreated control cells following 3 days of continuous treatment with Khelma Longevity™. These results support the notion that formulas rich in natural compounds from the Mediterranean area possess anti-ageing properties.

Proteasome activation: An innovative promising approach for delaying aging and retarding age-related diseases.

Aging is a natural process accompanied by a progressive accumulation of damage in all constituent macromolecules (nucleic acids, lipids and proteins). Accumulation of damage in proteins leads to failure of proteostasis (or vice versa) due to increased levels of unfolded, misfolded or aggregated proteins and, in turn, to aging and/or age-related diseases. The major cellular proteolytic machineries, namely the proteasome and the lysosome, have been shown to dysfunction during aging and age-related diseases. Regarding the proteasome, it is well established that it can be activated either through genetic manipulation or through treatment with natural or chemical compounds that eventually result to extension of lifespan or deceleration of the progression of age-related diseases. This review article focuses on proteasome activation studies in several species and cellular models and their effects on aging and longevity. Moreover, it summarizes findings regarding proteasome activation in the major age-related diseases as well as in progeroid syndromes.

Optimization of in vitro measurement of proteasome activity in mammalian cells using fluorogenic substrates.

The proteasome is the major multi-catalytic machinery responsible for protein degradation and maintenance of the proteome. The 26S proteasome is an ATP-dependent proteolytic complex, dedicated to the degradation of poly-ubiquitinated proteins. It consists of a 20S proteolytic core and one or two flanking 19S regulatory complexes. The three catalytic subunits harboring chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L; also termed PGPH) activities respectively reside in the 20S proteasome that can also exist in a free form and degrade oxidized and unfolded proteins. Impaired proteasome function has been implicated in the pathogenesis of a number of diseases including Alzheimer's disease, diabetes, cancer and aging. The emerging interest in proteasome function as diagnostic marker of various human pathologies and therapeutic target necessitates the development of accurate, sensitive and reliable methodologies for the assessment of proteasome activity. Herein, we describe an optimization procedure for the measurement of CT-L, T-L and C-L activities in cell lysates of fibroblasts (HFL-1), melanocytes (B16F10) and peripheral blood mononuclear cells (PBMCs) using fluorogenic peptide substrates in a mid-throughput 96-well plate format. Optimization involves the composition of cell lysis and assay buffers, and the determination of the concentrations of specific fluorogenic substrates and protein content in the reaction to attain appropriate linear catalytic response during measurement. Additional parameters assessed include the concentration of the cell lysate and of ATP in the cell lysis and assay buffers. Our methodological analysis provides useful guidelines for the accurate and rapid determination of proteasome activity in various cell types.