Inflammatory and oxidative stress biomarkers in alkaptonuria: data from the DevelopAKUre project.

OBJECTIVE: The aim of this work was to assess baseline serum levels of established biomarkers related to inflammation and oxidative stress in samples from alkaptonuric subjects enrolled in SONIA1 (n = 40) and SONIA2 (n = 138) clinical trials (DevelopAKUre project). METHODS: Baseline serum levels of Serum Amyloid A (SAA), IL-6, IL-1ß, TNFα, CRP, cathepsin D (CATD), IL-1ra, and MMP-3 were determined through commercial ELISA assays. Chitotriosidase activity was assessed through a fluorimetric method. Advanced Oxidation Protein Products (AOPP) were determined by spectrophotometry. Thiols, S-thiolated proteins and Protein Thiolation Index (PTI) were determined by spectrophotometry and HPLC. Patients' quality of life was assessed through validated questionnaires. RESULTS: We found that SAA serum levels were significantly increased compared to reference threshold in 57.5% and 86% of SONIA1 and SONIA2 samples, respectively. Similarly, chitotriosidase activity was above the reference threshold in half of SONIA2 samples, whereas CRP levels were increased only in a minority of samples. CATD, IL-1ß, IL-6, TNFα, MMP-3, AOPP, thiols, S-thiolated protein and PTI showed no statistically significant differences from control population. We provided evidence that alkaptonuric patients presenting with significantly higher SAA, chitotriosidase activity and PTI reported more often a decreased quality of life. This suggests that worsening of symptoms in alkaptonuria (AKU) is paralleled by increased inflammation and oxidative stress, which might play a role in disease progression. CONCLUSIONS: Monitoring of SAA may be suggested in AKU to evaluate inflammation. Though further evidence is needed, SAA, chitotriosidase activity and PTI might be proposed as disease activity markers in AKU.

Cysteinylation and homocysteinylation of plasma protein thiols during ageing of healthy human beings.

The purpose of the present study was to determine the relative amount of S-thiolated proteins (i.e. S-homocysteinylated, S-cysteinylglycinylated, S-glutathionylated and S-cysteinylated proteins) to the total protein thiols (i.e. the sum of reduced protein sulphydryl groups (PSHs) and protein mixed disulphides with homocysteine [HcySH], cysteinylglycine, cysteine [CysSH] and glutathione) in the plasma of healthy individuals aged 20 to 93. After plasma separation, total protein thiols, S-thiolated proteins, as well as CysSH, cystine, HcySH and homocystine were measured by high-performance liquid chromatography (HPLC) with fluorescence determination of the thiol-monobromobimane conjugate. Determination of plasma levels of protein thiols was performed by spectrophotometry with 5,5'-dithiobis(2-nitrobenzoic acid) as a titrating agent. The present study demonstrates an age-dependent reduction in the amount of PSHs, and an age-dependent increase in cysteinylated and homocysteinylated plasma proteins in healthy human beings. This indicates that the efficiency of the reduced protein thiol pool as an antioxidant defence system decreases with age, possibly causing an increased risk of irreversible oxidation (i.e. further oxidation to sulphinic and sulphonic acids, which are usually not reducible by thiol reducing agents) of sulphydryl groups of plasma proteins. The drop in the plasma level of protein sulphydryl groups suggests depletion and/or impairment of the antioxidant capacity of plasma, likely related to an alteration of the delicate balance between the different redox forms of thiols.

Altered thiol pattern in plasma of subjects affected by rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease which involves the synovial membrane of multiple diarthroidal joints causing damage to cartilage and bones. The damage process seems to be related to an overproduction of oxygen reactive species inducing an oxidative perturbation. Since sulfhydryl groups are primary antioxidant factors, we were interested in investigating the balance of plasma sulfhydryl/disulfides in patients with active RA compared to healthy control subjects. METHODS: Twenty-one patients with RA and 15 age-matched controls were studied. Plasmatic sulfhydryl groups and their disulfide form concentrations were measured by spectrophotometry or HPLC. RESULTS: RA patients showed significantly lower levels of plasma protein sulfhydryls and cysteinyl-glycine compared to healthy controls (p < 0.001). Conversely, cystine and homocystine, and protein-bound cysteine and homocysteine were significantly increased (p < 0.005 in disulfides forms and p < 0.05 in protein mixed disulfides forms). There was a significant correlation between some clinical data (ESR, number of tender/swollen joints) and some of the parameters studied. CONCLUSION: The results of this study indicate a biochemical disturbance of plasma sulfhydryl/disulfides balance in patients with RA compared to controls with an increase in some oxidised forms (disulfides and protein mixed disulfides) and a decrease in free thiols. The increase in total homocysteine, correlated to the higher risk of cardiovascular diseases in RA patients, is associated with higher levels of the oxidised forms, disulfides and protein-thiol mixed disulfides.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation.

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this "depowered" GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators.

The oxidation produced by hydrogen peroxide on Ca-ATP-G-actin.

We report here that in vitro exposure of monomeric actin to hydrogen peroxide leads to a conversion of 6 of the 16 methionine residues to methionine sulfoxide residues. Although the initial effect of H2O2 on actin is the oxidation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, and Met355 are the other sites of the oxidative modification. Met44 and Met47 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medium and are found in all four subdomains of actin. The conformations of subdomain 1, a region critical for the functional binding of different actin-binding proteins, and subdomain 2, which plays important roles in the polymerization process and stabilization of the actin filament, are changed upon oxidation. The conformational changes are deduced from the increased exposure of hydrophobic residues, which correlates with methionine sulfoxide formation, from the perturbations in tryptophan fluorescence, and from the decreased susceptibility to limited proteolysis of oxidized actin.

Reversible S-glutathionylation of Cys 374 regulates actin filament formation by inducing structural changes in the actin molecule.

S-glutathionylation, the reversible formation of mixed disulphides of cysteinyl residues in target proteins with glutathione, occurs under conditions of oxidative stress; this could be a posttranslational mechanism through which protein function is regulated by the cellular redox status. A novel physiological relevance of actin polymerization regulated by glutathionylation of Cys(374) has been recently suggested. In the present study we showed that glutathionylated actin (GS-actin) has a decreased capacity to polymerize compared to native actin, filament elongation being the polymerization step actually inhibited. Actin polymerizability recovers completely after dethiolation, indicating that S-glutathionylation does not induce any protein denaturation and is therefore a reversible oxidative modification. The increased exposure of hydrophobic regions of protein surface observed upon S-glutathionylation indicates changes in actin conformation. Structural alterations are confirmed by the increased rate of ATP exchange as well as by the decreased susceptibility to proteolysis of the subtilisin cleavage site between Met(47) and Gly(48), in the DNase-I-binding loop of the actin subdomain 2. Structural changes in the surface loop 39-51 induced by S-glutathionylation could influence actin polymerization in view of the involvement of the N-terminal portion of this loop in intermonomer interactions, as predicted by the atomic models of F-actin.

Methionine oxidation as a major cause of the functional impairment of oxidized actin.

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer's disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimer's disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.

Responses of thiols to an oxidant challenge: differences between blood and tissues in the rat.

Treatment of rats with diamide (100 mg/kg i.p.) altered the thiol components of the blood to a very different extent than in tissues (liver, kidney, lung, spleen, heart and testis). A total consumption (10 min) and regeneration (120 min) of blood glutathione (GSH), matched by a parallel increase and decrease in glutathione-protein mixed disulfides (GS-SP) was observed. In contrast, no modification of non-protein SH groups (NPSH) and protein SH groups (PSH), GS-SP and malondialdehyde (MDA) was observed in liver, kidney, lung, testis spleen and heart within same time range. In particular, only glutathione disulfide (GSSG) levels and some activities of antioxidant enzymes were modified to a small extent and in an opposite direction in some organs. For example, GSSG, and glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase (CAT) activities appeared up-regulated in one tissue and down-regulated in another. The least modified organ was the liver, whereas lung and spleen were the most affected (lung, GSSG, significantly increased whereas G-6-PDH, glutaredoxin (GRX), GPX, superoxide dimutase (SOD) levels were significantly lowered; spleen, GSSG and the activity of glutathione reductase (GR), G-6-PDH and glutathione transferase (GST) were significantly decreased). The different responses of erythrocytes and organs to diamide were explained by the high affinity of hemoglobin and by the relatively high potential of thiol regeneration in organs. The rapid reversibility of the process of protein S-thiolation in blood and the small effects in organs leads us to propose the existence of an inter-organ cooperation in the rat that regulates protein S-thiolation in blood. Plasma thiols may well play a role in this process.

S-NO-actin: S-nitrosylation kinetics and the effect on isolated vascular smooth muscle.

We describe the modification of reactive actin sulfhydryls by S-nitrosoglutathione. Kinetics of S-nitrosylation and denitrosylation suggest that only one cysteine of actin is involved in the reactions. By using the bifunctional sulfhydryl cross-linking reagent N,N'-1,4-phenylenebismaleimide and the monofunctional reagent N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine, we identified this residue as Cys374. The time course of filament formation followed by high-shear viscosity changes revealed that S-nitrosylated G-actin polymerizes less efficiently than native monomers. The observed decrease in specific viscosity at steady state is due mainly to a marked inhibition of filament end-to-end annealing and, partially, to a reduction in F-actin concentration. Finally, S-nitrosylated actin acts as nitric oxide donor showing a fast, potent vasodilating activity at unusually low concentrations, being comparable with that of low molecular weight nitrosothiols.

The role of cysteine in the regulation of blood glutathione-protein mixed disulfides in rats treated with diamide.

The kinetics of GSH, GSSG, and thiol-protein mixed disulfides (RS-SP) of GSH (GS-SP) and cysteine (CYS-SP) were studied in rat blood and liver in the time range 0-120 min after treatment with 100 and 200 mg/kg i.p. of diamide. Total consumption (10 min) and regeneration (120 min) of blood GSH, matched by parallel increases and decreases in RS-SP, were observed. GSSG did not change appreciably. No dose-effect relationship was obtained with either treatment. On the contrary, in vitro treatment of blood with 0.75 mM diamide provoked the same trends of GSH and RS-SP as in vivo (e.g., reversible modifications), whereas treatment with 1.5 mM caused drops and rises in GSH and RS-SP, respectively, without any subsequent return to control values. The presence of a hematic factor responsible for RS-SP regulation is hypothesized in the in vivo experiment. Successive experiments involving in vitro pretreatment with 2 mM diamide and treatment with 0.5 mM of various thiols indicated that cysteine (CYS), but not GSH or N-acetylcysteine, rapidly restored erythrocyte GSH and RS-SP to their basal levels. No evident sign of hemolysis was observed in these experiments. These results indicate that CYS is a diffusible thiol important for RS-SP regulation. Analysis of whole blood of rats treated with 100 mg/kg i.p. diamide and the presence of two reversible peaks (about 10 times the corresponding control level) of CYS-SP and free CYS confirmed the plausible role of CYS in maintaining the reversibility of the process. Preliminary results in liver of rats treated with 100 mg/kg diamide indicated that CYS may act by metabolic cooperation between organs. We suggest that CYS may have a role in the regulation of the intracellular redox state of rat erythrocytes during oxidative stress.

Role of protein -SH groups in redox homeostasis--the erythrocyte as a model system.

The reactivities of the sulfhydryl groups of rat, turkey, human, and calf hemoglobin were studied together with the enzyme activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutaredoxin in lysed erythrocytes to evaluate their roles in regulating redox homeostasis. The results of -SH reactivity showed rate constants spanning four orders of magnitude (k2, calf, 6.67 M-1 s-1; rat -SH fast reacting, 2.8 x 10(4) M-1 s-1). Enzyme activities of glucose-6-phosphate dehydrogenase ranged from 0.402 U/ml (calf) to 0.900 U/ml (rat), glutathione reductase from 0. 162 U/ml (rat) to 0.381 U/ml (human), glutaredoxin from 0.778 U/ml (rat) to 2.28 U/ml (turkey), and glutathione peroxidase from 2.07 U/ml (human) to 27.3 U/ml (rat). Blood samples of the four species were also treated with 0.5-1.5 mM tert-butyl hydroperoxide (t-BOOH) or diamide, and levels of glutathione-derived species [GSH, GSSG, and glutathione-protein mixed disulfides (GS-SP)] were determined within 120 min and related to the corresponding protein -SH group (PSH) reactivities and enzyme repertoires. In all cases t-BOOH rapidly transformed GSH into GSSG by the action of glutathione peroxidase; GSSG was in turn transformed into GS-SP, according to the reaction GSSG + PSH --> GS-SP + GSH, or reduced back to GSH by glutathione reductase. The GSSG reduction was more efficient in rat and human blood, due to the contribution of the fast-reacting -SH of hemoglobin, in the rat, and to the efficiency of the enzyme repertoire of human blood. Calf blood showed a relatively low capacity to restore normal values after oxidative stress, due to its low PSH reactivity and the weak contribution of its enzymes. Diamide treatment, which is known to react nonenzymatically with thiols, gave increased GS-SP levels in rat and turkey, but not in human and calf blood, as expected from the different corresponding PSH reactivities. Species with relatively high PSH reactivity and glucose 6-phosphate dehydrogenase activity, such as the rat, therefore had a higher antioxidant capacity than species (calf) in which these parameters were relatively low.

A method to study kinetics of transnitrosation with nitrosoglutathione: reactions with hemoglobin and other thiols.

The rate of protein S-nitrosylation, a reversible process by which S-nitroso thiol (RS-NO) compounds exchange the NO+ moiety with protein SH groups, is essentially governed by two factors, the pK alpha and the accessibility of the protein sulfhydryl. A useful method of following transnitrosation kinetics of various protein and nonprotein SH compounds with GS-NO is described. When the reaction is carried out in the presence of 1-chloro-2,4-dinitrobenzene and glutathione transferases, the rate of RS-NO formation (RSH + GS-NO-->RS-NO + GSH) can be monitored by spectrophotometry at 340 nm in terms of the enzymatic conversion of GSH to a GS conjugate. Unlike methods based on NO release from the S-NO bond, this procedure is rapid and accurate and requires relatively small amounts of thiols. The second order rate constants of S-nitrosylation of human and rat oxy- and deoxyhemoglobin of BSA and other thiols were calculated by this method which confirmed previous results reported in the literature.

Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress.

Changes in the concentrations of protein-mixed disulfides (XS-SP) of glutathione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in human platelets treated with diamide and t-BOOH in timecourse experiments (time range, 1-30 min) in order to understand the contribution of minor thiols CSH and CGSH to the regulation of glutathione-protein mixed disulfides (GS-SP). Diamide was much more potent than t-BOOH in altering the platelet thiol composition of XS-SP (threshold dose: diamide, 0.03 mM; t-BOOH, 0.5 mM) and caused reversible XS-SP peaks whose magnitude was related to the concentration of free thiols in untreated cells. Thus maximum levels of GS-SP (8 min after 0.4 mM diamide) were about 16-fold higher than those of controls (untreated platelets, GS-SP = 0.374 nmol/10(9) platelets), whereas those of CS-SP and CGS-SP were only 4-fold increased (untreated platelets, CS-SP = 0.112 nmol/10(9) platelets; CGS-SP = 0.024 nmol/10(9) platelets). The greater effects of diamide with respect to t-BOOH were explained on the basis of the activities of fast reactive protein SH groups for diamide and glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t-BOOH. The addition of cysteine (0.3 mM, at 4 min) after treatment of platelets with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to normal values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased gamma-glutamyltranspeptidase activity was found in platelets treated with diamide. Moreover, untreated platelets were found to release and hydrolyze GSH to CGSH and CSH. Ratios of thiols/disulfides (XSH/XSSX) and activities of GR and G-6PDH were also related to a high reducing potential exerted by GSH but not by minor thiols. The lower mass and charge of minor thiols is a likely requisite of the regulation of GS-SP levels in platelets.

Different metabolizing ability of thiol reactants in human and rat blood: biochemical and pharmacological implications.

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position beta125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.

Studies on the biological effects of ozone: 9. Effects of ozone on human platelets.

During the course of ozonated autohaemotherapy (O3-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O3) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O3 can promote platelet aggregation. In contrast, after Ca(2+) chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H2O2) in the presence of Ca2+ with the possible expression of adhesion molecules is briefly discussed.

Altered glutathione anti-oxidant metabolism during tumor progression in human renal-cell carcinoma.

It has been proposed that oxidative stress develops in tumors, with important consequences for growth and progression. To investigate this hypothesis, we measured low m.w. thiols, disulfides, protein-mixed disulfides and a pool of major anti-oxidant enzymes in renal-cortex as well as renal-cell carcinoma (RCC) specimens at stages I-II and III. Our data showed (i) a significant increase in the levels of total intracellular glutathione at both tumor stages (levels were 2.6-2.8 fold higher than those in the normal renal cortex), (ii) a marked lowering of the GSH/GSSG ratio in stage I-II accompanied by a significant decrease of many GSH-dependent enzymes (i.e., GPX, GST, GGT, GR) and (iii) unchanged GSH/GSSG ratio and GSH-dependent enzyme activity in stage III with respect to normal renal cortex. These results indicate that relevant variations exist in the glutathione antioxidant system in the different stages of RCC and support the hypothesis that oxidative stress plays an important role in RCC growth and progression.