Oxidized Albumin: Evaluation of Oxidative Stress as a Marker for the Progression of Kidney Disease

Abstract

Oxidative stress has been reported to be associated with the progression of renal pathology as well as with the onset of complications associated with this condition. Bardoxolone methyl, a nuclear factor-erythroid 2-related factor 2 (Nrf2) activator with anti-oxidative and inflammatory modulation effects, has been reported to improve renal function in clinical trials. As of this writing, there have been no systems for the quantitative evaluation of oxidative stress that could be applied as a clinical test. We recently reported on post-translational modifications of albumin using electrospray-ionization time-of-flight mass spectrometry (ESI-TOF MS) and the results indicated that oxidized albumin (cysteinylated albumin: a molecule that was oxidatively cysteinylated at Cys34) was found to increase with the progression of renal pathology. A clinical study demonstrated that monitoring the levels of oxidized albumin could be useful for the early diagnosis of diabetic kidney disease and for predicting renal prognosis. Higher levels of serum oxidized albumin were also associated with cardiovascular complications or sarcopenia in patients who were undergoing hemodialysis. The overall findings indicate that oxidized albumin could also be an index of therapeutic efficacy against kidney disease. Oxidized albumin is not only a marker for kidney disease but also a nephrotoxic substance. This suggests that measuring the levels of oxidized albumin could be a biomarker for diagnosing the progression of and complications associated with renal disease in addition to evaluating the efficacy of therapeutic drugs.

1. INTRODUCTION

Chronic kidney disease (CKD) is a risk factor for end-stage renal disease and its complications such as cardiovascular disease. Approximately 13% of the Japanese adult population is estimated to have CKD.¹⁾ Oxidative stress, a state in which excessively produced reactive oxygen species (ROS) exceed antioxidant capacity, has attracted interest as a factor that might be associated with the progression of CKD and its complications.²⁾ Inducers of oxidative stress in renal pathology and their mechanisms remain largely unknown. In addition to the decrease in intracellular antioxidant factors such as superoxide dismutase and an increase in ROS production caused by endothelial damage associated with increased blood pressure and blood glucose, it has been shown that uremic substances that accumulate in conjunction with decreased renal function are involved in the increased oxidative stress.^2–5) It has also been reported that bardoxolone methyl, a nuclear factor-erythroid 2-related factor 2 (Nrf2) activator with anti-oxidative and inflammatory modulator characteristics, improves renal function in clinical trials in Japan and overseas.^6–9) Based on the above findings, the progression of renal pathology and oxidative stress appear to be closely related, and a method for the quantitative evaluation of oxidative stress in clinical practice is extremely important for diagnosing renal pathology and evaluating the efficacy of possible therapeutic agents. Here, we mainly review the usefulness of monitoring the levels of oxidized albumin as a marker for the progression of renal disease.

2. POST-TRANSLATIONAL MODIFICATIONS OF ALBUMIN

Albumin is the most abundant protein in serum, accounting for 60–65% of total serum proteins.¹⁰⁾ In addition to maintaining colloidal osmotic pressure, albumin plays a role as a carrier for long-chain fatty acids and drugs in serum, and as an antioxidant by scavenging radicals. The liver of a healthy adult produces 0.2 g/kg of body weight of albumin per day, and the albumin that is produced in the liver is quickly transferred to the systemic circulation, where 30–40% is distributed in the blood and 60–70% in the interstitium.^11,12) Quantitative changes in serum albumin levels have been widely used in clinical practice as an index of liver function and nutritional status.

Since albumin has a long elimination half-life of about 20 d, it undergoes various post-translational modifications such as glycation, oxidation, partial deletion, and carbamylation, sensitively reflecting the in vivo environment.¹³⁾ Such post-translational modifications also affect the functions of albumin, such as its ligand-transporting ability and anti-oxidative ability. The qualitative heterogeneity due to post-translational modifications as well as quantitative changes are reported as possible diagnostic markers of various pathological conditions.^11,14,15) In fact, glycated albumin, in which glucose is added to the lysine residue of albumin, is used in clinical practice for blood glucose control in diabetes.

3. OXIDATIVE STRESS EVALUATION METHODS FOCUSING ON POST-TRANSLATIONAL MODIFICATIONS OF ALBUMIN

Albumin is a simple non-glycosylated protein consisting of 585 amino acids and contains 17 disulfide bonds in the molecule. Among the 35 cysteine residues in albumin, the cysteine at position 34 (Cys34), which has a free thiol group accounts for about 80% of the free thiol content in serum,¹⁶⁾ is the major site of anti-oxidative activity that is responsible for about 40% of total anti-oxidative activity of albumin¹⁷⁾ (Fig. 1). It is well known that Cys34 forms not only reversible disulfide bonds by reaction with low-molecular-weight thiols such as cysteine (Cys), homocysteine and glutathione but also is irreversibly modified by sulfinic acid and sulfonic acid, which are more highly oxidized forms.^18–20) Such oxidized forms of albumin can be used as biomarkers that serve as indicators of the state of oxidative stress in vivo. Terawaki et al. investigated the redox state of Cys34 in predialysis CKD patients using an HPLC method.²¹⁾ As a result, it was shown that oxidized albumin was increased with the deterioration of renal function, and that the deterioration of renal function and oxidative stress are closely related. We recently measured the post-translational modifications of albumin using electrospray-ionization time-of-flight mass spectrometry (ESI-TOF MS), which is capable of high-sensitivity and for producing high-throughput measurements, and found that oxidized albumin (cysteinylated albumin: an albumin molecule that is oxidatively cysteinylated at Cys-34) was found to increase with the progression of renal pathology^11,22) (Fig. 2).

Fig. 1. The Location of Cys34 of HSA

(PDB ID: 1E78.)

Fig. 2. Deconvoluted ESI-TOF MS Spectra of HSA

(a) Spectrum of HSA from a healthy subject. (b) Spectrum of HSA from a patient with CKD. The peaks correspond to the following: (1) Asp-Ala truncation from N-terminal of HSA, (2) Leu truncation from C-terminal of HSA, (3) unmodified HSA (reduced HSA), (4) Cys-Cys34-HSA (cysteinylated HSA: oxidized HSA), (5) glycated HSA and (6) glycated Cys-Cys34-HSA.

4. USEFULNESS OF OXIDIZED ALBUMIN MEASUREMENT

4-1. Relationship between Serum Free Thiol Group Content and CKD Onset in the General Population

Bourgonje et al. recently followed the association between serum free thiol group (-SH group) content and CKD development over a 10 year period using 4745 subjects from the general population without CKD (median estimated glomerular filtration rate (eGFR): 96 mL/min/1.73 m²) that were enrolled in The Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study in the Netherlands.²³⁾ The results indicated that residents with a low serum free thiol group content at the time of entry had a higher risk of developing CKD (eGFR less than 60 mL/min/1.73 m² or daily urinary albumin excretion of 30 mg or more). Considering that about 80% of the free thiol group content in serum is derived from Cys34 of albumin, this result suggests that the decrease in reduced albumin (unmodified albumin), that is, the increase in the levels of oxidized albumin, could be associated with the onset of CKD.

4-2. Diabetic Kidney Disease and Oxidized Albumin

Diabetic kidney disease (DKD) is a typical complication associated with type 2 diabetes, and is the primary disease leading to renal replacement therapy. Proteinuria and albuminuria are currently used as standard diagnostic markers, but it has become clear that there are many patients who show a rapid deterioration of renal function even at the stage of normal to microalbuminuria and atypical nephropathy in the absence of overt albuminuria. This suggests that there is a need for the development of markers capable of diagnosing disease progression and renal prognosis more accurately as compared to measurements of proteinuria and albuminuria.

We recently investigated the association between DKD and oxidative stress in patients with type 2 diabetes.²⁴⁾ Post-translational modifications of albumin in 257 outpatients with type 2 diabetes mellitus were analyzed, and the relationship between the progression of DKD and extent of post-translational modifications was evaluated. The results indicated that an increase in the percentage of oxidized albumin (cysteinylated albumin) was observed with increasing progression of the nephropathy stage based on the eGFR classification of the CKD clinical practice guideline (G1: 20.76% ± 0.47, G2: 24.51% ± 0.31, G3a: 26.67% ± 0.43, G3b: 28.84% ± 0.75, G4: 32.11% ± 0.80). A receiver operating characteristic (ROC) curve analysis demonstrated that, regarding the discrimination of nephropathy stages G2 to G3a, the diagnostic performance of cysteinylated albumin (area under the curve (AUC) = 0.665, cutoff value = 25.69% (sensitivity = 0.655, specificity = 0.657)) was higher than that of the urinary albumin-to-creatinine ratio (AUC = 0.500). A multiple regression analysis demonstrated that the levels of cysteinylated albumin were most strongly associated with decline in eGFR among cysteinylated albumin, age, hemoglobin A1C, diabetes duration, systolic blood pressure, body mass index, plasma albumin concentration, or urinary albumin-to-creatinine ratio as independent variables in patients with type 2 diabetes. In addition, a Kaplan–Meier analysis also demonstrated that, in the case of patients who exceeded the cut-off value for cysteinylated albumin, that the nephropathy stage progressed rapidly in 2 years as compared with the patients whose values did not exceed the cut-off value. Thus, monitoring the levels of cysteinylated albumin could be useful for the early diagnosis of DKD and for predicting renal prognosis.

4-3. Oxidized Albumin in Dialysis Patients

Terawaki et al. investigated the relationship between the redox state of albumin and cardiovascular complications in 86 dialysis patients. The redox state of Cys34 was measured using an HPLC method. The results showed that patients with lower levels of reduced albumin (higher levels of oxidized albumin) had a higher odds ratio of developing cardiovascular complications and a higher risk of death associated with cardiovascular complications.²⁵⁾ Lim et al. also investigated the relationship between oxidized albumin levels and cardiovascular mortality in 248 dialysis patients with normal serum albumin levels.²⁶⁾ The results showed that patients with higher oxidized albumin levels had a higher proportion of mortality due to cardiovascular complications, and also a higher rate of mortality including factors other than cardiovascular complications.

4-4. Oxidized Albumin as an Index of Therapeutic Efficacy

Yamamoto et al. reported that when spherical activated charcoal (AST-120: Kremezin^®), which suppresses the accumulation of uremic substances, was administered to dialysis patients, both uremic substances and oxidized albumin levels were reduced.²⁷⁾ We also found that the administration of AST-120 decreased the levels of oxidized albumin in 5/6-nephrectomized CKD rats.²⁸⁾ These data indicated that the administration of AST-120 has an indirect anti-oxidative effect through lower uremic substances. In addition, using mice with an acute kidney injury, we found that long-acting thioredoxin, an anti-oxidative and inflammatory modulator, reduced the level of oxidized albumin, showing therapeutic effects.²⁹⁾

There is a concern that the clinical use of iron-containing preparations induces an increase in oxidative stress via free iron-related Fenton reactions. Oral ferric citrate, a drug used to treat hyperphosphatemia in dialysis patients, is an iron-containing preparation. We showed that it does not increase oxidized albumin levels when administered at clinical doses.³⁰⁾ Based on the above findings, it would appear that monitoring the levels of oxidized albumin could be applied, not only to the evaluation of oxidative stress in renal pathology, but also to evaluate the therapeutic effects of drugs such as bardoxolone methyl, an anti-oxidative and inflammatory modulator that is used to treat kidney disease, or in studies of the appropriate dosage of iron preparations.

4-5. Oxidized Albumin as an Index for the Quality Evaluation of Antioxidant Drugs and Albumin Preparations

Some drugs have pleotropic antioxidant activities that differ from their main pharmacological activity. Anti-hypertensive agents such as angiotensin receptor blockers and hypoglycemic agents such as dipeptidyl peptidase-4 (DPP-4) inhibitors or sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to exert antioxidant effects directly or indirectly.^31–33) We also demonstrated that the administration of cinacalcet, a calcium-sensing receptor agonist, for the treatment of secondary hyperparathyroidism under maintenance dialysis has an antioxidant effect by lowering the level of oxidized albumin.³⁴⁾

Albumin preparations are clinically used for the treatment of hypoalbuminemia and hemorrhagic shock associated with burns, nephrotic syndrome and liver cirrhosis. The origin of the serum pool for purifying albumin, its purification conditions and storage/transport conditions are thought to affect oxidative modification and it is presumed that such a modification would also affect the function of albumin. We measured the post-translational modifications of albumin and evaluated the ligand-binding ability and antioxidant activity of albumin for five albumin preparations that are currently available in Japan.³⁵⁾ As a result, although there was no significant difference between the formulations in terms of the glycated form and the N-terminal truncated form, it was found that the ligand-binding ability and antioxidant ability differed among the formulations. Interestingly, formulations with a lower ligand-binding capacity and lower antioxidant capacity contained higher levels of oxidized albumin. We therefore conclude that analyzing the post-translational modifications of albumin preparations can be used for evaluating the quality of albumin preparations.

5. OXIDIZED ALBUMIN AS A RENAL EXACERBATING FACTOR

Is oxidized albumin simply a biomarker? Or, is it a progression factor? Hypochlorous acid (HOCl), which is produced by myeloperoxidase in neutrophils that infiltrate tissues accompanied by increasing oxidative stress or inflammation, produces advanced oxidation protein products (AOPPs) by the oxidative modification of serum proteins (mainly albumin). We recently clarified that AOPPs are involved in the development of renal tubular disorders by inducing abnormal fatty acid metabolism in renal tubular cells.³⁶⁾ Regarding the muscle atrophy and muscle weakness (sarcopenia) observed in CKD, AOPPs were found to show skeletal muscle atrophy and mitochondrial dysfunction effects.³⁷⁾ Interestingly, we also found that serum AOPPs and oxidized albumin levels were higher in dialysis patients with sarcopenia than in non-sarcopenia patients, suggesting that AOPPs and oxidized albumin may be applicable for use as diagnostic markers for sarcopenia in CKD patients.³⁷⁾

6. CONCLUSION

Oxidative stress has been reported to be associated with the progression of renal pathology as well as with the onset of complications. As of this writing, there was no quantitative system for evaluating oxidative stress that could be applied as a clinical test. The measurement of oxidized albumin (cysteinylated albumin) is a high-throughput measurement method that can be applied to clinical practice. It has the potential for being used as a biomarker for diagnosing renal disease progression and its complications in addition to the efficacy of therapeutic drugs (Fig. 3).

Fig. 3. Oxidized Albumin as an Index for Oxidative Stress Evaluation in CKD

Acknowledgments

I am grateful to Dr. Toru Maruyama and Dr. Hitoshi Maeda, Kumamoto University, Kumamoto, Japan; Dr. Masafumi Fukagawa, Tokai University School of Medicine, Kanagawa, Japan and Dr. Takashi Wada, Kanazawa University, Ishikawa, Japan; Dr. Motoko Tanaka and Dr. Kazutaka Matsushita, Akebono Clinic, Kumamoto, Japan; Dr. Hideaki Jinnouchi and Dr. Akira Yoshida, Jinnouchi Hospital Diabetes Care Center, Kumamoto, Japan; Dr. Kentaro Oniki and Dr. Junji Saruwatari, Kumamoto University; Dr. Masaki Otagiri, Dr. Shigeyuki Miyamura, Dr. Daisuke Kadowaki and Dr. Makoto Anraku, Sojo University, Kumamoto, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI 16H05114, 20H03406).

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2022 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.

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