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Abstract

Objectives

Oxidative stress is associated with the development and progression of chronic kidney disease (CKD) in humans. The aim of this study was to evaluate the concentrations of oxidative stress markers, 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA), in the plasma and urine of cats with different stages of CKD.

Methods

Plasma and urine samples were collected from cats with CKD that were referred to the Veterinary Medical Center of the University of Tokyo between April 2019 and October 2022. Plasma and urine samples were collected from healthy cats (n = 6 at most), cats with stage 2 CKD (n = 8) and stage 3–4 CKD (n = 12), and cats with idiopathic cystitis (disease control, n = 5). Plasma and urine concentrations of 8-OHdG and MDA were measured using ELISA and thiobarbituric acid reactive substances assay kits, respectively.

Results

The median plasma 8-OHdG concentrations were 0.156 ng/ml (<0.125–0.210 ng/ml) in the healthy group, <0.125 ng/ml (range <0.125 ng/ml) in the idiopathic cystitis group, 0.246 ng/ml (range 0.170–0.403 ng/ml) in cats with stage 2 CKD and 0.433 ng/ml (range 0.209–1.052 ng/ml) in cats with stage 3–4 CKD. Concentrations in stage 3–4 CKD were significantly higher than those in the healthy and disease control groups. Plasma MDA concentrations were low in the healthy and disease control groups and significantly higher in cats with stage 3–4 CKD. In every cat with CKD, plasma 8-OHdG and MDA concentrations were positively correlated with plasma creatinine concentrations (8-OHdG, r_s = 0.68; MDA, r_s = 0.67). Urinary 8-OHdG/urinary creatinine (u-CRE) and urinary MDA/u-CRE levels did not differ significantly between the groups; however, it was difficult to evaluate them because of the small sample size.

Conclusions and relevance

This report shows that plasma 8-OHdG and MDA concentrations increase with the severity of feline CKD. These markers may be useful for assessing oxidative stress in cats with CKD.

Keywords

8-OHdG chronic kidney disease oxidative stress malondialdehyde reactive oxygen species

Introduction

There are several reports of oxidative stress contributing to the development and exacerbation of renal interstitial injury in rodent models with chronic kidney disease (CKD).^1–3 Oxidative stress in the kidney is also an important factor in the progression of CKD in cats.⁴ Several biomarkers have been reported to reflect oxidative injury and pathogenesis of CKD in cats, for example liver-type fatty acid-binding protein (L-FABP), F2-isoprostane and transforming growth factor beta 1 (TGF-β1). A detailed review of these markers is available elsewhere.⁵

Major oxidative stress markers, such as 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA), have been evaluated in human patients with CKD. One of the oxidative products of DNA, 8-OHdG, is also a sensitive biomarker of oxidative stress that reflects oxidative DNA damage.⁶ However, serum 8-OHdG concentrations increase with the deterioration of renal function.⁷ The concentration of 8-OHdG was measured in cats in a previous report, which found that there was no significant difference in serum 8-OHdG concentrations between healthy cats and those with CKD.⁸ Since most cats with CKD in that report were classified as International Renal Interest Society (IRIS) stage 2, the concentrations of 8-OHdG in cats with late-stage CKD remain unknown. The concentration of MDA is an indicator of lipid peroxidation and oxidative tissue injury. MDA is also known as a thiobarbituric acid reactive substance⁹ and is widely measured using the thiobarbituric acid assay. Serum MDA concentrations are elevated in rats with significant kidney tissue damage.¹ In human patients with CKD, plasma MDA concentrations are higher than those in healthy groups.¹⁰ However, there have been no reports of MDA being measured in cats with different stages of CKD. In addition, there are no reports on the urinary concentrations of these markers in cats with CKD.

The purpose of this study was to determine the association between plasma and urinary 8-OHdG and MDA concentrations and the different stages of CKD in cats, and whether 8-OHdG or MDA is more useful in assessing oxidative stress in feline CKD.

Materials and methods

Cats

Blood plasma and urine supernatant samples were collected from cats with CKD, idiopathic cystitis (disease control) and healthy controls at the Veterinary Medical Center, The University of Tokyo, between April 2019 and October 2022. Samples that remained after routine clinical tests were obtained with informed consent from the owners. The diagnosis of CKD was based on the IRIS guidelines,¹¹ which were used as the inclusion criteria for this study. Briefly, all the cats with CKD were diagnosed based on the results of a history, physical examination, blood examination (complete blood count [ProCyte Dx Hematology Analyzer; IDEXX Laboratories], blood urea nitrogen (BUN) and plasma creatinine (p-CRE), calcium, inorganic phosphorus and electrolytes (DRI-CHEM 7000V; Fujifilm VET Systems), urinalysis (urine specific gravity, the urine dipstick test [Arkray urine test strips; Arkray] and sediment observation) and imaging using abdominal radiography and ultrasound. Cats with no abnormalities on physical examination, blood tests or urinalyses were considered healthy. Cats with idiopathic cystitis were used as disease controls. Cats with clinical signs of irritative voiding, such as pollakiuria or dysuria, and the absence of neoplasia or bacteriuria were diagnosed with idiopathic cystitis by exclusion diagnosis.¹² All cats with idiopathic cystitis were diagnosed based on the results of the history, physical examination, blood examination, urinalysis and imaging. They did not have any uroliths. The urine supernatant samples were collected only from cases in which urinalysis was performed. The samples were centrifuged at 500 × g for 15 mins, and the supernatants were used. The collected plasma and urine were stored in a freezer at −80 °C until use. The urine protein:creatinine ratio (UPC) was measured using the enzymatic colorimetric method provided by Fujifilm VET Systems. Sample collection was approved by the Animal Ethical Committee of the University of Tokyo (P22-009).

Measurement of 8-OHdG and MDA in plasma or urine

The concentrations of 8-OHdG in the plasma and urine were measured using a highly sensitive ELISA kit for 8-OHdG (Japan Institute for the Control of Aging). MDA concentrations in the plasma and urine were measured using a malondialdehyde assay kit (Japan Institute for the Control of Aging). For validation assessment, a spike and recovery test, dilutional linearity test, and intra- and inter-assay CV were performed. Feline plasma and urine pool samples for validation were prepared by mixing surplus specimens from several cats and healthy cats. The additive for the spike and solution for the inter- and intra-assays were prepared from the standard diluent and pooled samples. For the dilutional linearity test, feline pool samples were diluted 1:2 and 1:4 using phosphate-buffered saline. All samples were analysed in more than triplicate for the validation tests, and five aliquots were measured for intra- and inter-assay coefficient of variation (CV). The specimens were analysed in duplicate. The lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) based on the standard solution were in the range of 0.125–10.0 ng/ml and 1.00–8.00 µM, respectively. When the measured concentrations of the specimens were higher than the ULOQ, they were measured again after appropriate dilution. Urinary 8-OHdG and MDA concentrations were adjusted according to the urinary creatinine (u-CRE) concentrations. The u-CRE concentrations were measured using a DRI-CHEM 7000V analyser by diluting 100 times with distilled water.

Statistical analyses

Statistical analyses were performed using Prism 5 version 5.02 (GraphPad Software). Statistical significance was evaluated using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. Correlation between two variables was evaluated using Spearman’s rank correlation coefficient, rs. Statistical significance was defined as a P value of <0.05.

Results

Validation of the assays

The 8-OHdG and MDA kits were validated using mixed plasma and urine samples derived from several cats. For the spike and recovery analyses, the recovery rates were 96–104% and 95–125% for the 8-OHdG and MDA kits, respectively (see Table 1 in the supplementary material). The dilutional linearity assessment was not performed perfectly, because several diluted samples were lower than the LLOQ; however, the recovery rates were 84–122% and 88–110%, respectively (Supplementary Table 2). The intra-assay CVs were 3.0–8.1% and 17.6–23.0% (Supplementary Table 3). For the inter-assay CVs, standard solutions were used for quality checks in the kits, giving values of 12.9% and 7.0%.

Biomarkers of kidney function

Cats with CKD had significantly higher BUN and p-CRE concentrations than healthy or disease controls. No significant changes were observed in the BUN or p-CRE levels in healthy or disease controls (Table 1). The UPC levels were >0.4 in 4/13 cases, 0.2–0.4 in 3/13 cases and <0.2 in 6/13 cats with CKD, respectively.

Table 1

The levels of biomarkers and oxidative markers in cats

	Healthy	Disease control	CKD stage 2	CKD stage 3–4	RI
Blood urea nitrogen (mg/dl)	21.1 (14.4–33.2), 6	21.2 (20.3–25.4), 5	45.2 (34.0–69.0), 8	84.5 (45.1–361.2)**, ^†††, 12	17.6–32.8
Plasma creatinine (mg/dl)	0.91 (0.73–1.36), 6	1.22 (1.20–1.55), 5	2.43 (1.69–2.70)*, 8	4.32 (2.34–17.49)**, ^††, 12	0.80–1.80
Plasma 8-OHdG (ng/ml)	0.156 (<0.125–0.210), 6	<0.125 (<0.125), 5	0.246 (0.170–0.403)^†, 8	0.433 (0.209–1.052)*, ^†††, 8
Urinary 8-OHdG/urinary creatinine (ng/mg)	3.17 (0.03–5.22), 6	1.78 (1.50–4.22), 5	4.00 (2.58–6.71), 6	4.83 (1.56–7.87), 7
Plasma MDA (µM)	2.00 (1.54–2.54), 5	2.36 (1.53–4.39), 4	3.14 (2.06–4.61), 5	4.79 (2.18–8.67)*, 7
Urinary MDA/urinary creatinine (µmol/g)	2.59 (1.98–3.34), 5	2.46 (1.80–3.49), 5	3.97 (1.38–6.41), 6	3.64 (2.86–6.69), 6

Values are median (range), n

P <0.05, **P <0.001 vs healthy cats

†

P <0.05, ^††P <0.01, _†††P <0.001 vs disease controls

CKD = chronic kidney disease; RI = reference interval; 8-OHdG = 8-hydroxy-2’-deoxyguanosine; MDA = malondialdehyde

Biomarkers of oxidative stress

Plasma 8-OHdG concentrations were measured in eight cats with stage 2 CKD, eight cats with stage 3–4 CKD, six healthy cats and five cats with idiopathic cystitis as disease controls. The median plasma 8-OHdG concentration in healthy cats was 0.156 ng/ml (range <0.125–0.210 ng/ml, n = 6), with similarly low levels in the disease controls (<0.125 ng/ml, range <0.125, n = 5). Plasma 8-OHdG concentrations in stage 2 CKD (0.246 ng/ml, range 0.170–0.403, n = 8) tended to be higher than those in healthy cats; however, no significant changes were observed. Plasma 8-OHdG concentrations in cats with stage 3–4 CKD (0.433 ng/ml, range 0.209–1.052, n = 8) were significantly higher (P <0.05) than those in healthy and disease control cats (Figure 1a). Urinary 8-OHdG/u-CRE levels were measured in six cats with stage 2 CKD, seven cats with stage 3–4 CKD, six healthy cats and five cats with idiopathic cystitis. Urinary 8-OHdG/u-CRE levels showed a similar trend; however, no significant changes were observed in healthy cats or cats with CKD (Figure 1b).

Figure 1

(a) Plasma and (b) urinary 8-OHdG concentrations in healthy cats, cats with idiopathic cystitis as disease controls, and cats with IRIS stages 2 and 3–4 CKD. Median values are indicated by horizontal lines. The lower limit of quantitation is indicated by the dotted line. *P <0.05, ***P <0.001; Kruskal–Wallis test followed by Dunn’s test. 8-OHdG = 8-hydroxy-2′-deoxyguanosine; CKD = chronic kidney disease; u-CRE = urinary creatinine

Plasma MDA concentrations were also measured in five cats with stage 2 CKD, seven cats with stage 3–4 CKD, five healthy cats and four cats with idiopathic cystitis. The median plasma MDA concentration in healthy cats was 2.00 µM (range 1.55–2.54, n = 5), with similarly low levels in the disease controls (2.36 µM, range 1.53–4.39, n = 4). Plasma MDA concentrations in cats with stage 2 CKD (median 3.14 µM, range 2.06–4.61, n = 5) tended to be higher than those in healthy cats, but no significant differences were observed. Plasma MDA concentrations in cats with stage 3–4 CKD (median 4.79 µM, range 2.18–8.67, n = 7) were significantly higher (P <0.05) than those in healthy cats (Figure 2a). Urinary MDA/u-CRE concentrations were measured in six cats with CKD stage 2, six cats with CKD stage 3–4, five healthy cats and five cats with idiopathic cystitis. No significant changes were observed in the urinary MDA/u-CRE levels in healthy cats and cats with CKD (Figure 2b). In addition, when all 20 cats with CKD and others were compared, the plasma 8-OHdG concentrations of cats with CKD were significantly higher than those of the healthy and disease control groups, and the concentrations of MDA in cats with CKD were significantly higher than those in healthy cats (Supplementary Figure 1).

Figure 2

(a) Plasma and (b) urinary MDA concentrations in healthy cats, cats with idiopathic cystitis as disease controls, and cats with IRIS stages 2 and 3–4 CKD. Median values are indicated by horizontal lines. *P <0.05, Kruskal–Wallis test followed by Dunn’s test. CKD = chronic kidney disease; MDA = malondialdehyde; u-CRE = urinary creatinine

Plasma 8-OHdG concentrations were weakly positively correlated with p-CRE but not with BUN (Figure 3a, p-CRE, r_s = 0.68, P <0.01; Figure 3b, BUN, r_s = 0.48, P = 0.07). Plasma MDA concentrations were weakly and positively correlated with both p-CRE and BUN levels in cats with CKD (Figure 3c, p-CRE, r_s = 0.67, P <0.01; Figure 3d, BUN, r_s = 0.56, P <0.05). However, no statistical correlation was observed between plasma 8-OHdG and MDA concentrations (Figure 3e). There was no significant correlation between the urine 8-OHdG/u-CRE, MDA/u-CRE and p-CRE levels (data not shown). In addition, UPC levels were compared with these oxidative markers in the cats with CKD, but no significant associations were observed.

Figure 3

Correlation between (a) plasma creatinine and plasma 8-OHdG concentrations, (b) blood urea nitrogen and plasma 8-OHdG concentrations, (c) plasma creatinine and plasma MDA concentrations, (d) BUN and plasma MDA concentrations and (e) plasma 8-OHdG and MDA concentrations in cats with CKD. Spearman rank correlation coefficient. 8-OHdG = 8-hydroxy-2′-deoxyguanosine; BUN = blood urea nitrogen; CKD = chronic kidney disease; CRE = plasma creatinine; MDA = malondialdehyde

Discussion

In the present study, plasma 8-OHdG and MDA concentrations in cats with CKD were significantly higher than those in healthy cats and correlated with p-CRE. To the best of our knowledge, this is the first study to report an association between plasma 8-OHdG and p-CRE concentrations and plasma MDA and p-CRE concentrations in cats with CKD. In a previous report, no significant differences were observed in the baseline concentrations of plasma 8-OHdG between healthy cats and cats in the early stages of CKD.⁸ In that report, the sample size was small and weakly powered for the analysis, similar to this study. Their study differed from ours in that they collected specific samples of stage 2 CKD. In this study, plasma 8-OHdG and MDA concentrations in the stage 3–4 CKD group were especially high, which could explain this discrepancy. As mentioned in the Introduction, oxidative stress is reportedly related to the deterioration of cats with CKD.^1,8,13,14 The evaluation of oxidative markers could have a deeper significance if there is an association between mortality and the levels of oxidative markers in cats, just as in humans.⁷

In this study, 8-OHdG and MDA were identified as markers of oxidative stress. These oxidative markers exhibit different characteristics. 8-OHdG concentrations are associated with DNA oxidation and cellular damage.^6,15 In a previous study on humans, serum 8-OHdG concentration was associated with increased all-cause mortality risk in individuals with a wide range of estimated glomerular filtration rates, which are independent of inflammation.⁷ The MDA concentrations are associated with the oxidation of lipids and reportedly increase with severity in human non-dialysis patients with CKD.¹⁰ In this study, both 8-OHdG and MDA concentrations were elevated in the plasma of cats with IRIS stage 3–4 CKD, suggesting that CKD may lead to oxidation of both DNA and lipids. However, further studies with larger numbers of cats in different IRIS stages are required to confirm whether one of these biomarkers is superior for the detection of oxidation in cats with CKD.

In this study, plasma 8-OHdG and MDA concentrations were elevated in cats with CKD, but not significantly in urine. Urinary 8-OHdG/u-CRE is elevated in human patients with CKD and proteinuria compared to those without proteinuria.¹⁶ In the present study, there was no correlation between UPC and urinary 8-OHdG/u-CRE or MDA/u-CRE. Future studies should assess urinary 8-OHdG and MDA concentrations in cats with CKD and proteinuria.

This study had several limitations. The validation results of both the 8-OHdG and MDA kits suggest that these two measurement systems should work sufficiently but not very accurately. Because the sample size was small and the groups were underpowered, there could be a risk of type 2 statistical errors. Other limitations include a lack of association between proteinuria and oxidative markers, lack of evaluation of antioxidants, such as superoxide dismutase and glutathione peroxidase, and lack of prognostic studies.

Conclusions

This is the first report describing the plasma 8-OHdG and MDA concentrations in cats at different stages of CKD. The plasma concentrations of these markers were increased in the IRIS stage 3–4 cats, and weakly but significantly correlated with BUN and p-CRE in cats with CKD. These findings suggest that these concentrations could be helpful for assessing the intensity of oxidative stress in cats with CKD.

Supplemental Material

Figure S1

(a) Plasma 8-OHdG; (b) urinary 8-OHdG/u-CRE; (c) plasma MDA; and (d) urinary MDA/u-CRE concentrations in healthy cats, cats with disease controls, and cats with CKD

Supplemental Material

Table S1

Spike and recovery analysis in a Highly Sensitive ELISA Kit for 8-OHdG and a Malondialdehyde Assay Kit

Supplemental Material

Table S2

Dilutional linearity assessment of a Highly Sensitive ELISA Kit for 8-OHdG and a MDA Assay Kit

Supplemental Material

Table S3

Intra- and inter-assay variation for a Highly Sensitive ELISA Kit for 8-OHdG and a MDA Assay Kit

Footnotes

Acknowledgements

The authors thank the cat owners for kindly enrolling the cats in this study.

Accepted: 15 April 2023

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by JSPS KAKENHI grant number 21H02355.

Ethical approval

The work described in this manuscript involved the use of non-experimental (owned or unowned) animals. Established internationally recognised high standards (‘best practice’) of veterinary clinical care for the individual patient were always followed and/or this work involved the use of cadavers. Ethical approval from a committee was therefore not specifically required for publication in JFMS. Although not required, where ethical approval was still obtained, it is stated in the manuscript.

Informed consent

Informed consent (verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (experimental or non-experimental animals, including cadavers) for all procedure(s) undertaken (prospective or retrospective studies). No animals or people are identifiable in this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Yasuyuki Momoi

Tomohiro Yonezawa

Supplementary material

The following files are available online:

Table S1: Spike and recovery analysis in a Highly Sensitive ELISA Kit for 8-OHdG and a Malondialdehyde Assay Kit

Table S2: Dilutional linearity assessment of a Highly Sensitive ELISA Kit for 8-OHdG and a MDA Assay Kit

Table S3: Intra- and inter-assay variation for a Highly Sensitive ELISA Kit for 8-OHdG and a MDA Assay Kit

Figure S1: (a) Plasma 8-OHdG; (b) urinary 8-OHdG/u-CRE; (c) plasma MDA; and (d) urinary MDA/u-CRE concentrations in healthy cats, cats with disease controls, and cats with CKD

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Supplementary Material

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8-Hydroxy-2′-deoxyguanosine and malondialdehyde in plasma and their association with disease severity in 20 cats with chronic kidney disease

Abstract

Objectives

Methods

Results

Conclusions and relevance

Keywords

Introduction

Materials and methods

Cats

Measurement of 8-OHdG and MDA in plasma or urine

Statistical analyses

Results

Validation of the assays

Biomarkers of kidney function

Biomarkers of oxidative stress

Discussion

Conclusions

Supplemental Material

Figure S1

Supplemental Material

Table S1

Supplemental Material

Table S2

Supplemental Material

Table S3

Footnotes

Acknowledgements

Conflict of interest

Funding

Ethical approval

Informed consent

ORCID iD

Supplementary material

References

Supplementary Material