Slov Vet Res | Vol 60 No 1 | 25 The Effect of a Specific Chicken Based Renal Diet as Monotherapy on Clinical, Biochemical, Urinary and Serum Oxidative Stress Parameters in Cats With CKD Stage 1 and 2 Key words clinical parameters, symmetric dimethylarginine, oxidative stress, renal diet, cats, chronic kidney disease, urinary protein electrophoresis Martina Krofič Žel1, Alenka Nemec Svete1, Breda Jakovac Strajn2, Katarina Pavšič Vrtač2, Tomaž Vovk3, Nataša Kejžar4, Darja Pavlin1* 1Small Animal Clinic, 2Institute of Food Safety, Feed and Environment, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, 3Chair of Biopharmacy and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, 4Institute for Biostatistics and Medical Informatics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia *Corresponding author: darja.pavlin@vf.uni-lj.si Abstract: The aim of the study was to investigate the effect of a therapeutic renal diet on selected clinical, biochemical, and urinary parameters and on selected parameters of oxidative stress in cats with early stages of chronic kidney disease (CKD). A prospective study of a 3-month duration was conducted to evaluate the effect of renal diet on se- lected clinical and laboratory parameters in client-owned cats with early stages of CKD. Of a total of 29 enrolled client-owned cats, nineteen (19) cats completed the study, ten receiving renal diet and nine receiving a diet of the owner’s choice. A clinical examina- tion was performed, and blood and urine samples were collected on the day of presen- tation and at regular check-ups after 3-4, 7-8, and 10-12 weeks. Serum creatinine and symmetric dimethylarginine (SDMA) concentrations and selected parameters of oxida- tive stress (plasma glutathione peroxidase (GPX) activity and plasma malondialdehyde (MDA) and serum selenium concentrations), were measured and electrophoresis of uri- nary proteins was performed. At inclusion, a significant positive correlation (p < 0.001) was found between serum selenium concentration and plasma GPX activity (Pearson correlation coefficient 0.83 (95% CI: [0.65 - 0.92]) and a significant negative correlation (p < 0.001) between serum SDMA and urine specific gravity (Pearson correlation coeffi- cient -0.70 (95% CI: [-0.87 - (-0.38)]). At the end of the 3-month feeding trial no significant difference was found in SDMA and creatinine concentrations. Received: 11 July 2022 Accepted: 6 February 2023 DOI 10.26873/SVR-1515-2023 UDC 636.8.09:616.61:613.24:637.54:616.152/.153 Pages: 25–35 Original Research Article Introduction Chronic kidney disease (CKD) in cats has an overall preva- lence of 2-3% of the feline population (1, 2). The prevalence is higher in older cats: 10% in cats older than ten years (3) and 28% in cats older than 12 years (4). A prevalence of 28% and a 67% is reported in the overall population of cats and in those older than 18 years old, respectively, based on the serum concentration of symmetric dimethylarginine (SDMA) (5). Chronic kidney disease is characterized by progressive loss of functional renal tissue, leading to renal fibrosis, which can cause uremic crisis and death (6). Early recognition of CKD is essential for prompt management of such patients, leading to a better long-term prognosis (7,8). Proteinuria is an important and independent predictor of worsening of CKD (9,10,11). In contrast to dogs, chronic in- terstitial nephritis is predominantly found in cats. Therefore, tubular proteinuria is more common than glomerular pro- teinuria (12). SloVetRes_NOTRANJOST ver2.indd 25 09/05/2023 10:14 26 | Slov Vet Res | Vol 60 No 1 Oxidative stress aids progression of CKD in human patients (13). Plasma glutathione peroxidase (GPX), synthesized by renal tubular cells, is the major reactive oxygen species scavenger in the kidneys (14). In human CKD patients, plas- ma GPX activity decreases as the disease progresses and its activity is already reduced in patients with mild chronic uremia (15,16). On the other hand, a significantly higher plasma GPX activity was found in cats with CKD IRIS stage 4 compared with healthy cats (17). Plasma malondialdehyde (MDA) is one of the most popular and reliable markers of the extent of lipid peroxidation and thus oxidative stress (18). Selenium is an integral part of selenoproteins, one of which is plasma GPX. This micro- element is present in protein-rich foods, and its excess is excreted via the kidneys (19). Unlike human uremic patients, uremic cats do not have a selenium deficiency (17). According to evidence-based veterinary medicine, renal diet is the therapy of choice in both feline and canine CKD patients from the International Renal Interest Society (IRIS) stage 2 (20,21,22). Hall and colleagues (23) reported that cats with IRIS CKD stage 1 and 2 benefit from a diet with increased caloric density and enhanced concentrations of carnitine and essential amino acids. The biomarkers of kid- ney function, body weight and lean muscle mass were sta- ble in cats consuming such a diet. However, a recent study reported that feeding a highly phosphorus-restricted diet to cats with early-stage CKD may lead to hypercalcemia and urolithiasis, while a diet moderately restricted in protein and phosphorus may be beneficial (24). The authors are aware of only a few studies that deal with oxidative stress in feline CKD (17, 25, 26, 27, 28, 29, 30). However, the results of these studies are inconclusive about the role of oxidative stress in the pathophysiology of CKD. Furthermore, there is a lack of data on the effects of renal diet on oxidative stress parameters in CKD cats. The aim of the present study was to investigate/explore the effect of a therapeutic renal diet on selected clinical, bio- chemical, and urinary parameters and on selected param- eters of oxidative stress in cats with early stages of CKD and to provide credible insight with monitoring of clinical and laboratory parameters. Materials and methods This prospective study was conducted on client-owned cats with early stages of CKD and lasted for three months. The inclusion criterion was CKD stage 1 or 2, according to the IRIS guidelines (31), with no previous treatment recorded. Cats with acute kidney injury, prerenal or postrenal azo- temia, nephropathy of toxic or infectious origin within the last 28 days, urinary tract obstruction, acute systemic in- flammation, liver disease, chronic heart failure, cancer or serologically positive for feline leukemia or feline immuno- deficiency were excluded from the study. All owners signed a consent form before enrolling the cats in the study. All procedures complied with the relevant Slovenian governmental regulations (Animal Protection Act, Official Gazette of the Republic of Slovenia, No. 43/2007). The cats were randomly divided into two groups: cats in the control group, which received a regular diet and cats in the experimental group, which received a renal diet. Simple randomization method with sequentially numbered sealed envelopes was used to group the patients (32). The cats in both groups received their diet ad libitum ac- cording to their habitual regime. Clinical examination in- cluding body weight monitoring, blood pressure measure- ment, routine hematological and biochemical analyses, and urinalysis with UPC (urine protein to creatinine ratio) were performed on the day of presentation and regular check- ups after 3-4, 7-8, and 10-12 weeks. In addition to routine laboratory parameters, measure- ments of SDMA concentration, and selected parameters of oxidative stress (GPX, MDA, selenium) were also performed at each check-up and will be described below. Composition of the diet The cats in the experimental group were fed Vet Life Feline Renal Formula (Farmina Pet Foods, Naples, Italy). The composition of the renal diet is shown in Table 1. The re- nal diet used in the study had the same lot number for all cats. The cats in the control group continued to receive the maintenance diet to which they were accustomed to prior to participation in the study. Blood and urine sample collection, processing, and analysis Blood samples were taken from the jugular vein and trans- ferred into serum separator tubes (Vacuette, Greiner Bio- One, Kremsmunster, Austria) for the determination of se- rum biochemical profiles, including SDMA, and antigen detection of feline leukemia virus (FeLV) and specific an- tibody against feline immunodeficiency virus (FIV). The tubes were stored for 30 minutes at room temperature to clot and then centrifuged at 1300 × g for 10 minutes at room temperature to separate the serum. Serum samples for the determination of routine biochemical parameters (urea, creatinine, alanine aminotransferase, alkaline phosphatase, total proteins, albumins, total calcium, inorganic phosphate, electrolytes (sodium, potassium, chloride)) were analysed on the day of blood collection. For measurement of SDMA concentration in serum, an aliquot of the serum sample was prepared and immediately stored at -80°C until anal- ysed in batch. SloVetRes_NOTRANJOST ver2.indd 26 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 27 Blood samples for hematological analysis were collected into 0.5 ml EDTA-containing tubes (BD Microtainer Tubes, Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). Urine samples were collected by cystocentesis and ana- lyzed within 1 to 2 hours. Biochemical profiles (urea, creatinine, alanine aminotrans- ferase, alkaline phosphatase, total proteins, albumins, to- tal calcium, inorganic phosphate), except electrolytes and SDMA, were determined with an automated biochemistry analyser RX Daytona (Randox, Crumlin, UK). The electro- lytes were determined with an Ilyte electrolyte analyzer (Instrumentation Laboratory, Lexington, Massachusetts, USA). Hematological analyses were performed with an au- tomated laser hematology analyzer ADVIA 120 (Siemens, Munich, Germany) using species-specific software. ELISA for the detection of antigen against FeLV and spe- cific antibody against FIV were carried out according to the instructions of the manufacturer (IDEXX, Lenexa, Kansas, USA) on the day of collection. Determination of the MDA concentration Blood samples for the determination of plasma MDA con- centration were collected into 2 ml EDTA-containing tubes (Vacuette, Greiner Bio-One, Kremsmunster, Austria). All samples were immediately centrifuged at 1500 × g for 15 min at 4°C. The plasma was separated and immediately frozen at -80°C until analysis. The total plasma concentration of MDA was determined by a gentle alkaline saponification and derivatization method (33). MDA was derivatized with 2,4-dinitrophenylhydrazine to a pyrazole derivative and determined with an Agilent 1200 series high performance liquid chromatography sys- tem (Agilent, Waldbronn, Germany). The derivatized sam- ples were separated on an Agilent Eclipse XBD-C18 column by gradient elution with acetonitrile, water and acetic acid and the MDA derivative was detected with the diode array detector. The plasma MDA concentration was expressed as µmol per L (µmol/L). Determination of the SDMA concentration All serum SDMA concentrations were measured in batch at the end of the study by IDEXX Laboratories in Germany (IDEXX SDMA Test, IDEXX Laboratories INC., Leipzig, Germany). Determination of GPX activity Plasma GPX activity was measured spectrophotometri- cally with an automated biochemistry analyzer RX-Daytona (Randox, Crumlin, UK) using the commercial Ransel kit (Randox Laboratories, Crumlin, UK) which is based on the Table 1: The composition of the renal diet Raw protein 26.00% Raw oils and fats 20.00% Raw fiber 2.40% Raw ashes 7.30% Calcium 0.80% Phosphorus 0.60% Sodium 0.35% Potassium 0.90% Magnesium 0.07% Omega 3 fatty acids 0.40% Omega 6 fatty acids 3.90% EPA 0.10% DHA 0.15% Energy value 3965 kcal/kg – 16.6 MJ/kg Nitrogen-free extract/1000 kcal 11.77 g/1000 kcal Selenomethionine 60 mg per kg corresponding to 13.5 mg selenium/kg dry matter) Legend: EPA eicosapentaenoic acid; DHA docosahexaenoic acid Composition: pea starch, potatoes, chicken fat, hydrolyzed fish proteins, dehydrated whole eggs, hydrolyzed chicken proteins, dehydrated chicken meat, quinoa seed extracted, dehydrated fish, fish oil, calcium carbonate, inulin, fructooligosaccharides, mannanoligosaccharides, potassium chloride, sodium chloride, glucosamine (500 mg/kg), Marigold extract (source of lutein) Additives per kg Nutritional additives: Vitamin A 15000 IU; Vitamin D3 600 IU; Vitamin E 550 mg; niacin 125 mg; pantothenic acid 42 mg; Vitamin B2 17 mg; Vitamin B6 7 mg; Vitamin B1 8 mg; Vitamin H 1.3 mg; folic acid 1.3 mg; Vitamin B 12 0.08 mg; choline chloride 2500 mg; beta-carotene 1.5 mg; zinc chelate of the analogous methionine hydroxylase 725 mg; manganese chelate of the analogous methionine hydroxylase 385 mg; ferrous chelate of glycine hydrate 185 mg; copper chelate of the analogous methionine hydroxylase 54 mg; selenomethionine 60 mg; calcium iodate anhydrous 2.4 mg; taurine 2000 mg; DL methionine 5000 mg; L-lysine HCl 2000 mg; L-tryptophan 2000 mg; L-carnitine 250 mg. Technological additives: potassium citrate 3000 mg. Antioxidants: tocopherol-rich extracts of natural origin 10 mg. SloVetRes_NOTRANJOST ver2.indd 27 09/05/2023 10:14 28 | Slov Vet Res | Vol 60 No 1 29 cats met the inclusion criteria and were randomised 15 cats in the experimental group 10 cats completed the study in the experimental group 5 cats excluded 1 cat: renal lymphoma 2 cats: urethral obstruction 2 cats: less than 3 sample collections due to uncompliant owner 14 cats in the control group 9 cats completed the study in the control group 5 cats excluded 1 cat: urethral obstruction 4 cats: less than 3 sample collections due to uncompliant owner Figure 1: The flow diagram of cats with CKD during the study Paglia and Valentine method (34). The activity of plasma GPX was expressed as units per L (U/mL). Determination of the selenium concentration The microwave digestion of serum samples was performed with a Start D Microwave Acceleration Reaction System (Milestone, Sorisole, Italy). 0.4 to 1 mL of the samples were transferred into a 100 mL Teflon vessel and 3 mL 65% nitric acid, 0.5 mL 30% hydrogen peroxide and 4.5 mL Milli-Q wa- ter were added. The samples were digested in a closed 10- vessel microwave system at 200°C for 30 min. After cooling to room temperature, the solutions were diluted with Milli-Q water, and the concentrations of selenium were determined by inductively coupled plasma mass spectrometry (Varian 820-MS, Mulgrave, Australia). Argon was used as the car- rier gas, and the isotope 78Se was selected as the analyti- cal mass in ICP-MS normal sensitivity mode. For measure- ments of selenium, a Collision Reaction Interface (CRI) was used to reduce common polyatomic interferences. Urinalysis Urinalysis included the measurement of specific grav- ity with a refractometer, the use of a standard multitest urine dipstick (Multistix 10SG, Siemens, Munich, Germany) and microscopic examination of the urine sediment. The urine samples were centrifuged at 800 × g for 10 minutes at room temperature. Urine supernatants were used to determine protein and creatinine concentrations to calcu- late the UPC. Protein and creatinine concentrations were measured with an automated biochemistry analyzer RX Daytona (Randox, Crumlin, UK) using the pyrogallol red and picric acid methods, respectively. Protein concentrations were not determined if the urine samples were grossly con- taminated with blood. Gel electrophoresis was performed routinely by a commercial laboratory Euregio Laboratory Services (Kerkrade, Netherlands) in batch at the end of the study. Statistical analysis Based on the sample size, five keynote parameters were selected for statistical analysis (body weight, creatinine, SDMA, MDA and GPX). The differences between the first and the last (4th) measurement time-point were compared. The remaining parameters were presented in the form of descriptive statistics (median and interquartile range (IQR)) and with boxplots over time (Supplementary material). Basic characteristics and baseline measurements of sys- tolic blood pressure selected hematological, biochemical, oxidative stress, and urinalysis parameters in both groups were compared using the Fisher exact test (categorical) and the Mann-Whitney U test (numerical variables). Since a slight deviance towards older cats in the experimental group was observed, the comparison of the difference in keynote parameters was adjusted for age by the use of linear regression. The P-values for the group comparison from the linear models were corrected by Holm procedure (5%). The Pearson product-moment correlation was used to in- vestigate the possible correlation between the parameters (scatter plots are presented in the Supplementary material). SloVetRes_NOTRANJOST ver2.indd 28 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 29 Results Patients’ baseline characteristics A total of 29 cats of different breeds were enrolled, all of which were neutered. Of 29 cats enrolled in the study, 19 cats (ten cats in the ex- perimental group and nine cats in the control group) com- pleted the study. They represented the sample that we used for all analyses. The flow diagram of cats included in the study is presented in Figure 1. Blood and urine samples were collected at all four sched- uled check-ups from most cats enrolled. The missing blood and urine samples were not obtained due to problems with owners’ compliance. In all animals the first and the last (10- 12 weeks later) check-up were performed. Baseline characteristics (demographic data) are shown in Table 2. At the point of randomization in terms of age, sex, and body weight, there were no significant differences be- tween the experimental group and the control group; how- ever, the cats in the experimental group that completed the study were slightly older. Clinical signs and baseline clinical and laboratory parameters Clinical signs at presentation were mild in all included cats; the owners usually reported polyuria/polydipsia. At the end of the study, most owners of cats in the experimental group reported an improvement in the clinical status of the cats with reduced vomiting. At inclusion, all cats of both groups were reported to have normal appetite, which remained un- changed during the whole observation period. On each occasion, the owners were asked to evaluate their cats’ appetite and acceptance; the information on the ap- petite and acceptance of the diet was recorded at each check-up. The cats were given the amount of the diet that was appropriate for their weight as recommended by the manufacturer. The new diet was accepted 100 % by all cats in the experimental group; and the diet change was achieved in two weeks. During the first two weeks, the new diet was mixed with the usual diet and the amount of the new diet was gradually increased until the meal consisted only of the new diet. The owners reported that the entire amount of the meal was eaten by all included cats both during the transition period and during the study. Furthermore, the amount of the diet eaten during the study remained the same as before being enrolled into the study. Medians (IQR) for baseline and final (4th) measurement of laboratory parameters are presented in Supplementary material. The distributions of none of the parameters mea- sured at baseline significantly differed between groups. Time monitoring of all measured parameters is also pre- sented graphically in Supplementary material. Serum creatinine concentration During the study, median serum creatinine concentrations decreased in both the experimental and control groups (Supplementary material). The age adjusted difference in mean serum creatinine concentration decrease between both groups was not significant (Figure 2 A, Table 3). With one exception, all the included cats had stable CKD and re- mained at the same IRIS stage during the study. However, one cat in the experimental group was reclassified from IRIS 2 to 3 at the end of the study. The exact cause of the increase in serum creatinine concentration in that cat was not found. Seven out of 19 cats with elevated serum SDMA and creatinine concentrations, abnormal renal imaging findings and pathological urinary sediment, consistent with CKD, that were classified to IRIS stage 2, retained their urine concentration ability (Supplementary material). Serum SDMA concentration After the 3-month feeding trial, the median serum SDMA concentration decreased numerically in the Table 2: Baseline demographic and laboratory characteristics of cats in the experimental and the control group Group Control group (n = 9) Experimental group (n = 10) p value F/M 4/5 3/7 0.65 Age (months) Median (IQR) 78 (64−107) 116 (94−166) 0.066 Body weight (kg) Median (IQR) 5.9 (3.3−6.5) 4.9 (3.6−6.2) 0.842 Creatinine Median (IQR) 140.2 (130.0−178.8) 168.9 (161.1−178.8) 0.441 UPC (unitless) Median (IQR) 0.14 (0.12−0.19) 0.17 (0.11−0.26) 0.755 Legend: F-female cats; M-male cats; IQR-interquartile range SloVetRes_NOTRANJOST ver2.indd 29 09/05/2023 10:14 30 | Slov Vet Res | Vol 60 No 1 experimental group but remained the same in the control group (Supplementary material). There was no significant difference in age-adjusted de- crease of serum SDMA concentration between both groups (Table 3). Plasma MDA concentration At inclusion there was no significant difference in median plasma MDA concentrations between the sampled cats in the control group and those in the experimental group (Supplementary material). After the 3-month feeding trial, the mean age-adjusted difference in MDA was negligible in both groups (Table 3). Serum selenium concentration and plasma GPX activity In sampled cats receiving renal diet, median values of plasma GPX activity and serum selenium concentra- tion did not differ from the median values in the control group (Supplementary material). There was a negligible mean age-adjusted difference in plasma GPX activity after 3-month feeding trial in both groups (Table 3). A significant positive correlation (Pearson correlation coef- ficient 0.83 (95% CI: [0.65 - 0.92]) was found between the selenium concentration and plasma GPX activity at the be- ginning of the study. Urinary Protein electrophoresis At the beginning of the study, four cats in the control group and three cats in the experimental group had microalbu- minuria. Furthermore, more cats in the experimental group had non-zero fractions of protein in the urinary sample. The leading protein fractions at the beginning of the study were alpha 1 and albumin in the control group and beta in the experimental group. At the end of the study, beta fraction predominated in both groups. The median per cent fraction of the urinary protein at the beginning of the study in com- parison to the end of the study is shown in Table 4. Moreover, at the beginning of the study, a significant nega- tive correlation (Pearson correlation coefficient -0.70 (95% Figure 2: The changes in the concentrations of serum creatinine, serum SDMA, plasma MDA, and plasma GPX activity from the beginning to the end of the study in cats receiving renal diet in comparison to the control group A – change in serum creatinine concentration (P = 0.92), B – change in serum SDMA (symmetric dimethylarginine) concentration (P = 0.85), C – change in plasma MDA (malondialdehyde) concentration (P = 0.18), D – change in plasma GPX (glutathione peroxidase) activity (P = 0.65); P-values stand for comparisons of age-adjusted differences SloVetRes_NOTRANJOST ver2.indd 30 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 31 CI: [-0.87 - (-0.38)]) between SDMA and USG in the sampled cats were found. Discussion Cats with CKD IRIS stage 1 and 2 were monitored during a prospective 3-month feeding trial. All cats tolerated the diet change well in the experimental group and had normal ap- petite. During the study, the SDMA concentrations did not significantly change in any of the groups studied. Previously published data in cats with CKD IRIS stage 1 and 2 report a gradual increase in serum SDMA concentration regardless of the diet used (23). In the mentioned study, serum SDMA concentration increased from baseline at the first check- up after one month and continued to increase after three months in both the experimental and control groups (23). According to the published data, SDMA has a lower index of individuality than creatinine in cats (35) and dogs (36). Furthermore, consecutive measurements were performed in our study, and an individual value for each cat deter- mined. A gradual increase in SDMA in successive measure- ments can therefore be due to a gradual decrease in kidney function (35,37). However, the difference in results of the mentioned studies might be ascribed to different renal pa- thologies of the patients that were included in both studies. Since the IRIS classification is applied to all patients suffer- ing from CKD regardless of their cause, a variety of patients can be included. The progression of CKD and its response to treatment may be more variable at early stages than later when the majority of nephrons are lost. Although the se- rum SDMA concentration is correlated with the glomerular filtration rate (GFR) (38), further studies are warranted to assess the effect of the renal diet on the GFR. Serum selenium concentration and plasma GPX activity measured in the present study were generally consistent with previously reported values (13, 39). No cat was seleni- um deficient at the beginning nor at the end of the 3-month feeding trial. In addition, a significant positive correlation between serum selenium concentration and plasma GPX activity was found, which contrasts with previously pub- lished data in cats (39). The study mentioned above found a correlation between serum selenium concentration and plasma GPX activity only in the case of selenium deficien- cy. When selenium concentrations continued to increase, plasma GPX activity reached its plateau, which was not ob- served in our study. Unlike in human patients, selenium is not a limiting factor in feline CKD (16, 17, 39). In addition, the correlation between the above-mentioned parameters is inconsistent in human CKD patients (16). The median plasma MDA concentrations measured in our study were slightly higher, but in general agreement with the previously published values in healthy and CKD cats (40). We found no significant difference in mean change (final measurement – baseline) of plasma MDA concentration or Table 3: Results of five linear regression models, modelling difference (measurement 4 – measurement 1) between control and experimental group; the model controls for the age of cats Linear regression coefficient at group P value Adjusted P value Weight (kg) 0.401 0.054 0.272 Creatinine (µmol/L) -2.907 0.919 > 0.999 SDMA (µg/dL) 0.373 0.854 > 0.999 MDA (µmol/L) 0.891 0.175 0.699 GPX (U/mL) 0.875 0.654 > 0.999 Legend: Adjusted P-values are adjusted by Holm procedure for multiple comparisons; SDMA-Serum symmetric dimethylarginine; MDA-Plasma malondialdehyde; GPX-Plasma glutathione peroxidase Table 4: Median per cent fraction of the urinary protein at the beginning of the study in comparison to the end of the study (after 3 months) albumin Alpha 1 Alpha 2 Beta gamma Beginning Control group 4.0 5.8 0 2.4 0 Experimental group 3.1 4.1 0 14.7 0 After 3-month diet Control group 3.8 7.3 0 54.1 0 Experimental group 1.9 9.4 0 12.7 0 SloVetRes_NOTRANJOST ver2.indd 31 09/05/2023 10:14 32 | Slov Vet Res | Vol 60 No 1 plasma GPX activity between the experimental group and the control group. Our results suggest that the renal diet had no significant effect on the parameters of oxidative stress measured in our study. In contrast to previously published studies (39, 41), we found that three out of ten cats staged to IRIS 1 with elevated se- rum SDMA concentration, abnormal renal imaging findings as well as pathological urinary sediment, consistent with CKD, had a normal urinary specific gravity. The loss of the ability to concentrate urine is one of the first clinical signs of CKD and occurs when two-thirds of the nephrons are not functional. Apparently, the serum SDMA concentration increased before the ability of the kidneys to concentrate urine was impaired and proved valuable in the clinical evalu- ation of feline CKD patients at risk of developing CKD. Furthermore, it was observed that some cats (seven out of 19) with elevated serum SDMA and creatinine concen- trations, abnormal renal imaging findings and pathological urinary sediment, consistent with CKD, that were classified to IRIS stage 2, retained their urine concentration ability. The USG in these cats was up to 1.070 without showing clinical signs of heart failure, dehydration or hypovolemia. The literature data on this topic are scarce; Watson (42) reported that in contrast to dogs, USG values may remain normal (up to 1.045) in some cats with CKD and azotemia and that kidney disease may therefore still be suspected in a cat if these values are accompanied by persistent azo- temia. Furthermore, cats often retain some concentrating ability in IRIS stages 2 and 3 CKD (43). The authors assume that the high USG in the CKD cats included in the present study could partially be caused by eating dry food. Further research is warranted to address this topic. Cats of both groups had early kidney disease. Most of them exhibited borderline proteinuria, some were non-protein- uric. Except for one cat in the experimental group, the kid- ney disease was stable and did not deteriorate during the study. Two cats in the experimental group ended the study with a marked decrease of UPC. However, in one of these cats the serum creatinine and SDMA concentration rose to such extent that the cat was restaged from IRIS 2 to 3. The reason for this progression remained unknown. As serum SDMA and creatinine concentrations are negatively corre- lated to glomerular filtration, it might be assumed that sub- clinical dehydration, lower glomerular filtration rate as well as lower glomerular pressure may have led to a decreased UPC in that cat. Though not expected, we found a negative correlation between SDMA and USG in the sampled cats. A similar finding has already been reported in dogs with decreased glomerular filtration rate (44). Cats with CKD have and im- paired GFR; SDMA in such patients is increased (38). With a concurrent impairment of urine concentration ability, a de- crease in USG is observed. A negative correlation between SDMA and USG in the sampled cats that all had CKD might therefore reflect the pathogenesis of CKD or it might be a consequence of a stochastic chance. At the beginning of the study, four cats in the control group and three cats in the experimental group had microalbu- minuria. According to Giraldi and colleagues (45), micro- albuminuria is found in cats at risk for developing CKD. Overall, urinary protein electrophoresis and the UPC val- ues indicate that tubular processes rather than glomerular disease were present in the cats that were enrolled in the study. Furthermore, we observed more cats in the experi- mental group to have non-zero fractions of protein in the urinary sample which might be partially ascribed to a higher systolic blood pressure or to a different predominant renal pathology. At the end of the study, beta fraction predomi- nated in cats of both groups of our sample. However, when compared to the beginning of the study, there is an increase in the median percent beta fraction in cats in the control group, while it remained similar in the experimental group. Furthermore, the per cent albumin fraction decreased in cats in the experimental group while it remained similar in the control group. The presence of a leading beta frac- tion at the end of our study in both groups suggests tubu- lar kidney damage, although the cats were non-azotemic, non-proteinuric or borderline proteinuric. Thus, we may as- sume that the tubular inflammatory processes progressed in both groups of cats, but to a different extent, although the UPC values remained grossly unchanged (45). Furthermore, the results of our sample show no effect of renal diet on USG and UPC. The results are similar to the study published by Hall and colleagues (23), where no change in UPC or USG are reported. Therefore, we may conjecture that the tested renal diet had no effect either on electrophoretic pattern of urinary proteins or on halting the progression or development of proteinuria. Urine protein electrophoresis seems to be a valuable tool in assessing the progression of CKD. In order to provide better insight into the dynamics of CKD, we suggest urine protein elec- trophoresis to be added into monitoring scheme of feline CKD. Furthermore, recent recommendations in dogs with CKD include urinary electrophoresis, especially in those where renal biopsy is not indicated or not possible to be performed. The same recommendations may also be pro- posed in cats (46). The main limitation of our study was the small number of patients who completed the study. In addition, the study lasted for a relatively short period of time, which may be an additional reason for the lack of significant differences in the measured parameters between the groups. Further studies with greater number of animals and with the as- sessment of GFR and urinary protein typization including the LMW (low molecular weight) spectrum are needed to get a thorough insight of the effect of renal diet on renal pathology. SloVetRes_NOTRANJOST ver2.indd 32 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 33 Another limitation of the study is the fact that the diet of the cats in the control group was not standardized. As some cats do not tolerate any changes in their feeding regime including the diet change, the data gathered in this study give insight into the natural progression of early CKD where no medical intervention is possible. Moreover, some nu- tritional studies in human medicine follow similar design, where only experimental group receives diet, and the con- trol group consists of individuals who continue with their habitual diet (47). The study was performed on client-owned cats. Due to this fact, some cats were not brought to every scheduled check- up and some samples could therefore not be collected. The compliance of the owners in clinical studies like the present one tends to be a common problem. Conclusions After the 3-month feeding trial, no significant change in dif- ference of body weight, serum creatinine or serum SDMA concentrations between experimental and control group was observed. Renal diet did not significantly increase the level of lipid peroxidation and decrease the activity of GPX, indicative of increased oxidative stress. Furthermore, our study demonstrated a significant positive correlation be- tween serum selenium concentration and plasma GPX ac- tivity and a significant negative correlation between SDMA and USG in all CKD cats at inclusion. Acknowledgements The study was supported by Farmina Pet Foods. The funders were not involved in the study design, sample collection, analysis, data interpretation, or writing the manuscript. References 1. Conroy M, Brodbelt DC, O’Neill D, Chang YM, Elliott J. Chronic kid- ney disease in cats attending primary care practice in the UK: a VetCompass (TM) study. Vet Rec 2019; 184(17): e526. Available from: doi: 10.1136/vr.105100 2. O’Neill DG, Church DB, McGreevy PD, et al. Prevalence of disorders recorded in cats at-tending primary-care veterinary practices in Eng- land. Vet J 2014; 202: 286–91. 3. 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Comp Clin Pathol 2021; 30: e1–11. doi: 10.1007/s00580-021-03231-2 47. Markhus MW, Kvestad I, Midtbø LK, et al. Effects of cod intake in pregnancy on iodine nutrition and infant development: study protocol for Mommy’s Food - a randomized controlled trial. BMC Nutr 2018; 4: e7. doi: 10.1186/s40795-018-0215-1 SloVetRes_NOTRANJOST ver2.indd 34 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 35 Učinek monoterapevtske ledvične diete na klinične, biokemijske, urinske in serumske parametre oksidativnega stresa pri mačkah s KLB stopnje 1 in 2 M. Krofič Žel, A. Nemec Svete, B. Jakovac Strajn, K. Pavšič Vrtač, T. Vovk, N. Kejžar, D. Pavlin Izvleček: Namen študije je bil raziskati učinek terapevtske ledvične diete na izbrane klinične, biokemijske in urinske parametre ter parametre oksidativnega stresa pri mačkah v začetnih stopnjah kronične ledvične bolezni (KLB). Raziskava je bila zasnovana kot prospektivna, tri mesece trajajoča klinična študija, v katero je bilo vključenih 29 lastniških mačk. Devetnajst mačk je zaključilo študijo, od teh jih je deset prejemalo ledvično dieto, devet pa vzdrževalno dieto po izboru lastnika. Pri vseh mačkah smo izvedli klinični pregled in odvzem krvnih ter urinskih vzorcev na dan vključitve v študijo in pri treh kontrolnih pregledih, ki so bili izvedeni 3–4, 7–8 in 10–12 tednov kasneje. Določili smo serumsko koncentracijo kreatinina, simetričnega dimetilarginina (SDMA) in izbrane parametre oksidativnega stresa (aktivnost plazemske gluta- tion peroksidaze (GPX) in plazemsko koncentracijo malondialdehida (MDA) ter serumsko koncentracijo selena). Poleg tega smo izvedli elektroforezo urinskih proteinov. Ob vključitvi mačk v raziskavo smo ugotovili značilno pozitivno kore- lacijo (p < 0,001) med serumsko koncentracijo selena in aktivnostjo plazemske GPX (Pearsonov korelacijski koeficient 0,83, 95 % CI: [0,65–0,92]) ter značilno negativno korealcijo (p < 0,001) med koncentracijo SDMA in specifično težo urina (Pearsonov korelacijski koeficient –0,70 (95 % CI: [–0,87–(–0,38)]). Trimesečno hranjenje s terapevtsko hrano ni privedlo do značilnih sprememb v serumski koncentraciji SDMA in kreatinina pri vključenih mačkah. Ključne besede: klinični parametri; simetrični dimetilarginin; oksidativni stres; ledvična dieta; mačke; kronična ledvična bolezen; elektroforeza urinskih proteinov SloVetRes_NOTRANJOST ver2.indd 35 09/05/2023 10:14