Nephroprotective Effect of the Virgin Olive Oil Polyphenol Hydroxytyrosol in Type 1-like Experimental Diabetes Mellitus: Relationships with Its Antioxidant Effect

antioxidants Article Nephroprotective Effect of the Virgin Olive Oil Polyphenol Hydroxytyrosol in Type 1-like Experimental Diabetes Mellitus: Relationships with Its Antioxidant Effect María Dolores Rodríguez-Pérez 1, Juan Antonio López-Villodres 2, María Monsalud Arrebola 3, Esther Martín-Aurioles 4,África Fernández-Prior 5, Alejandra Bermúdez-Oria 5, María Carmen Ríos 2, JoséPedro De La Cruz 1,*,† and JoséAntonio González-Correa 1,†   Citation: Rodríguez-Pérez, M.D.; López-Villodres, J.A.; Arrebola, M.M.; Martín-Aurioles, E.; Fernández-Prior, Á.; Bermúdez-Oria, A.; Ríos, M.C.; De La Cruz, J.P.; González-Correa, J.A. Nephroprotective Effect of the Virgin Olive Oil Polyphenol Hydroxytyrosol in Type 1-like Experimental Diabetes Mellitus: Relationships with Its Antioxidant Effect. Antioxidants 2021, 10, 1783. https://doi.org/10.3390/ antiox10111783 Academic Editor: Catalina Alarcòn de-la-Lastra Received: 14 October 2021 Accepted: 5 November 2021 Published: 8 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Pharmacology, Faculty of Medicine, Biomedical Research Institute (IBIMA), University of Malaga, 29010 Málaga, Spain; [email protected] (M.D.R.-P.); corr[email protected] (J.A.G.-C.) 2Area of Human Histology, Faculty of Medicine, University of Malaga, 29010 Málaga, Spain; [email protected] (J.A.L.-V.); [email protected] (M.C.R.) 3Clinical Laboratory, Clinical Management Unit, Hospital Axarquía, AGSEMA, 29740 Málaga, Spain; [email protected] 4Clinical Management Unit La Roca, Distrito Sanitario, AGSEMA, 29740 Málaga, Spain; esther[email protected] 5Department of Food Phytochemistry, Instituto de la Grasa (Spanish National Research Council, CSIC), 41013 Seville, Spain; [email protected] (Á.F.-P.); aleber[email protected] (A.B.-O.) *Correspondence: jpcr[email protected]; Tel.: +34-952131567 † These authors have contributed equally to this work. Abstract: The aim of this study was to determine whether hydroxytyrosol administration prevented kidney damage in an experimental model of type 1 diabetes mellitus in rats. Hydroxytyrosol was administered to streptozotocin-diabetic rats: 1 and 5 mg/kg/day p.o. for two months. After hydroxytyrosol administration, proteinuria was significantly reduced (67–73%), calculated creatinine clearance was significantly increased (26–38%), and the glomerular volume and glomerulosclerosis index were decreased (20–30%). Hydroxytyrosol reduced oxidative and nitrosative stress variables and thromboxane metabolite production. Statistical correlations were found between biochemical and kidney function variables. Oral administration of 1 and 5 mg/kg/day of hydroxytyrosol produced an antioxidant and nephroprotective effect in an experimental model of type 1-like diabetes mellitus. The nephroprotective effect was significantly associated with the systemic and renal antioxidant action of hydroxytyrosol, which also influenced eicosanoid production. Keywords: hydroxytyrosol; diabetes; nephropathy; virgin olive oil 1. Introduction Diabetes mellitus is the most prevalent endocrine disease globally, affecting 6.1% of the world population [ 1 ]. Sustained uncontrolled hyperglycemia over time leads to vascular complications, most notably macroangiopathy and diabetic microangiopathy, including diabetic retinopathy, neuropathy, and nephropathy. Diabetic nephropathy is the most serious microangiopathic complication that can occur in the evolution of a patient with diabetes mellitus. Individuals with diabetes have a nearly twofold greater likelihood of developing chronic kidney disease than those without diabetes. It is estimated that approximately 180 million patients with diabetes mellitus worldwide have some form of kidney injury [2]. Although diabetic nephropathy remains silent throughout life in some of these patients [ 3 ], when this complication is not well controlled, most patients will eventually need dialysis and, possibly, kidney transplantation. Moreover, in the United States, mortality among patients with diabetic nephropathy is four times higher than in patients with nondiabetic chronic kidney disease [ 2 ]. Therefore, in Antioxidants 2021,10, 1783. https://doi.org/10.3390/antiox10111783 https://www.mdpi.com/journal/antioxidants Antioxidants 2021,10, 1783 2 of 13 addition to maintaining proper control of hyperglycemia, prophylactic compounds are also being studied. Persistently high glucose concentrations alter renal cell morphology, mainly in the glomeruli [ 4 ]. At the biochemical level, several mechanisms are involved in diabetic nephropathy, which basically coincide with those affecting diabetic vasculopathy [ 4 ] including oxidative stress, nitrosative stress, inflammation, induction of growth mediators, and activation of the renin–angiotensin system [ 5 ]. In the pathophysiology of diabetic vasculopathy and diabetic nephropathy, oxidative stress caused by persistent hyperglycemia is one of the first biochemical events that initiate these lesions and enhance other pathways of cellular damage [ 6 ]. Accordingly, the potential use of antioxidant compounds in the prevention of diabetic nephropathy has been postulated [7]. One of the most important sources of antioxidant compounds is the Mediterranean diet, which has been proposed as beneficial in the prevention of diabetic nephropathy [ 8 ]. Extra virgin olive oil (EVOO) is the main source of fats and antioxidant compounds in the Mediterranean diet [ 9 ]. The renal protective effect of EVOO has been studied in experimental models of nephropathy, in which its antioxidant action plays an important role [ 10 , 11 ]. The polyphenolic compounds in EVOO are the main components responsible for its antioxidant action, with the most important of these being hydroxytyrosol [12]. In studies carried out in an experimental model of type 1 diabetes, hydroxytyrosol has been shown to slow retinal damage [ 13 ] and reduce certain cardiovascular biomarkers [ 14 ]. The main objective of this study was, therefore, to assess whether the administration of hydroxytyrosol prevents kidney damage in an experimental model of type 1 diabetes mellitus in rats. As secondary objectives, we considered whether these modifications were associated with changes in renal and serum parameters of certain biomarkers of oxidative and nitrosative stress. 2. Material and Methods 2.1. Material Thiobarbituric acid reactive substances, total antioxidant capacity colorimetric kits, 3-nitrotyrosine, 8-iso-prostaglandin F 2α (8-isoprostane), and 8-hydroxy-2-deoxyguanosine enzyme immunoassay kits were obtained from Cell Biolabs Inc. (Bionova Científica S.L., Madrid, Spain). Glutathione peroxidase and total glutathione colorimetric kits were obtained from Abcam (Cambridge, UK) and 11-dehydro-tromboxane B2 and 6-ketoprostaglandin F1 α enzyme immunoassay kits from Cayman Chemical Co., (Ann Arbor, MI, USA). Oxidized low-density lipoprotein immunoassay kits were obtained from Abyntec Biopharma S.L., Bizkaia, Spain. All other reagents were from Sigma Chemical Corp. (St. Louis, MO, USA). Hydroxytyrosol was isolated by hydrothermal treatment of the liquid phase obtained from alperujo (a by-product of the two-phase olive oil separation system) at 160 ◦C for 60 min [15] . The liquid was extracted by two-step chromatography fractionation. The final yield reached 99.6% purity relative to dry matter, according to the process described by Fernández-Bolaños et al. [ 16 ]. The phenols were quantified using a Hewlett-Packard 1100 liquid chromatography system with an ultraviolet/visible detector. A Mediterranea Sea C18 analytical column (250 × 4.6 mm i.d.; particle size = 5 µ m) (Teknokroma, Barcelona, Spain) was used at room temperature. The system was equipped with Rheodyne injection valves (20 µ L loop). The mobile phases were 0.01% trichloroacetic acid in water and acetonitrile, with the following gradient during a total run time of 55 min : 95% initially, 75% at 30 min, 50% at 45 min, 0% at 47 min, 75% at 50 min, and 95% at 52 min until the run was complete. Quantification was carried out by peak integration at 280 nm wavelength with reference to calibrations obtained with external standards. 2.2. Study Design The animals were 2-month-old adult male Wistar rats (body weight 200–250 g). All rats were used in accordance with current Spanish legislation for animal care, use and housing Antioxidants 2021,10, 1783 3 of 13 (EDL 2013/80847, BOE-A-2013-6271). The recommendations of the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985) were followed, as well as the Spanish Law on the Protection of Animals, where applicable. The study protocol was approved by the University of Malaga Ethics Committee for the Use of Animals ( Ref. CEUMA31-2018-A ) and the Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible, Junta de Andalucía (Department of Agriculture, Livestock, Fisheries, and Sustainable Development of the Regional Government of Andalusia) (Ref. 9/07/2019/124). The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org, (accessed on 17 March 2021)). The animals (n= 40, 10 rats per group) were allocated into four groups (a single animal in each cage): (1) control nondiabetic rats treated with saline (NDR), (2) control diabetic rats (DR) treated with saline, (3) DR rats treated with 1 mg/kg/day p.o. hydroxytyrosol (HT-1), and (4) DR treated with 5 mg/kg/day p.o. hydroxytyrosol (HT-5). These doses were chosen based on previous results with hydroxytyrosol to analyze certain biomarkers in the present study [ 13 , 14 ]. Hydroxytyrosol was administered in the drinking water once daily for 7 days before diabetes was induced and continued daily until the end of the diabetic period (2 months). Experimental diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg). Blood glucose concentration was measured by placing a FreeStyle glucometer (Abbot Laboratories S.A., Madrid, Spain) in contact with blood from the saphenous vein. Animals were considered to have diabetes when blood glucose was higher than 200 mg/dL for two consecutive days. Rats in the nondiabetic control group received a single intraperitoneal injection of isotonic saline solution, and blood glucose was measured in the same way as in the diabetic animals. During the follow-up period, the diabetic animals were treated with 4 IU/day s.c. of a soluble long-acting basal insulin analog (Levemir ® , Novo Nordisk A/S, Bagsværd, Denmark) to reduce mortality due to the high levels of blood glucose. Control animals received the same volume of isotonic saline solution s.c. The daily amount of food and water intake was recorded throughout the follow-up period. The animals were weighed weekly to adjust the dose of hydroxytyrosol in mg/kg. At the end of the follow up, all rats were anesthetized with pentobarbital sodium (40 mg/kg i.p.) and then decapitated with a guillotine. No rats were euthanized whilst conducting the study. 2.3. Analytical Techniques All techniques were run in a single-blind manner, i.e., the individuals who performed the assays were unaware of the origin and nature of the samples. 2.3.1. Samples The following samples were extracted from each animal: − Whole blood, collected in tubes without anticoagulants and with coagulation activator gel. The samples were centrifuged at 3500 × gfor 10 min and the supernatant was separated and frozen in aliquots at − 80 ◦ C until determination of the corresponding variables. − Kidneys: Both kidneys were perfused with isotonic saline by cannulation of the renal artery to eliminate the blood in the renal vessels. Subsequently, they were weighed, the cortex was separated from the medullary tissue, and the left renal cortex was homogenized in 50 mM phosphate-buffered saline, pH 7.0 (1/15 w/v), centrifuging the resulting sample at 13,000 × gfor 15 min at 4 ◦ C, separating the supernatant, and freezing the aliquots at − 80 ◦ C until determination of the corresponding variables. The right kidney was used for histological analysis. − Urine. Rats were individually placed in modular metabolic cages (Tecniplast S.p.A., Buguggiate, Italy) and 24 h urine was collected. Total diuresis was measured, and the samples were centrifuged at 3500 × gfor 10 min at 4 ◦ C and frozen at − 80 ◦ C in aliquots until the corresponding analytical determinations were made. Antioxidants 2021,10, 1783 4 of 13 2.3.2. Serum and Urine Biochemistry All biochemical parameters were analyzed using the Atellica ® CH autoanalyzer from Siemens Healthineers (Erlangen, Germany). Glucose concentration was determined by an enzymatic method using hexokinase and glucose-6-phosphate dehydrogenase. Creatinine determination was based on a reaction with picric acid in an alkaline medium. Total proteins were measured using cupric sulfate in alkaline solution (biuret method). Albumin was measured with bromocresol purple. Urine pH was determined with commercial Sysmex ® test strips (Sysmex España, S.L., Barcelona, Spain), using the UC-3500 autoanalyzer (Sysmex España, S.L., Barcelona, Spain), and pH was measured by reflectance photometry. Creatinine clearance was calculated using the following formula [17]: Urine creatinine (mg/dL)×urine volume (mL) Serum creatinine (mg/dL)×1000 Body weight (g)×1 1440 Values are expressed in mL/min/kg body weight. 2.3.3. Oxidative and Nitrosative Stress Malondialdehyde is the main product of reaction with thiobarbituric acid (TBARS) and was used as an index of serum and tissue lipid peroxide concentration. Serum oxidized low-density lipoprotein was measured as an index of oxidative status caused by free radicals. Urinary 8-isoprostane was determined as a global index of oxidative stress [ 18 ]. Serum and kidney glutathione concentration, glutathione peroxidase activity, and total antioxidant capacity were determined as a global index of antioxidant defense. Serum and kidney 8-hydroxy-2-deoxyguanosine concentrations were determined as an index of oxidative stress/DNA damage. Serum and kidney 3-nitrotyrosine concentrations were determined as an index of peroxynitrite formation. All these determinations were carried out according to the manufacturers’ protocols. 2.3.4. Eicosanoids Urinary 11-dehydro-thromboxane B 2 and 6-keto-prostaglandin F 1α concentrations were measured as an index of the global production, including that from the kidney, of thromboxane and prostacyclin. These determinations were made according to the manufacturer’s protocols. 2.3.5. Morphological Procedure The right kidney from each rat was fixed in 10% neutral buffered formalin and processed for 5 µ m paraffin sections for standard hematoxylin and eosin and periodic acid–Schiff (PAS) reaction staining methods. Morphometric measurements of stained sections were performed in an image analysis system: histological images were taken with a virtual slide microscope (Olympus BXUCB, with VS-ASW FL software, Hamburg, Germany). Glomerular images were then obtained with the QuPach-0.2.3 program and morphometric parameters with the FIJI ImageJ program (https://imagej.nih.gov/ij/download.html, (accessed on 5 June 2021)). To determine glomerular volume (GV), the glomerular area (GA) of 50 glomeruli was measured as follows [19]: GV = (GA)3/2 ×β/d where β is a dimensionless shape coefficient ( β = 1.0 for perfect spheres), and d is a size distribution coefficient used to adjust for variations in glomerular size. The rate of glomerulosclerosis was calculated from the PAS-stained slices in 50 glomeruli per slice. The area of each glomerulus was quantified, and the PAS-positive area was calculated in these glomeruli using the image analysis program, as follows: GMS = [PAS(+)A (µm2)/GA (µm2)] ×100 Antioxidants 2021,10, 1783 5 of 13 where GMS is the percentage of glomerular area with PAS(+) material, PAS(+)A is the area occupied with PAS(+) material in a glomerulus, and GA is the area of this glomerulus. 2.4. Statistical Analysis The data in the text, tables, and figures are expressed as the mean ± standard error of the mean (SEM) of 10 animals. All statistical analyses were done with the Statistical Package for Social Sciences v. 25.0 (SPSS Co., Chicago, IL, USA). One-way analysis of variance followed by Bonferroni transformation and unpaired Student’s t-tests were used. To establish a possible relationship between aortic morphological data and other biochemical variables, Pearson correlation coefficients were calculated. In all cases, statistical significance was assumed at a value of p< 0.05. 3. Results Diabetic animals showed a lower body weight evolution than the nondiabetic ones, the administration of hydroxytyrosol did not modify this variable significantly (Figure 1). On the other hand, all diabetic animals presented blood glucose levels much higher than nondiabetic animals; only in the group treated with the dose of 5 mg/kg/day p.o., blood glucose levels were reduced in the first month of treatment, but no significant differences were observed at the end of the study period (Figure 1). Finally, diabetic animals, controls, and those treated with hydroxytyrosol, ingested a greater quantity of feed and water daily (Figure 1). The kidneys of the diabetic control animals showed a higher relative weight (100 × kidney weight/body weight) than the healthy controls ( 0.6 ±0.04 vs. 0.8 ±0.04, p< 0.05 ), hydroxytyrosol did not modify this parameter even with 1 mg/kg/day p.o. (0.7 ±0.03) or with 5 mg/kg/day p.o. (0.7 ±0.02). Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 14 6-keto-PGF1α (pg/mg creatinine) 13.8 ± 2.1 7.0 ± 0.5 0.045 8.4 ± 0.7 n.s. 11.8 ± 1.3 0.01 n.s. = no significant differences. 6-keto-PGF1α: 6-keto-prostaglandin F1α; 8-isoprostane: 8-iso-prostaglandin F2α; 11-dH-TxB2: 11-dehydro-tromboxane B2. Figure 1. Time-course curves of the evolution of body weight, blood glucose, mean daily chow ingestion, and drinking water (mean ± standard error of the mean) in control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). N = 10 rats per group. Urine samples (Table 1) showed a significant level of glucosuria, which was unchanged after hydroxytyrosol treatment. Urinary creatinine levels were significantly reduced in diabetic control animals, increasing after hydroxytyrosol treatment. Proteinuria (Table 1 and Figure 2) was significantly higher in diabetic animals (increase factor = 5.9), decreasing after the administration of 1 mg/kg/day p.o. (67.8%) and 5 mg/kg/day p.o. (72.9%) of hydroxytyrosol. Calculated creatinine clearance (Figure 2) was significantly lower in diabetic control animals (reduction of 49.6% compared to healthy animals), increasing after treatment with 1 mg/kg/day p.o. (38.0% increase compared to diabetic controls) and 5 mg/kg/day p.o. (26.7% increase compared to diabetic controls) of hydroxytyrosol. Glomerular volume (Figures 2 and 3) increased significantly in diabetic control animals (69.1% compared to healthy controls), decreasing after the administration of 1 mg/kg/day p.o. (22.1% decrease) and 5 mg/kg/day p.o. (32.8% decrease) of hydroxytyrosol. Similarly, the glomerulosclerosis index (Figures 2 and 4) increased in the diabetic control animals (21.2% higher than in healthy control animals), decreasing after the administration of 1 mg/kg/day p.o. (13.9% decrease) and 5 mg/kg/day p.o. (18.1% decrease) of hydroxytyrosol. Figure 1. Time-course curves of the evolution of body weight, blood glucose, mean daily chow ingestion, and drinking water (mean ± standard error of the mean) in control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). N = 10 rats per group. Antioxidants 2021,10, 1783 6 of 13 The serum biochemical profile (Table 1) showed a significant increase in glucose levels in all groups of diabetic animals. Creatinine levels doubled in the diabetic animals and then decreased after hydroxytyrosol administration (42.8% reduction with 1 mg/kg/day p.o. and 28.5% with 5 mg/kg/day p.o.). The rest of the variables showed no significant changes. Table 1. Serum and urine variables (mean ± standard error of the mean) of nondiabetic rats (NDR), diabetic control rats (DR), and DR treated with hydroxytyrosol (HT) 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). N = 10 rats per group. Variable NDR DR pvs. NDR DR + HT-1 pvs. DR DR + HT-5 pvs. DR Serum Blood glucose (mg/dL) 90.0 ±5.5 471 ±9.9 0.0001 442 ±30.5 n.s. 451 ±42.7 n.s. Creatinine (mg/dL) 0.3 ±0.01 0.7 ±0.03 0.0001 0.4 ±0.04 0.001 0.5 ±0.04 0.0001 Protein (g/dL) 5.7 ±0.07 5.5 ±0.1 n.s. 5.2 ±0.05 n.s. 5.6 ±0.2 n.s. Albumin (g/dL) 1.5 ±0.08 1.4 ±0.1 n.s. 1.4 ±0.08 n.s. 1.5 ±0.1 n.s. Urine Creatinine (mg/dL) 103 ±3.7 60.6 ±3.2 0.0001 72.5 ±3.1 0.01 74.7 ±3.8 0.001 Proteinuria (mg/L) 13.1 ±0.8 91.9 ±4.7 0.0001 57.8 ±5.8 0.004 37.7 ±3.4 0.0001 Proteinuria (mg/24 h) 31.1 ±8.1 185 ±17.5 0.005 59.4 ±7.0 0.004 50.0 ±2.65 0.008 Glucosuria (mg/L) 0.0 ±0.0 4065 ±1611 0.0001 1958 ±643 n.s. 4752 ±1803 n.s. pH 7.8 ±0.6 7.3 ±0.8 n.s. 6.9 ±1.1 n.s. 7.5 ±0.9 n.s. 8-isoprostane (ng/mg creatinine) 6.9 ±0.6 49.1 ±0.6 0.0001 5.2 ±0.5 0.0001 5.5 ±0.5 0.0001 11-dH-TxB2(ng/mg creatinine) 4.1 ±0.8 9.8 ±0.6 0.003 6.4 ±1.0 0.045 4.3 ±0.8 0.009 6-keto-PGF1α (pg/mg creatinine) 13.8 ±2.1 7.0 ±0.5 0.045 8.4 ±0.7 n.s. 11.8 ±1.3 0.01 n.s. = no significant differences. 6-keto-PGF 1α : 6-keto-prostaglandin F 1α; 8-isoprostane: 8-iso-prostaglandin F 2α ; 11-dH-TxB 2 : 11-dehydrotromboxane B2. Urine samples (Table 1) showed a significant level of glucosuria, which was unchanged after hydroxytyrosol treatment. Urinary creatinine levels were significantly reduced in diabetic control animals, increasing after hydroxytyrosol treatment. Proteinuria ( Table 1and Figure 2 ) was significantly higher in diabetic animals (increase factor = 5.9), decreasing after the administration of 1 mg/kg/day p.o. (67.8%) and 5 mg/kg/day p.o. (72.9%) of hydroxytyrosol. Calculated creatinine clearance (Figure 2) was significantly lower in diabetic control animals (reduction of 49.6% compared to healthy animals), increasing after treatment with 1 mg/kg/day p.o. (38.0% increase compared to diabetic controls) and 5 mg/kg/day p.o. (26.7% increase compared to diabetic controls) of hydroxytyrosol. Glomerular volume (Figures 2and 3) increased significantly in diabetic control animals (69.1% compared to healthy controls), decreasing after the administration of 1 mg/kg/day p.o. (22.1% decrease) and 5 mg/kg/day p.o. (32.8% decrease) of hydroxytyrosol. Similarly, the glomerulosclerosis index (Figures 2and 4) increased in the diabetic control animals (21.2% higher than in healthy control animals), decreasing after the administration of 1 mg/kg/day p.o. (13.9% decrease) and 5 mg/kg/day p.o. (18.1% decrease) of hydroxytyrosol. Antioxidants 2021,10, 1783 7 of 13 Antioxidants 2021, 10, x FOR PEER REVIEW 7 of 14 Figure 2. Mean values (mean ± standard error of the mean) of the proteinuria/urine creatinine ratio, calculated creatinine clearance, glomerular volume, and glomerulosclerosis index after eight weeks of follow up in control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT5). N = 10 rats per group. * p = 0.04, ** p = 0.001, *** p = 0.004, **** p < 0.0001, with respect to DR. Figure 2. Mean values (mean ± standard error of the mean) of the proteinuria/urine creatinine ratio, calculated creatinine clearance, glomerular volume, and glomerulosclerosis index after eight weeks of follow up in control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). N = 10 rats per group. * p= 0.04, ** p= 0.001, *** p= 0.004, **** p< 0.0001, with respect to DR. All oxidative stress variables both in serum and kidney tissue (Table 2) were altered in the diabetic control animals. The markers of oxidative damage were significantly increased, and those of antioxidant defense were decreased. Likewise, the concentration of 3-nitrotyrosine (nitrosative stress) was also increased. Hydroxytyrosol administration reduced this imbalance significantly, except for the serum concentration of oxidized lowdensity lipoprotein with 1 mg/kg/day (Table 2) and the decrease in antioxidant defense quantified in the renal tissue of the diabetic controls, which did not change with either of the doses used. Finally, the concentration of 3-nitrotyrosine, which was elevated in the diabetic controls, decreased significantly, both in serum and in kidney tissue, with both doses of hydroxytyrosol. The production of eicosanoids and 8-isoprostane was also altered in the diabetic control animals (Table 1), showing an increase in 11-dH-TxB 2 and urinary 8-isoprostane and a decrease in 6-keto-PGF 1α . The administration of hydroxytyrosol reduced the production of 11-dH-TxB 2 and 8-isoprostane. However, only the dose of 5 mg/kg/day p.o. reduced the decrease in 6-keto-PGF1αquantified in the diabetic control animals. Antioxidants 2021,10, 1783 8 of 13 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 14 Figure 3. Representative examples of glomerular images from control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). Hematoxylin–eosin (×10). Figure 4. Representative examples of glomerular images from control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). PAS (×10). Figure 3. Representative examples of glomerular images from control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). Hematoxylin–eosin (×10). Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 14 Figure 3. Representative examples of glomerular images from control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). Hematoxylin–eosin (×10). Figure 4. Representative examples of glomerular images from control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). PAS (×10). Figure 4. Representative examples of glomerular images from control nondiabetic rats (NDR), control diabetic rats (DR), and DR treated with hydroxytyrosol 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). PAS (×10). Antioxidants 2021,10, 1783 9 of 13 Table 2. Serum and kidney variables (mean ± standard error of the mean) of oxidative and nitrosative stress of nondiabetic rats (NDR), diabetic control rats (DR), and DR treated with hydroxytyrosol (HT) 1 mg/kg/day p.o. (HT-1) or 5 mg/kg/day p.o. (HT-5). N = 10 rats per group. Variable NDR DR pvs. NDR HT-1 pvs. DR HT-5 pvs. DR Serum TBARS (nmol/mL) 4.2 ±0.4 8.44 ±0.4 0.0001 6.9 ±0.8 0.023 4.3 ±0.3 * 0.0001 oxLDL (ng/mL) 14.6 ±1.6 24.4 ±0.7 0.0001 21.5 ±1.7 n.s. 13.3 ±0.5 * 0.0001 8-OHdG (ng/mL) 16.1 ±0.2 26.3 ±0.8 0.0001 19.6 ±1.7 0.010 15.4 ±0.7 0.0001 GHS (nmol/mL) 127 ±3.9 91.3 ±3.9 0.0001 109 ±5.7 0.030 117 ±7.8 0.02 GSHpx (nmol/min/mL) 7.8 ±0.6 19.0 ±1.8 0.0001 11.3 ±1.3 0.005 11.6 ±1.7 0.02 TAC (U/mL) 17.9 ±0.3 13.2 ±0.4 0.0001 16.6 ±0.3 0.001 16.6 ±0.7 0.01 3-nitrotyrosine (pg/mL) 1.5 ±0.05 6.4 ±0.3 0.0001 3.2 ±0.1 0.0001 3.5 ±0.2 0.0001 Kidney TBARS (nmol/mg protein) 35.7 ±3.4 135 ±14.2 0.001 61.0 ±4.4 0.002 44.9 ±1.8 0.002 8-OHdG (ng/0.1 g tissue) 7.1 ±0.3 12.6 ±0.3 0.0001 9.0 ±0.3 0.0001 8.2 ±0.3 0.0001 GHS (µmol/0.1 g tissue) 475 ±12.8 150 ±10.1 0.0001 289 ±25.1 0.002 365 ±20.5 0.0001 GSHpx (nmol/min/0.1 g tissue) 91.4 ±3.4 65.0 ±3.1 0.0001 56.4 ±4.3 n.s. 63.9 ±3.2 n.s. TAC (U/0.1 g tissue) 87.2 ±3.0 40.2 ±7.6 0.001 58.5 ±7.0 n.s. 70.1 ±15.1 n.s. 3-nitrotyrosine (pg/0.1 g tissue) 20.7 ±0.7 117 ±6.1 0.0001 81.6 ±12.0 0.032 41.6 ±7.6 * 0.001 n.s. = no significant differences. * p< 0.05 with respect to HT-1. 8-OHdG: 8-hydroxy-2-deoxyguanosine; GSH: reduced glutathione; GSHpx: glutathione peroxidase activity; oxLDL: oxidized low-density lipoprotein; TAC: total antioxidant capacity; TBARS: thiobarbituric acid reactive substances. Table 3shows the correlations between the main variables and three fundamental parameters of renal function: glomerular volume, creatinine clearance, and proteinuria (protein/creatinine ratio in urine). Table 3. Pearson correlations between glomerular volume (GV), creatinine clearance (CrCl), and the proteinuria/urine creatinine ratio (Prot/Create) and biochemical variables in serum, kidney, and urine. Variable GV CrCl Prot/Creat Pc pPc pPc p Serum TBARS 0.846 0.0001 −0.686 0.005 0.732 0.003 8-HdG 0.888 0.0001 −0.587 0.021 0.764 0.001 oxLDL 0.767 0.0001 −0.560 0.030 0.597 0.024 GSH −0.829 0.0001 0.639 0.010 −0.810 0.0001 GSHpx 0.820 0.0001 −0.736 0.002 0.786 0.001 TAC −0.833 0.0001 0.723 0.002 −0.889 0.0001 3-NTy 0.913 0.0001 −0.875 0.0001 0.960 0.0001 Kidney TBARS 0.926 0.0001 −0.681 0.005 0.918 0.0001 8-HdG 0.948 0.0001 −0.780 0.001 0.935 0.0001 GSH −0.953 0.0001 0.816 0.0001 −0.861 0.0001 GSHpx −0.546 0.035 0.724 0.002 −0.478 0.084 TAC −0.783 0.001 0.707 0.003 −0.709 0.004 3-NTy 0.844 0.0001 −0.719 0.003 0.769 0.001 Urine 8-isoprostane 0.856 0.0001 −0.596 0.015 0.859 0.0001 11-dHTxB20.831 0.0001 −0.602 0.023 0.700 0.005 6-keto-PGF1α−0.636 0.015 0.595 0.025 −0.546 0.043 Pc: Pearson coefficient; p:pvalue; TBARS: thiobarbituric acid reactive substances; 8-OHdG: 8-hydroxy-2oxyguanosine; oxLDL: oxidized low-density lipoprotein; GSH: reduced glutathione; GSHpx: glutathione peroxidase activity; TAC: total antioxidant capacity; 3-Nty: 3-nitrotyrosine; 8-isoprostanes: 8-iso-prostaglandin F 2α; 11-dHTxB2: 11-dehydro-tromboxane B2; 6-keto-PGF1α: 6-keto-prostaglandin F1. 4. Discussion The results of this study show that hydroxytyrosol administered orally for 8 weeks to rats with experimental type 1-like diabetes mellitus reduced the main variables related to kidney damage caused by persistent hyperglycemia over time. An association between the protective effect and antioxidant action of hydroxytyrosol was also demonstrated both in kidney tissue and in serum.