Alterations in renal Na,K-ATPase activity and protein expression in rat models of pressure and volume overload

Alterations in renal Na,K-ATPase activity and protein expression in rat models of pressure and volume overload Norbert Vrbjar 1, Katarina Ondrejak Andelova 1, Matus Sykora 1, Ivona Kovacicova1, Denisa Snurikova 1, Ludek Cervenka 2, Jana Radosinska 3 & BarbaraSzeiffovaBacova 1 Purpose: To explore an unexamined mechanism of cardiorenal pathophysiology by assessing renal Na, K-ATPase kinetics in rat models of pressure overload, volume overload, and their combination. Methods: Tworatmodelswithdifferingrenin-angiotensin-aldosteronesystemactivitywereused: controlHannoverSpragueDawley(HAN)ratsandtransgenicTGR(mREN2)27rats,thelatermodeling pressureoverload.Eachmodelincludedshamandaortocavalfistula(ACF)-operatedgroupstoinduce volume overload. The kinetic parameters of Na,K-ATPase were determined: maximal velocity of enzyme reaction (Vmax), and the Michaelis constant (Km), representing the ATP concentration at halfmaximalvelocityandreflectingtheenzyme’saffinityforATP. Results: Histological studies, along with assessment of selected markers of renal injury and remodeling,confirmedkidneytissuealterationsinbothTGR(mREN2)27ratsandanimalssubjectedto ACF-surgery.RegardingNa,K-ATPase,Vmaxwashigherintransgenicrats,asrevealedby2-wayANOVA (F(1,80)=39.06,p<0.0001).FollowingACF,Vmax remained unchanged in both control and transgenic rats.Incontrary,ACFhadopposingeffectsonKm in the two rat models: it decreased in HAN rats , but increasedinTGR(mREN2)27ratsaftersurgery. Conclusion: With regard to functional properties of the Na, K-ATPase, an increased number of substrate molecules converted to products per active site per unit time (indicated by Vmax) was detectedinthekidneyofTGR(mREN2)27rats.AlthoughthecreationofACFdidnotaltertheVmax parameter,anotableimpairmentintheenzyme’sabilitytobindATPsubstratewithinphysiologically relevantconcentrationswasobservedinTGR(mREN2)27rats,butnotinHANrats. Keywords Renin-angiotensin-aldosterone system, Transgenic TGR(mREN2)27 rat, Aortocaval fistula, Renal na, K-ATPase The renin-angiotensin-aldosterone system (RAAS) is an important player not only in cardiovascular, but also in renal diseases1,2. Molecules generated within the RAAS cascade increase blood pressure through vasoconstriction, stimulation of thirst, and retention of water and sodium. The latter is mediated in large part by sodium-potassium ATPase (Na, K-ATPase, also known as the sodium pump), an aldosterone-stimulated active transport mechanism in renal tubular cells. By exporting sodium ions from the cell in exchange for potassium ions, Na, K-ATPase significantly contribute to the maintenance of water and sodium balance. Although this enzyme is expressed in all living cells, it is particularly abundant in the kidney. Na, K-ATPase significance lies not only in sodium homeostasis but also in the reabsorption of many other substances via secondary active transport mechanism driven by the sodium concentration gradient it generates. Renal Na, K-ATPase is subject to tight control, regulated in opposite directions by natriuretic and antinatriuretic hormones. A shift in this balance can promote salt retention, impair renal salt handling, and contribute to hypertension3. The importance of renal Na, K-ATPase for blood pressure regulation is well illustrated in the Milan hypertensive rat strain, in which enhanced expression of Na, K-ATPase in the basolateral membrane of tubular cells and the resulting abnormal sodium reabsorption contribute to the development of salt-sensitive hypertension4. Aging provides another illustration of the link between Na, K-ATPase and hypertension. In rats, aging was associated with reduced capacity for 1Institute for Heart Research, Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovak Republic. 2Institute for Clinical and Experimental Medicine, Centre for Experimental Medicine, Prague, Czech Republic. 3Institute of Physiology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia. email: [email protected] OPEN ScientificReports | (2025) 15:39604 1 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports sodium reabsorption in the proximal tubule due to declining Na, K-ATPase activity, while activity in the renal medulla increases. Although more distal nephron segments could compensate for impaired proximal Na,KATPase reabsorption, allowing sodium homeostasis and renal function up to 91 weeks of age, progressive decline of proximal Na, K-ATPase may eventually compromise glucose and amino acid reabsorption, which depend on the sodium gradient generated by Na, K-ATPase. At the same time, increased medullary Na, K-ATPase activity with age may favor sodium retention and contribute to the development of hypertension5. Beyond its transport function, Na, K-ATPase also acts as a signaling molecule. Through interactions with endogenous cardiotonic steroids (digitalis-like substances) and various signaling proteins, it can trigger pathways that results in impaired salt handling, promotion of inflammation, oxidative stress, and renal fibrosis6,7. For example, impairment of Na,K-ATPase in the renal proximal tubule, with consequent regulation of Src, NLRP3 and IL-1β, has been shown to contribute to hyperuricemia-induced renal tubular injury, providing a molecular link between cellular energy disturbance and downstream inflammatory processes8. Among various experimental models used to study in detail the individual components of RAAS, the creation of transgenic rats broadened the possibilities of in vivo investigations. The transgenic rat strain TGR(mREN2)27, created by introducing the mouse Ren-2 renin gene into the rat genome, was originally introduced by Mullins et al.9 and later characterized as a model of angiotensin II-dependent hypertension10. In this model, kidney tissue alterations were attributed to hypertension11, while the resulting glomerular damage may be linked to the observed albuminuria in TGR(mREN2)27 rats12. Additionally, these animals exhibited increased renal tubular sodium reabsorption, requiring higher renal perfusion pressure to excrete the same amount of sodium and water as the controls under identical conditions13. Structural damage to proximal tubular cells was also noted, likely associated with the oxidative stress response to angiotensin II14. Despite the well-established role of the kidney in blood pressure regulation in this transgenic rat model, limited information is available regarding the functionality of renal Na, K-ATPase enzyme. In TGR(mRen2)27 rats, Na, K-ATPase activity and cytosolic sodium levels were measured in blood elements. While sodium concentration was higher in lymphocytes of transgenic rats compared with controls, Na,K-ATPase activity was lower in erythrocytes15. As blood elements do not directly regulate sodium homeostasis, but rather reflect it, it seems to be important to understand how Na,K-ATPase in kidneys is affected in these transgenic rats. Renal dysfunctions can result not only from hypertension (i.e. pressure overload) but also from volume overload, as demonstrated in both human and animals studies16, or they may themselves contribute to these conditions. In animal studies, the volume overload can be induced by creating the aortocaval fistula (ACF). In addition to cardiac manifestations, this model allows to study mechanisms involved in cardiorenal pathophysiology17. Significant hemodynamic changes following ACF surgery lead to neurohumoral activation, including stimulation of the RAAS cascade, which likely influences Na,K-ATPase enzyme activity in various tissues. For example, Na,K-ATPase protein and activity increased in alveolar epithelial cells to increase alveolar fluid reabsorption. Its inhibition after propranolol administration suggested a role for norepinephrine in stimulating active sodium transport18. However, information on renal Na,K-ATPase alterations that may be related to the observed alterations in salt and water retention in rats with ACF17, remains missing. The significance of Na, K-ATPase in both renal physiology and disease has become increasingly recognized in recent years. Given the need for novel therapeutic strategies - such as development of Na, K-ATPase receptor agonists and antagonists - understanding its role in animal models of disease is highly desirable. Building on the data presented above, this study investigates a previously unexamined aspect of pathophysiology by analyzing changes in renal Na,K-ATPase activity under conditions of pressure overload (using TGR(mREN2)27 rats), volume overload (induced by ACF surgery), and their combination. Na,K-ATPase, being largely responsible for active reabsorption processes in the tubular system of the kidney, requires ATP as an energy source. In fact, Na,K-ATPase is the dominant energy consumer in the kidney, and it has been estimated that 90% of the oxygen extracted by the kidney is used for active sodium transport19,20. Therefore, we also focused on the enzyme’s affinity for its energy substrate, ATP, under the pathophysiological conditions described above. Methods Ethical approval The study was performed in accordance with the guidelines and practices established by the Animal Care and Use Committee of the Institute for Clinical and Experimental Medicine (IKEM), Prague, which accord with the European Convention on Animal Protection and Guidelines on Research Animal Use, and were approved by this committee and subsequently by the Ministry of Health of the Czech Republic (project number: 17078/20225/OVZ). This study was conducted in compliance with the ARRIVE 2.0 Guidelines for Reporting Animal Research21. All animals used in the study were bred in IKEM, which is accredited by the Czech Association for Accreditation of Laboratory Animals. Rats were originally obtained from Max Delbrück Center. Experimental design Male rats of two strains were used in this study: transgenic TGR(mREN2)27 rats and Hannover Sprague Dawley (further referred to as HAN when indicating the strain) rats, which served as controls. The experimental groups were as follows: 1. Sham-operated HAN rats (referred to as HAN-C) (n = 8). 2. Sham-operated TGR(mREN2)27 rats (referred to as TGR-C) (n = 8). 3. ACF-operated HAN rats (referred to as HAN-ACF) (n = 6). 4. ACF-operated TGR(mREN2)27 rats (referred to as TGR-ACF) (n = 6). ScientificReports | (2025) 15:39604 2 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ Rats were housed (two or four per cage) under standard conditions, maintained at 22 ± 1°C with a 12-hour light/ dark cycle, and had ad libitum access to standard rat chow and tap water. At the beginning of the experiment, eight-weeks-old male rats were used. At this age, TGR(mREN2)27 rats are characterized by sustained phase of hypertension (systolic blood pressure around 180 mmHg) and substantial activation of endogenous RAAS22. The model of volume overload was induced by creating an ACF between the abdominal aorta and inferior vena cava, using a needle technique routinely performed in our laboratory23, as previously described by Abassi et al.17. In short, after exposing both vessels below the renal arteries, an 18-gauge needle (1.2mm diameter) was inserted through the abdominal aorta into the vena cava, forming a direct connection between them. The needle was then removed, and the puncture site was sealed with cyanoacrylate tissue glue. A successful fistula was confirmed by the presence of visible, pulsatile blood flow from the aorta into the vena cava. Sham-operated animals underwent the same procedure without creating the fistula. Animals were excluded from the study if some technical errors occurred during the ACF creation, such as uncontrolled bleeding or undetected pulsatile flow in the inferior vena cava indicating a non-functional fistula. In addition, animals developing early postoperative complications were also excluded. These situations, which typically affect about 5–10% of animals, are handled carefully, and such animals were excluded from further experiments to ensure consistent study outcomes. Rats were sacrificed three weeks after the ACF operation, as mortality in TGR(mREN2)27 rats rises significantly beyond this period22. Euthanasia was performed under deep anesthesia induced by intraperitoneal administration of ketamine (Calypsol, Gedeon Richter, Hungary, 160mg/kg) and midazolam (Dormicum, Roche, France, 160mg/kg). Once the animals were deeply anesthetized, the thoracic cavity was surgically opened, the heart was excised, and kidneys were harvested. The left kidney was used for further analyses. Histological analysis of collagen in the renal tissue To stain collagen deposition, a histological Van Gieson technique was performed24. Briefly, 10μm thick kidney tissue cryosections were fixed within 4% buffered formaldehyde, incubated in a mixture of saturated picric acid and 1% aqueous acid fuchsine, and subsequently covered by Canada balsam and coverslips. Collagen-stained areas (red colour) were observed and photographed by the camera system of a light microscope (Zeiss Apotome 2 microscope, Carl Zeiss, Jena, Germany). For quantification of collagen deposition, 15 randomly selected fields of view were analyzed per tissue section and the average value per animal was calculated. The researcher who performed the histochemical image analysis was fully blinded to the allocation of experimental groups. The threshold for red color (representing collagen deposition) was initially set in the HSB color space, using a hue range of 30–255, optimized to capture red regions in the image based on pixel distributions. The brightness range was set to 0–213 to include both darker and moderately bright regions. Images were then converted from RGB format to 8-bit grayscale to facilitate quantitative analysis. Collagen deposition was quantified by measuring the area of pixels with a value below 128 on the 8-bit grayscale (ranging from black [0] to white [255]), using Image-Pro Plus (Media Cybernetics, Rockville, MD, USA)25. Determination of thiobarbituric acid reactive substances Approximately 100mg of frozen kidney tissue was homogenized in ice-cold lysis buffer (180 mM KCl, 4 mM EDTA, pH 7.4) with a protease inhibitor cocktail (Sigma-Aldrich, St.Louis, MO, USA, #P8340) added, as described in details in our previous work26. Thiobarbituric acid reactive substances (TBARS), considered as a standard marker for oxidative stress induced by lipid peroxidation, were analyzed according to Shlafer and Shepard27 with modifications28. Briefly, standards, tissue homogenates, and 20% trichloroacetic acid solution were mixed with TBARS reagent (37 mmol/l C4H4N2O2S; 500 mmol/l NaOH; 15% v/v CH3COOH) and incubated at 100°C for 70min. Next, the samples were transferred into another tube with n-butanol and pyridine (14:1, v/v) mixture, and centrifuged at 5000×g for 10min. The organic phase was subsequently used for measurement of absorbance at 535nm by Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, Vermont, USA). Determinationofmatrix-metalloproteinase-2activity Matrix-metalloproteinase-2 (MMP-2) activity was evaluated by gelatin zymography using SDS–polyacrylamide gels containing 2mg/ml gelatin. Renal tissue was homogenized following the same procedure as for TBARS assessment. Laemmli buffer without 2-mercaptoethanol was added to the protein samples, and non-heated samples were subjected to electrophoresis. After separation, gels were washed twice (20min) in 50 mmol/l Tris– HCl (pH 7.4) containing 2.5% Triton X-100, then incubated overnight at 37°C in activation buffer (50 mmol/l Tris–HCl, 10 mmol/l CaCl2, 1.25% Triton X-100, pH 7.4). Gels were stained with 1% Coomassie Brilliant Blue G-250 and destained in 40% methanol/10% acetic acid. Enzymatic activity was visualized as clear bands against a dark blue background28. Assay of Na, K-ATPase activity The membrane fraction enriched in plasmalemma was isolated from kidneys following the method of Jorgensen29 with slight modification as described previously30. The protein concentration was determined according to Lowry31 using bovine serum albumin as a standard. The kinetic measurements were executed similarly to method described previously32. All enzyme assays were carried out at 37°C using 10µg of membrane protein. The Na, K-ATPase activity was estimated in an assay buffer containing (in mmol·l− 1): 4 MgCl2, 100 NaCl, 10 KCl and 50 TRIS (pH = 7.4). Following 20min of preincubation in a substrate-free medium, the enzyme reaction was initiated by increasing amount of ATP in the range of 0.16–8.00 mmol l− 1. After 20min, the reaction was stopped by adding 12% ice–cold trichloracetic acid. The inorganic phosphorus derived from ATP hydrolysis was estimated according to the method of Taussky ScientificReports | (2025) 15:39604 3 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ and Shorr33. For calculation of Na, K-ATPase activity, the ATP hydrolysis that occurred in the presence of Mg2+ only, was subtracted. Regarding the kinetic parameters of Na, K-ATPase enzyme, the obtained data were evaluated using direct non-linear regression. The maximal velocity of enzyme reaction (Vmax) indicates the number of substrate molecules converted to products per active site per unit time, while the Michaelis constant (Km), representing the ATP substrate concentration at half-maximal velocity, reflects the affinity of enzyme for the substrate. For more precise statistical processing of the Na, K-ATPase kinetic measurements, we used the averages of Vmax or Km values, along with their upper and lower variance limits, for each determination of 6–8 individual samples in each experimental group. Protein analysis in the renal tissue In addition to the Na, K-ATPase kinetic assay, protein levels of its subunits were analyzed by western blotting after electrophoretic protein separation. The same approach was also used to assess neutrophil gelatinaseassociated lipocalin (NGAL) and protein kinase C delta (PKC-δ) levels to evaluate potential renal injury in the experimental groups. Renal tissue homogenates were prepared using the same method as for TBARS determination. Samples were diluted in Laemmli sample buffer under reducing conditions and equal amounts of protein (35µg) per lane were loaded and separated in 10% SDS–polyacrylamide gels (Mini-Protean TetraCell, Bio-Rad, Hercules, CA, USA). Subsequently, samples were electrically transferred to a nitrocellulose membrane (0.2m pore size, Advantec, Tokyo, Japan), blocked with 5% low-fat milk and incubated with the appropriate primary and secondary antibodies (Supplementary Table 1.). Proteins were detected by the enhanced chemiluminescence detection method using Amersham Imager 600 and quantified by densitometric analysis using Carestream Molecular Imaging Software (version 5.0, Carestream Health, New Haven, CT, USA). Beta-actin and GAPDH were used as loading controls for protein normalization, depending on the subcellular localization of the target protein (soluble or membrane-associated). Statistical analysis All results are expressed as means ± standard deviations. A two-way analysis of variance (ANOVA) was conducted to evaluate the effects of two factors: RAAS genetic modification and ACF surgery. Tukey’s multiple comparisons test was applied to identify significant differences between individual groups. The D’Agostino & Pearson test was used to check data normality. For nonparametric data, we worked with logarithmically transformed data to induce the normal distribution appropriate for ANOVA analysis. Differences were considered significant when the p-value was less than 0.05. Results Measured data – body, kidney, heart and lung weight and their normalization against the tibia length with statistical analysis are presented in Table1. The effect of ACF on all parameters was significant (F(1,24) = 9.688, p = 0.0047 for body weight; F(1,24) = 15.43, p = 0.0006 for kidney weight, F(1,24) = 88.01, p < 0.0001 for heart weight, F(1,24) = 52.66, p < 0.0001 for lung weight), while it remained significant after normalization to tibial length. ACF surgery was associated with reduced kidney weight and increased heart and lung weights in both rat strains. RAAS genetic modification had a significant effect on both heart (F (1, 24) = 4.687, p = 0.0406) and lung (F (1, 24) = 52.66, p < 0.0001) weight; however, after adjusting for tibial length, the effect remained significant only for lung weight. Multiple comparison tests showed that, in sham-operated rats, heart and lung weights were significantly higher in TGR-C rats compared with the HAN-C group. ACF surgery induced a significant decrease in kidney weight HAN-C (n = 8) TGR-C (n = 8) HAN-ACF (n = 6) TGR-ACF (n = 6) GEN ACF INT BW (g) 433.0 ± 27.2 422.3 ± 27.8 398.0 ± 20.9 399.3 ± 17.4 ** BW/TL (g/mm) 12.1 ± 0.8 11.6 ± 0.7 11.2 ± 0.5 11.0 ± 0.5 ** KW (g) 2.9 ± 0.2 2.9 ± 0.2 2.6 ± 0.2 a 2.6 ± 0.2 b *** KW/TL (mg/mm) 81.9 ± 6.3 78.5 ± 4.3 74.5 ± 4.8 71.2 ± 5.0 ** HW (g) 1.3 ± 0.1 1.6 ± 0.1 a 2.1 ± 0.3 aaaa 2.1 ± 0.2 bbb * **** * HW/TL (mg/mm) 36.0 ± 1.5 44.1 ± 2.5 a 59.0 ± 10.2 aaaa 57.9 ± 4.5 bbb **** * LW (mg) 916.1 ± 48.69 1195 ± 67.49 aaa 1294 ± 200.8 aaaa 1479 ± 135.8 bb **** **** LW/TL (mg/mm) 50.0 ± 1.4 53.6 ± 2.86 79.7 ± 9.2 aaaa 65.6 ± 2.5 bbb ** **** **** Table 1. General characteristics of the experimental animals. Body weight (BW), kidney weight (KW), heart weight (HW), lung weight (LW) and their normalization against the tibia length (TL) in Hannover Sprague Dawley rats (HAN), transgenic rats TGR(mREN2)27 (TGR), with or without aortocaval fistula (ACF) operation. ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Data represent means ± standard deviations. For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN) and ACF, as well as their interaction (INT). Statistical significance: *p<0.05, **p<0.01, ***p<0.001, *****p<0.0001. To identify significant differences between individual groups, Tukey’s post−hoc test was used. Statistical significance: a p<0.05, aaa p<0.001, aaaa p<0.0001 vs.. HAN−C, b p<0.05, bbb p<0.001 vs.. TGR−C. ScientificReports | (2025) 15:39604 4 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ and an increase in heart and lung weights in both HAN and TGR(mREN2)27 rats. Significant interactions in the two-way ANOVA analysis (Table1) suggest that HAN and TGR(mREN2)27 rats responded differently to ACF intervention. Normalized heart and lung weights increased in both TGR-ACF and HAN-ACF rats, but the increase was more pronounced in the HAN-ACF group compared with their respective controls. Renal injury and structural remodeling To assess the effects of pressure and volume overload on kidney tissue, a panel of markers was evaluated in experimental animals. Two-way ANOVA revealed that collagen deposition, as visualized by Van Gieson staining, was significantly affected by both RAAS genetic modification (F(1, 24) = 11.74, p = 0.0022) and ACF surgery (F(1, 24) = 6.12, p = 0.0208) (Fig.1A, C). Multiple comparison tests showed that Van Gieson staining was more intense in TGR-C rats than in HAN-C rats. Oxidative damage to lipids was evaluated by measuring the levels of thiobarbituric acid reactive substances (TBARS). In two-way ANOVA analysis, the effect ACF (F(1, 24) = 21.64, p = 0.0001) was shown to be significant. ACF surgery induced a significant increase in TBARS levels in HAN rats (Fig.1D). When focusing on protein levels of pro-fibrotic PKC-δ, ACF surgery (F(1, 24) = 50.09, p < 0.0001), as well as the interaction between both factors (F(1, 24) = 25.75, p < 0.0001) were significant. PKC-δ protein levels were Fig. 1. Histological demonstration of collagen deposition (pink color) by Van Gieson staining (Panel A). Black arrows point to vascular deposition of collagen, while white arrows point to the deposition of collagen in the area of the glomerular basement membrane. Bar: 200μm. Representative western blot images and zymogram (Panel B). Quantification of collagen deposition (Panel C). Renal thiobarbituric acid reactive substances (TBARS) levels (Panel D). Protein levels of renal protein kinase C delta (PKC−δ, Panel E). Protein levels of renal neutrophil gelatinase−associated lipocalin (NGAL, Panel F). Matrix−metalloproteinase−2 activity (MMP−2, Panel G). Results are presented for Hannover Sprague Dawley rats (HAN), transgenic rats TGR(mREN2)27 (TGR), with or without aortocaval fistula (ACF) operation (n=6–8/each group). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Glyceraldehyde 3−phosphate dehydrogenase (GAPDH) was used as loading control for PKC−δ and NGAL protein normalization. Data represent means ± standard deviations. For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN.) and ACF, as well as their interaction (INT.). Statistical significance: *p<0.05, **p<0.01, ***p<0.001, *****p<0.0001. To identify significant differences between individual groups, Tukey’s post−hoc test was used. Statistical significance: a: p<0.05 vs. HAN−C, b: p<0.05 vs. TGR−C. ScientificReports | (2025) 15:39604 5 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ significantly elevated in TGR-C rats when compared with HAN-C rats (by 26%). ACF creation increased protein expression of PKC-δ (by 46%) only in the HAN rats (Fig.1B, E). Protein levels of NGAL, generally accepted as a marker of kidney injury and a key mediator of vascular fibrosis, showed significant effect of genetic modification (F(1, 24) = 221.7, p < 0.0001), ACF (F(1, 24) = 14.06, p = 0.0010) as well as their interaction (F(1, 24) = 7.755, p = 0.0103). NGAL protein levels were significantly elevated in TGR-C rats when compared with HAN-C rats (by 47%). ACF creation increased NGAL protein expression (by 17%) only in the HAN rats (Fig.1B, F). Data on matrix-metalloproteinase-2 (MMP-2) activity, a key indicator of extracellular matrix remodeling, revealed statistically significant effects for genetic modification (F(1, 20) = 7.828, p = 0.0111), the presence of ACF (F(1, 20) = 4.466, p = 0.0473), as well as their interaction (F(1, 20) = 5.467, p = 0.0299). Direct measurement of MMP2 activity in renal tissue showed elevated levels in HAN-ACF rats and in TGR-C rats, compared with HAN-C controls (Fig.1B, G). To summarize the changes in renal injury and remodeling markers, RAAS genetic modification was mainly associated with enhanced collagen deposition and increased NGAL levels and MMP-2 activity, whereas ACF surgery was linked to increases in all markers, including collagen deposition, TBARS, PKC-δ, NGAL, and MMP2 activity. In both pressure and volume overload, renal tissue underwent significant alterations; although, twoway ANOVA indicates that increases in PKC-δ, NGAL, and MMP-2 activity were more pronounced after ACF surgery in control rats than genetically modified TGR rats. Kinetic parameters of renal Na,K-ATPase Vmax values were consistently higher in both groups of TGR rats (21.49±1.86 in TGR, 22.02 ± 2.50 in TGR-ACF) compared with HAN rats (18.90±2.61 in HAN-C, 17.36±3.52 in HAN-ACF), and this effect was independent of ACF surgery (two-way ANOVA: main effect of genetic modification, F(1, 80) = 39.06, p < 0.0001). Km values (0.59 ± 0.12 in HAN-C, 0.49 ± 0.05 in HAN-ACF, 0.57 ± 0.11 in TGR-C, 0.66 ± 0.09 in TGR-ACF) were also influenced by genetic modification (two-way ANOVA: main effect of genetic modification, F(1, 80) = 12.23, p = 0.0008). Moreover, a significant interaction between strain and surgery was observed (two-way ANOVA: significant interaction, F(1, 80) = 21.32, p < 0.0001) with ACF decreasing the Km value in HAN rats but increasing it in TGR(mREN2) 27 rats (Fig.2). To better visualize changes in Na, K-ATPase activity resulting from genetic modification and ACF surgery, the results are presented separately for each experimental intervention within the individual rat groups. When activating renal Na, K-ATPase with increasing concentrations of ATP in sham-operated rats, TGR(mREN2)27 rats exhibited higher enzyme activities throughout the applied ATP concentration range compared with the HAN group (shown in Fig.3). Evaluation of the activity data by the method of nonlinear regression resulted in a moderately but significantly higher Vmax by 14% in TGR(mREN2)27 rats compared with the HAN group. The Km values between both sham-operated groups were not statistically different (Fig.2). Fig. 2. Kinetic parameters of the Na, K−ATPase enzyme during activation by the ATP substrate in Hannover Sprague Dawley rats (HAN) and TGR(mREN2)27 rats (TGR), both sham−operated (C) and subjected to aortocaval fistula (ACF) surgery. ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. The parameter Vmax represents the maximal velocity of enzyme reaction, the Km value refers to the concentration of ATP necessary for half maximal activation of the enzyme. Data represent means ± standard deviations, (n = 6–8 animals/group, technical replicates 3/animal). For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN.) and ACF, as well as their interaction (INT.). Statistical significance: ***p < 0.001, *****p < 0.0001. To identify significant differences between individual groups, Tukey’s post−hoc test was used. Statistical significance: a: p < 0.05 vs. HAN−C, b: p < 0.05 vs. TGR−C. ScientificReports | (2025) 15:39604 6 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ In kidney samples from rats subjected to ACF, the enzyme activities were higher in the TGR(mREN2)27 group compared with the HAN group (shown in Fig.4). Evaluation of kinetic parameters showed statistically significant increase of Vmax by 27% like in sham-operated animals, however, the Km value was increased by 35% in TGR(mREN2)27 rats when compared with the HAN group (Fig.2). When the renal Na, K-ATPase from HAN rats was activated with increasing concentrations of its energy substrate, rats subjected to ACF showed activities comparable to those of sham-operated rats (shown in Fig.5A). Although analysis of the activity data revealed similar Vmax values in both groups, the Km value was lower in HAN-ACF rats than in HAN-C rats (shown in Fig.2). When focusing on TGR(mREN2)27 rats, the rats subjected to ACF showed decreased Na,K-ATPase activities in the presence of lower concentration of ATP below the 0.8 mmol l− 1. Exceeding the concentration of 0.8 mmol l− 1 of ATP, the activity in ACF animals reached the level similar to sham-operated animals (shown in Fig.5). Evaluation of the activity data revealed similar Vmax values in both groups; however, a statistically significant increase in the Km value in rats was observed in rats after ACF surgery (Fig.2). Protein levels of Na,K-ATPase subunits in kidney tissue When focusing on the protein level of catalytic α1 subunit of Na.K-ATPase assessed by western blot analysis, two-way ANOVA revealed significant effect of genetic modification (F(1, 24) = 104.5, p < 0.0001), ACF surgery (F(1, 24) = 40.22, p < 0.0001), as well as their interaction (F(1, 24) = 7.084, p = 0.0137). The TGR-C group showed Fig. 4. Activation of the Na, K−ATPase in aortocaval fistula (ACF)−operated rats by low concentrations of energy substrate ATP. Inset: activation of the enzyme in the whole investigated concentration range in ACF−operated Hannover Sprague Dawley (HAN, n = 6) and transgenic rats TGR(mREN2) 27 (TGR, n = 6). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Data represent mean ± standard deviations. Fig. 3. Activation of the Na, K−ATPase in sham−operated rats by low concentrations of energy substrate ATP. Inset: activation of the enzyme in the whole investigated concentration range in sham−operated Hannover Sprague Dawley (HAN, n = 8) and transgenic rats TGR(mREN2)27 (TGR, n = 8). The animals underwent a sham operation at 8 weeks of age and were sacrificed at 11 weeks of age. Data represent means ± standard deviations. ScientificReports | (2025) 15:39604 7 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ higher presence of the α1 subunit by 24% compared with the HAN-C group (Fig.6A, B). Induction of ACF significantly decreased the protein expression of the α1 subunit in renal tissue in the HAN group by nearly 40% (p < 0.0001, shown in Fig.6A, B). Evaluation of β1 subunit expression revealed different results for its glycosylated and unglycosylated forms. When focusing on glycosylated β1 subunit of Na, K-ATPase, the effects of genetics (F(1, 24) = 50.88), ACF surgery (F(1, 24) = 36.26) and their interaction (F(1, 24) = 46.99) was shown significant (p < 0.0001 in all). The ACF surgery did not influence the expression of the glycosylated form of β1 subunit in the HAN rats. However, in the group of rats carrying the mouse Ren2 gene, its expression was more than 44% higher (p < 0.0001) compared with the corresponding sham-operated group (Fig.6A, C). Regarding the two-way ANOVA analysis of unglycosylated β1 subunit protein expression, only the interaction between genetics and ACF was statistically significant (F(1, 24) = 12.3, p = 0.0018) (shown in Fig.6A, D). Discussion The present study focused on renal tissue in two experimental rat models that differ in the status of RAAS: control HAN rats and transgenic TGR(mREN2)27 rats, which exhibit higher activity of the RAAS vasoconstrictor axis; while both were divided into a subgroup of shamor ACF-operated. Regarding basic characteristics of experimental animals, rats transfected with the mouse Ren2 gene displayed signs of cardiac hypertrophy, despite having body weight comparable with control HAN rats. This hypertrophy was evidenced by increased absolute heart weight and heart weight normalized to tibia length. These findings align with previous studies22,34,35. Although genetic background did not significantly influence body weight in the experimental animals, the ACF procedure led to a marked reduction in body weight specifically in ACFoperated HAN - an effect, to the best of our knowledge, not previously reported. In contrast, the pronounced cardiac hypertrophy observed following ACF surgery in both investigated lines of rats is in agreement with findings from previous studies across various rat models28,36. Our additional finding that TGR(mREN2)27 rats, as well as those subjected to ACF surgery, exhibited increased lung weight suggests the presence of pulmonary congestion and altered fluid dynamics in the lungs as it was documented by previous studies35,37. This pulmonary fluid overload, along with cardiac hypertrophy, has been associated with increased RAAS activation38,39, which is further supported by increased plasma and renal levels of angiotensin II observed in ACF-operated rats22,35. Although parameters such as arterial blood pressure, pressure-volume loops, renal function (including RAP–RBF relationship, urine output, and sodium excretion), echocardiography, and other organ weights were not assessed in this study. However, these models have been comprehensively characterized in our previously published articles22,40–44. In this context, it is worth mentioning that in ACF TGR rats, mean arterial pressure, renal blood flow, urine flow, and sodium excretion were significantly lower compared to sham-operated TGR. Notably, sham-operated HAN rats do not differ from sham-operated TGR in renal parameters, despite having significantly lower blood pressure. When focusing on kidney tissue, histopathological analysis revealed structural remodeling in rats with high activity of the RAAS vasoconstrictor axis. In the context of renal pathology, fibrosis frequently initiates within the perivascular regions, where damage to blood vessels triggers collagen deposition and scarring45,46. Over time, vascular fibrosis can subsequently extend to glomerular and tubular structures, contributing to functional Fig. 5. Activation of the Na,K−ATPase in sham− and ACF−operated rats by low concentrations of energy substrate ATP. Inset: activation of the enzyme in the whole investigated concentration range in Hannover Sprague Dawley (HAN, panel A) and transgenic rats TGR(mREN2)27 (TGR, panel B). ACF was induced in 8− week−old animals, and they were sacrificed at 11 weeks of age. Data represent mean ± standard deviations, (n = 6–8 each group). ScientificReports | (2025) 15:39604 8 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/ decline, and eventual onset of chronic kidney disease47,48. NGAL, a recognized biomarker of kidney injury, was elevated as the consequence of genetic modification and following ACF surgery. The renal structural remodeling observed in our study is further supported by the analysis of PKC-δ protein expression. PKC-δ plays a crucial role in mediating fibrotic response under stress conditions by stimulating extracellular matrix production, inflammation, and renal tubular injury. Expression of PKC-δwas markedly upregulated in our models of pressure and volume overload, mirroring observations in human chronic kidney disease and murine models of renal fibrosis49. Kidney fibrosis and increased oxidative stress are closely linked in the condition of chronic kidney disease as shown previously12,50,51. Our data align with these findings, as we observed the promotion of fibrosis and increase in lipid peroxidation, as evidenced by elevated TBARS levels, in both genetically modified and ACF-operated rats. Furthermore, activity of MMP-2, which plays a significant role in renal injury and fibrosis that eventually progress to chronic kidney disease52,53, underwent similar changes. The above analysis was necessary to characterize the status of the kidneys in the experimental animals and to provide context for the observed properties of Na, K-ATPase, which was the main focus of this study. This enzyme is responsible for maintaining the low intracellular sodium concentration, which is essential in the kidney for all sodium gradient-driven transport mechanisms, and at the whole-organism level for the regulation of extracellular fluid volume. Firstly, the results of the present study offer detailed, original information on Fig. 6. Representative western blot images (A) and protein levels of renal Na, K−ATPase subunits α1 (Panel B) and β1 (Panel C, D), measured in Hannover Sprague Dawley rats (HAN), transgenic rats TGR(mREN2)27 (TGR), with or without aortocaval fistula (ACF) operation (n = 6–8 in each group). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Beta−actin was used as loading controls for protein normalization. Data represent means ± standard deviations. For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN.) and ACF, as well as their interaction (INT.). Statistical significance: ***p<0.001, *****p<0.0001. To identify significant differences between individual groups, Tukey’s post−hoc test was used. Statistical significance: a: p<0.05 vs. HAN−C, b: p<0.05 vs. TGR−C. ScientificReports | (2025) 15:39604 9 | https://doi.org/10.1038/s41598-025-23241-2 www.nature.com/scientificreports/