Cholinergic stimulation after acute myocardial infarction improves hemodynamic parameters and modulates the renal injury in spontaneously hypertensive rats

Cholinergic stimulation after acute myocardial infarction improves hemodynamic parameters and modulates the renal injury in spontaneously hypertensive rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cholinergic stimulation after acute myocardial infarction improves hemodynamic parameters and modulates the renal injury in spontaneously hypertensive rats Manuella Silva Teixeira, Camilla Fanelli, Samuel Jesus-Junior, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7641684/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract This study examined the impact of pharmacological cholinergic activation on cardiac function and renal inflammatory responses in spontaneously hypertensive rats (SHRs) subjected to acute myocardial infarction (AMI). Adult male SHRs were allocated into three groups: sham-operated, AMI (infarcted), and AMI + PY (infarcted and treated with the cholinesterase inhibitor pyridostigmine bromide [PY] at 40 mg/kg, administered once daily for seven days). Animals were euthanized seven days after surgery by anesthetic overdose, and clinical parameters were evaluated the day prior to euthanasia. Following euthanasia, blood samples were collected and kidney tissues were processed for histological analysis to assess inflammation and injury. At seven days post-surgery, the AMI + PY group showed improvements in blood pressure regulation and autonomic dysfunction. In addition, treatment reduced plasma creatinine, proteinuria, cell proliferation, and collagen accumulation compared with both AMI and sham groups. These findings indicate that cholinergic stimulation with PY provides cardiac and renal protection by mitigating post-AMI injury and inflammation. Health sciences/Cardiology Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Physiology acute myocardial infarction renal inflammation autonomic nervous system cholinergic stimulation bromide of pyridostigmine renal injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cardiovascular diseases remain the leading cause of mortality worldwide and comprise a heterogeneous group of disorders affecting the heart and vascular system. Among them, acute myocardial infarction (AMI) is of clinical relevance due to its strong association with atherosclerosis. Following AMI, renal function is rapidly compromised as a consequence of cardiac ischemia. Reduced oxygen delivery leads to cardiomyocyte death and local inflammatory responses, which in turn decrease cardiac output and renal perfusion, ultimately impairing kidney function. Studies correlating kidney damage from AMI have shown that the post-AMI inflammatory response is likely systemic, as evidenced by increased circulating levels of cytokines/inflammatory markers in both human and experimental models ( 1 ). Another significant factor is renal interstitial fibrosis, a common feature of progressive kidney disease that leads to impaired function regardless of the nature of the initial injury ( 2 ). The inflammation, post-AMI, actively participates and contributes to the progression of renal injury( 3 ) and induces cell injury and death ( 4 , 5 ). Tubular cell death triggers an innate immune response through the release of damage-associated molecular patterns (DAMPs), which activate pattern recognition receptors such as Toll-like receptors on resident and recruited immune cells, leading to the secretion of pro-inflammatory cytokines and( 6 – 8 ) causing them to secrete pro-inflammatory cytokines and chemokines( 6 – 9 ). This mechanism continues and forms a vicious cycle of inflammation through cell death ( 10 , 11 ). Recently, it has been described that the immune system is modulated by neural circuits through the inflammatory reflex, in which afferent pathways detect injury or infection, and efferent vagus nerve signals, via acetylcholine, regulate cytokine synthesis and anti-inflammatory cell recruitment, constituting the cholinergic anti-inflammatory pathway ( 12 ). Experimental studies of cholinergic stimulation in myocardial infarction models demonstrated reduced pro-inflammatory cytokine production, apoptosis, and oxidative stress, along with Treg cell activation and NF-κB inhibition ( 13 , 14 ). Pyridostigmine (PY) is a reversible cholinesterase inhibitor that prevents acetylcholine degradation, thereby enhancing cholinergic signaling, including efferent vagus nerve activity ( 14 ). Its anti-inflammatory effects following acute myocardial infarction (AMI) have been previously documented ( 14 – 16 ). However, the renal-specific anti-inflammatory actions of PY in AMI-induced acute kidney injury (AKI) remain insufficiently explored. This knowledge gap is particularly relevant given the rising incidence of AMI-induced AKI and the limited availability of effective therapeutic options for this condition. In this study, we examined the role of pyridostigmine (PY) in modulating cardiac performance and renal inflammation following acute myocardial infarction (AMI), employing spontaneous hypertensive rats (SHRs) as a model of human-like pathophysiology. A 7-day course of PY treatment after AMI resulted in prompt cardioprotective benefits and attenuation of renal inflammation and injury. These observations underscore the relevance of PY-induced cholinergic activation as a potential strategy for future experimental and clinical investigations in AMI and kidney damage. Results Effects of Pyridostigmine on Hemodynamic Indices and Cardiovascular Variability after AMI As shown in Table 1 , hemodynamic parameters were assessed in the three groups 7 days after AMI or sham surgery. At this point, a significant decrease in systolic blood pressure (SBP) and mean blood pressure (MBP) was observed in the AMI group compared with the sham group, highlighting the early hemodynamic consequences of infarction. Interestingly, the AMI + PY group demonstrated a more marked reduction in SBP, DBP, and MBP relative to the sham group, as well as a greater decline in DBP compared with the AMI group, suggesting an additive effect of PY on these measures. However, the reduction in SBP in the AMI + PY group did not reach statistical significance when compared with the AMI group. Table 1 Hemodynamic parameters of the SHAM, AMI, and AMI + PY groups were evaluated at 7 days of the protocol. Hemodynamic Parameters SHAM (N = 10) AMI (N = 10) AMI + PY (N = 10) SBP (mmHg) Mean (SD) 204.0 (2.0) 190.0 * (4.3) 181.6 * (2.4) DBP (mmHg) Mean (SD) 143.6 (3.1) 139.5 (2.4) 127.7 *# (3.0) MBP (mmHg) Mean (SD) 173.0 (3.3) 160.1 * (3.3) 155.0 * (2.5) HR (bpm) Mean (SD) 377.0 (9.9) 372.0 (5.0) 373,4 (9.4) SBP = systolic blood pressure; DBP = diastolic blood pressure; MAP = mean arterial pressure; HR = heart rate; SD = standard deviation of the mean. * = group vs. SHAM # = group vs. AMI. As shown in Table 2 , the components of heart rate variability, along with baroreflex sensitivity, were also evaluated in the three groups 7 days post-AMI or sham surgery. Regarding the time domain, the AMI + PI group showed a significant increase in the RMSSD parameter compared to the AMI and SHAM groups, indicating a greater parasympathetic influence in the animals treated. Regarding the frequency domain, an increase in the high-frequency component (HF nu) was observed in the treated group, reinforcing the predominance of parasympathetic modulation compared to the AMI group. The treated group also showed a decrease in the normalized LF component, representing a sympathetic decrease compared to the AMY group. Furthermore, an increase in the alpha index component was observed, demonstrating greater baroreflex parasympathetic activity, in relation to the other groups. Table 2 Components of heart rate variability and baroreflex sensitivity of the SHAM, AMI, and AMI + PY groups evaluated at 7 days of the protocol. HRV SHAM (N = 10) AMI (N = 10) AMI + PY (N = 10) RMSSD (ms) SD 7.01 (0.43) 8.1 (0.5) 12.8 *# (1.7) LF ab (ms 2 ) SD 3.9 (0.62) 6.0 (0.3) 7.06 * (0.9) HF ab (ms 2 ) SD 15.4 (1.7) 19.4 (6.5) 17.8 # (2.9) LF (nu) SD 20.5 (1.61) 26.0 (1.7) 17.8 # (2.9) HF (nu) SD 79.5 (1.6) 76.7 (4.6) 85.1 # (2.5) LF/HF SD 0.27 (0.03) 0.29 (0.03) 0.21 (0.04) Alpha-index (ms 2 /mmHg 2 ) SD 0.49 (0.10) 0.55 (0.06) 0.98 *# (0.15) HRV = heart rate variability; RMSSD = root mean square of successive differences between normal heart beats; LF ab = absolute power of the low-frequency band (0.04–0.15 Hz); HF ab = absolute power of the high-frequency band (0.15–0.4 Hz); LF (nu) = Relative power of the low-frequency in normal units; HF (un) = relative power of the high-frequency band in normal units; LF/HF = ratio of LF-to-HF power; ALFA INDEX = power ratio of RR interval variability and of systolic arterial pressure series variability. * = p < 0.05 group vs. SHAM # = p < 0.05 group vs. AMI. Effects of Pyridostigmine on Renal Function Markers after AMI Plasma creatinine, urea, and proteinuria 24 hours concentration in the three groups of animals after 7 days post-surgery are shown in Fig. 1 . There is a significant difference in plasma creatinine concentrations (Fig. 1 A) between the infarcted groups compared to the SHAM group. While no differences were observed in the urea between the groups (Fig. 1 C). Furthermore, proteinuria concentration (Fig. 1 D) decreased in the AMI + PY group compared to the AMI group. Effects of Pyridostigmine on cellular proliferation activity in the kidney after AMI Representative microphotographs of renal proliferating cells are shown in Fig. 2 (b, c, d, f, g, h, j, k and l), and the bar graphs represent the quantitative data on cell proliferation (a, e and i), obtained by analysis of cells staining positively for PCNA. It was possible to observe that the treated group, compared to the SHAM group, presented a decrease in glomerular, interstitial, and tubular PCNA, suggesting a reduction in renal inflammation in animals undergoing treatment. While the AMI group presented less glomerular and tubular proliferation compared to the SHAM group. Moreover, no significant differences were observed between the infarcted groups. Effects of Pyridostigmine on renal injury after the AMI Representative microphotographs of renal cortical interstitial fibrosis, evaluated in Masson’s Trichrome-stained slides, are shown in Fig. 3 (c, d and e), and the bar graph representing the quantitative data of interstitial fibrosis (a) presents the analyses of glomerular fibrosis. It was possible to observe that the AMI + PY group, compared to the AMI and SHAM groups, presented a decrease in the percentage of interstitial fibrosis, evidenced in the Masson trichrome staining by the positive areas, stained in blue, demonstrating the efficacy of the treatment in reduces the inflammatory process in renal tissue. Glomerular structural alterations, characterized by the development of glomerulosclerosis and by the presence of collapsed glomeruli, were accessed by PAS staining. PAS staining highlights the extracellular matrix accumulation, as well as the thickening of basement membrane, in strong pink color. Representative microphotographs of glomerulosclerosis (f, g and h), and the bar graph represents the quantitative data of percentage of glomerulosclerosis (b). No differences were observed between the groups. As illustrated in Fig. 4 , renal collagen content was markedly elevated in the AMI group compared with the sham group at 7 days after surgery. In contrast, AMI rats treated with PY displayed significantly lower collagen levels than both AMI and SHAM animals. These findings suggest a beneficial lasting effect of PY treatment in reducing kidney injury and collagen deposition. Discussion In this study, we demonstrated the therapeutic potential of pyridostigmine (PY) in spontaneously hypertensive rats (SHRs) subjected to acute myocardial infarction (AMI). AMI triggered early renal injury, evidenced by impaired functional parameters, increased collagen deposition in the renal interstitium, and progressive histological fibrosis. These findings reinforce the concept that post-AMI renal damage develops rapidly and is multifactorial, combining impaired cardiac function with inflammatory cell recruitment. Importantly, a short-term 7-day PY treatment exerted acute cardioprotective effects and attenuated renal inflammation. The implications of AMI are strongly linked to autonomic dysfunction, characterized by worsening hemodynamic responses and reduced heart rate variability (HRV) ( 17 , 18 ). Nevertheless, HRV and BS are autonomic markers with independent prognostic value for mortality after myocardial infarction ( 19 ). Thus, changes caused by cardiovascular autonomic dysfunction can be prevented by increasing vagal activity. Previous studies demonstrated that 7-day treatment with pyridostigmine increased the baroreflex and HRV in intact rats without altering blood pressure dynamics ( 20 ). Consistent with our group’s previous protocols, PY-treated animals exhibited reduced blood pressure, improved HRV, and enhanced baroreflex sensitivity (BS) ( 14 , 15 ), the study revealed important findings regarding the hemodynamic response, in which animals subjected to treatment with pyridostigmine bromide showed reduced blood pressure when compared to the other groups studied, as well as improved modulatory components of heart rate variability, evidenced by greater modulation of the parasympathetic system and reduced sympathetic system, predicting a better prognosis in hypertensive animals after an ischemic event, such as an infarction ( 20 ). The present results showed that oral administration of pyridostigmine increased HRV and BS in spontaneously hypertensive rats, suggesting a parasympathetic modulating effect of the drug on cardiovascular function. The interaction between the nervous and immune systems, in which vagus nerve cholinergic signaling plays a critical role, has garnered attention for the development of novel anti-inflammatory therapies ( 19 ). Pyridostigmine is a reversible acetylcholinesterase inhibitor with peripheral effects, as it does not cross the blood-brain barrier. Its main site of action is the synaptic cleft, inhibiting the hydrolysis of acetylcholine released by cholinergic neurons and has been used as a pharmacological strategy to increase vagal tone ( 21 ). In the clinical setting, this drug is used to treat myasthenia gravis ( 22 ). In recent years, therapeutic strategies, including pharmacological ones using anticholinesterases such as pyridostigmine, have been tested to modulate the autonomic nervous system and, consequently, the inflammatory response in various pathophysiological conditions, such as kidney disease, cardiovascular disease, rheumatoid arthritis, and intestinal tract diseases ( 23 ). The anti-inflammatory properties of cholinergic stimulation, through the use of α7nAChR agonists like nicotine and GTS-21, have been demonstrated in multiple preclinical studies, mainly in models of sepsis-induced AKI and renal ischemia–reperfusion injury ( 24 ). Moreover, a central aspect of our work was to demonstrate that pyridostigmine attenuated these renal alterations. Treated animals showed functional preservation, reduced interstitial fibrosis, and decreased inflammatory markers, such as PCNA markers, suggesting that cholinergic modulation can interfere with the course of AMI-induced renal injury. Renal dysfunction following acute myocardial infarction (AMI) represents one of the main determinants of morbidity and mortality, even in patients with no prior history of kidney disease. Experimental and clinical studies demonstrate that a decrease in glomerular filtration rate, systemic inflammatory activation, and interstitial matrix deposition constitute an early manifestation of cardiorenal syndrome ( 25 ). In the present study, we demonstrated that AMI induced an early renal injury, evidenced by a decrease in functional parameters and increased collagen deposition in the renal interstitium. Furthermore, histological changes consist in progressive fibrosis. These results confirm the hypothesis that post-AMI renal injury is early and multifactorial, combining cardiac function and inflammatory cell recruitment. However, it is important to emphasize that pyridostigmine did not modify the glomerulosclerosis or urea levels observed after AMI. These findings indicate that the protective action of cholinergic modulation was most evident on inflammatory and fibrosing processes in the renal interstitium, without preventing already established glomerular alterations or significantly influencing global markers of nitrogen clearance. This result is consistent with the idea that glomerulosclerosis reflects a chronic structural change that is less reversible in the short term, while interstitial fibrosis and inflammation exhibit greater plasticity when subjected to pharmacological intervention. Taken together, these results demonstrate that short-term pyridostigmine treatment after AMI exerts cardioprotective and renoprotective effects by modulating autonomic function, reducing inflammation, and limiting fibrosis. Nonetheless, its actions appear more targeted to interstitial injury than to irreversible glomerular changes, supporting its role as an anti-inflammatory and antifibrotic strategy rather than a complete renoprotective agent. Methods Chemicals and Reagents . Pyridostigmine (Pyridostigmine bromide) was purchased from Sigma-Aldrich® (Saint Louis, MO) and dissolved in sterile, pyrogen-free PBS (Gibco®, Life Technologies, Grand Island, NY). Ketamine was purchased from Henry Schein Animal Health (Dublin, OH), and xylazine from Akron Animal Health (Lake Forest, IL, United States). Bioethical statement All experiments in this study were approved by the Animal Use Ethics Committee of the University of São Paulo Medical School (FMUSP, São Paulo, Brazil) under protocol number 1791/2022 and comply with the regulations published by the National Council for the Control of Animal Experimentation (CONCEA, Brazil) and the ARRIVE guidelines ( 26 – 28 ). Animals. Thirty male SHR rats, aged 10 to 12 weeks and weighing 250 to 300g, were obtained from the InCor animal facility at FMUSP and maintained under sanitary conditions of a conventional animal facility, with controlled humidity (50–60%), temperature (22 to 24°C), and light control (12 hours of light and 12 hours of dark). Water and feed (Nuvilab, Nuvital brand, pelleted) were offered unrestricted, and the diet was normal protein (12% protein). Experimental Protocol. Animals were randomly assigned to one of three groups, with 6–10 animals in each group: sham rats (Sham), untreated infarcted rats (AMI), and pyridostigmine-treated infarcted rats (AMI + PY). All animals were monitored for 7 days. The AMI + PY Group received pyridostigmine bromide, as described previously (40 mg/kg once a day, by gavage), started one hour after surgery and continued for seven days after this procedure. According to a prior study, the dose and period of pyridostigmine administration chosen were appropriate to inhibit approximately 40% of plasma acetylcholinesterase activity ( 29 ). The echocardiogram evaluated the cardiac parameters in the five day. Catheter Implantation was done on the same day as the echocardiogram. The hemodynamic records were done in the six day to evaluate the hemodynamic, autonomic modulation, and baroreflex sensitivity. The tissues were collected for histological and biochemical analysis after euthanizing the animal by anesthetic overdose. Myocardial infarction . Rats in the AMI and AMI + PY groups were anesthetized (80 mg/kg ketamine and 12 mg/kg xylazine intraperitoneal injected, I.P.) and underwent induction of AMI by surgical occlusion of the left coronary artery, as previously described. A left thoracotomy is performed by dissecting the third intercostal space and exposing the heart. Then, the left coronary artery was occluded with a single nylon (6.0 mm) suture 1 mm distal to the left atrial appendage. The chest was then sutured. The rats were maintained under ventilation until recovery. Arterial catheterization, hemodynamic measurements, and cardiovascular variability analysis . On the sixth experimental day, rats were anesthetized with 2.5% isoflurane gas (1 mL/mL), and a saline-filled catheter (0.06 mL) was implanted into the femoral artery for hemodynamic measurements. The arterial catheter was connected to a pressure transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT), and arterial pressure (AP) signals as well as pulse interval–derived heart rate (HR) were digitally recorded for 30 minutes in conscious, freely moving animals using a data acquisition system (WinDaq, 2 kHz; DATAQ, Springfield, OH) ( 30 ). These baseline recordings were used for heart rate variability (HRV) analysis. For both time- and frequency-domain analyses, three 5-minute pulse interval (PI) segments were extracted per animal, interpolated using cubic spline (250 Hz), resampled to obtain equally spaced time points, and detrended by linear trend removal. Power spectral density was estimated using Fast Fourier Transformation, and spectral power was quantified within low-frequency (LF: 0.20–0.75 Hz) and high-frequency (HF: 0.75–4.0 Hz) bands by integration of the spectral density (Cardioseries software). Time-domain HRV was assessed by the root mean square of successive differences (RMSSD). The α-index in the LF band was calculated only when squared coherence between PI and systolic arterial pressure (SAP) exceeded 0.5 (range: 0–1) ( 31 ) . Dosage of renal function markers . On day four of protocol, the rats were placed in individual metabolic cages, housed in a room with a controlled environment with temperatures ranging from 20 to 24°C, relative humidity of 50%, and a 12-hour light/12-hour dark photoperiod. They remained in cages for 24h, representing the period during which urine was collected. The measurement of urea and plasma creatinine concentration was performed according to the standardization of the Biochemistry Laboratory of the Central Laboratory of InCor - FMUSP using a commercially available Alinity c Urea Nitrogen Reagent kit and Alinity c Creatinine Reagent Kit, respectively. The proteinuria concentrations were analyzed using 24-hour urine samples using a commercially available Sensiprot kit from LabTest. Considering that rat urine is more concentrated than human urine, a 1:5 dilution was used in the samples. Immunohistochemistry for immune cells . The kidney tissue sections were paraffinized and cut at a thickness of 4 µm and fixed to glass slides for histology. The slides were kept for 30 minutes in an oven at 60°C and subjected to the deparaffinization and rehydration process. To remove the paraffin and rehydrate the tissue, the slides were passed through a series of three-phase baths of xylene for 9 minutes each, followed by two baths in absolute alcohol, and two baths in 96% alcohol for 5 and 3 minutes, respectively. After removing the alcohol through a 5-minute bath in distilled water, antigen retrieval was performed by exposure to a temperature of 95°C for 20 minutes. The slides were then immersed in a buffer solution (sodium citrate or EDTA) according to the standardization performed for each primary antibody and kept at room temperature for 20 minutes to cool. The sections were then immersed in an endogenous peroxidase blocking solution composed of 70% methanol, 10% hydrogen peroxide, and 20% distilled water, protected from light, for 30 minutes. After washing in wash buffer, the sections were traced around the tissue using a PAP Pen. To increase the blocking of nonspecific binding, the samples were subjected to a final blocking solution composed of 6% rehydrated milk (Nestlé Brasil LTDA, São Paulo, Brazil) and 0.5% BSA (bovine serum albumin). The primary antibody (50 µL) was diluted in the milk and BSA solution and left overnight in a humid chamber in a refrigerator at 4°C. After incubation with the primary antibody, they were incubated for 20 minutes with the secondary antibody (50 µL). Finally, development was performed with the chromogenic substrate DAB (3,3-Diaminobenzidine) (K346811; Dako Co, Denmark), and the slides were sealed with a coverslip and counterstained with hematoxylin. The primary antibody used was DAKO Anti-PCNA, code #M0879, at a concentration of 1:1000 in 1% BSA. The secondary antibody was DAKO Envision mouse + rabbit, polymer-conjugated, code #K4061. Quantitative analysis of positive cells was conducted blindly, counting them under 400x microscopic magnification. The results were expressed as the number of positive cells per 10 analyzed fields, using the image analysis program Image J version 1.53d 2020 (free software, NIH, Bethesda, Maryland, USA). Histological analysis in renal tissue . The kidney fragments were dehydrated and diaphanized. Subsequently, the tissues were embedded in paraffin blocks and used to prepare histological slides. The 4-µm-thick histological sections were cut with a microtome, mounted on previously silanized glass slides, and incubated at 60–65°C for 2 hours to melt the paraffin and adhere the fragments to the slides. Masson's Trichrome Reaction. Interstitial fibrosis percentage analyses were performed using histological Masson's Trichrome blue staining. First, conventional deparaffinization was performed, in which the slides were immersed in Weigert's iron hematoxylin solution for 10 minutes. They were then washed in running water for a few minutes to remove excess dye. Subsequently, the slides were immersed in Biebrich's scarlet solution for 10 minutes. They were washed again in running water. The slides were then placed in the differentiating solution (phosphotungstic-phosphomolybdic acid) for 5 minutes. After this step, they were washed again in running water. The slides were immersed in aniline blue solution for 10 minutes. A final wash was performed in running water. Finally, the slides were dehydrated in an ascending alcohol series, diaphanized in xylene, and coverslipped using an appropriate mounting medium. The fraction of renal interstitial fibrosis (positively stained with Masson's trichrome) was quantified using a point counting method. Thirty consecutive microscopic fields were evaluated at a final magnification of 200x, using a dotted screen containing 160 equidistant points, yielding a final value expressed as the percentage of interstitial fibrosis. Periodic Acid-Schiff Reaction (PAS). To analyze glomerular sclerosis by counting the percentage of sclerotic glomeruli (GS%), histological slides underwent a staining protocol according to the following steps: Initially, the slides were immersed in a 1% periodic acid solution for 10 minutes. The solution was prepared with 1 g of sodium periodate (Na₂H₃IO₆) or potassium periodate (KIO₄) dissolved in 100 mL of 1 N sulfuric acid (H₂SO₄). After incubation, the slides were washed repeatedly in running water to remove excess reagent. The slides were then immersed in Schiff's relight andch was performed in a fume hood and protected from light and incubated for 30 minutes. A further wash was performed in running water until the water remained completely colorless, indicating the completion of the reaction. The slides were then immersed in Carazzi's hematoxylin solution, protected from light, and incubated for 5 minutes. Subsequently, a wash was performed in 1X TBS buffer. Finally, the slides were dehydrated in an ascending series of alcohol, cleared in xylene, and coverslipped using an appropriate mounting medium. The slides stained by the PAS reaction were submitted for analysis by a pathologist, who evaluated 50 glomeruli from each animal at a final magnification of 400x. The percentage of sclerotic glomeruli (GS%) was assessed based on the total number of glomeruli analyzed. Quantitative analysis of collagen fibers in renal tissue . The images for analysis will be obtained by scanning histological slides stained with picrosirius red using the Scanscope CS System (Aperio Technologies, Ic., CA, USA) with an Olympus UPlanSApo 20x objective with 40x/0.75 specifications attached to the scanner. The scanned images will be transferred to the Aperio ImageScope View digital image analysis software (Aperio Technologies, Ic., CA, USA). From the scanned images, the areas of renal tissue will be manually delimited. From this delimitation, the software will automatically quantify the total tissue area and the amount of collagen present. Statistical analysis . The values of each parameter studied are presented as mean ± standard error or median ± minimum and maximum limits, after performing the Kolmogorov-Smirnov normality test. For parametric data, one-way analysis of variance (ANOVA) was used, followed by Tukey's post-hoc test for comparison between groups. For two samples, an unpaired t-test was performed with the GraphPad Prism program, version 8.00 for Windows, from GraphPad Software, San Diego. A p-value < 0.05 was considered significant. Declarations Competing interests The authors declare no competing interest. Funding This research was jointly funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq): process 407871/2023-3 and process 312962/2020-7. M.S.T recevived scolarship from the Coordenação de Aperfeiçoamento de Pessoa de Nível Superior (CAPES): process 88887.895276/2023-00. Author Contribution M.S.T., F.M.C.C and M.C.I contributed to the conception and design of the study. M.S.T., L.E.S., and M.B.S. performed the experiments. M.S.T., C.F., S.J.J., I.L.N. and F.T. performed the experiments and analyzed the data of figures 1D-3. M.S.T., F.M.C.C and M.C.I analyzed the data and wrote the main manuscript text and final revision. All authors reviewed the manuscrispt. Data Availability The datasets analysed during the current study are available from the corresponding author on reasonable request. References Lekawanvijit, S. et al. Myocardial infarction impairs renal function, induces renal interstitial fibrosis, and increases renal KIM-1 expression: implications for cardiorenal syndrome. Am. J. Physiol. Heart Circ. Physiol. 302 (9), H1884–H1893 (2012). Eddy, A. A. Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions. J. Am. Soc. Nephrol. 5 (6), 1273–1287 (1994). Vianna, H. R., Soares, C. M. B. M., Tavares, M. S., Teixeira, M. M. & Silva, A. C. S. e. Inflamação na doença renal crônica: papel de citocinas. Jornal Brasileiro de Nefrologia. ;33(3):351–64. (2011). Chawla, L. S., Eggers, P. W., Star, R. A. & Kimmel, P. L. Acute Kidney Injury and Chronic Kidney Disease as Interconnected Syndromes. N. Engl. J. Med. 371 (1), 58–66 (2014). Molitoris, B. A. Therapeutic translation in acute kidney injury: the epithelial/endothelial axis. J. Clin. Invest. 124 (6), 2355–2363 (2014). Kurts, C., Panzer, U., Anders, H. J. & Rees, A. J. The immune system and kidney disease: basic concepts and clinical implications. Nat. Rev. Immunol. 13 (10), 738–753 (2013). Leaf, I. A. et al. Pericyte MyD88 and IRAK4 control inflammatory and fibrotic responses to tissue injury. J. Clin. Invest. 127 (1), 321–334 (2016). Wu, H. et al. TLR4 activation mediates kidney ischemia/reperfusion injury. J. Clin. Invest. 117 (10), 2847–2859 (2007). Yang, D., Han, Z. & Oppenheim, J. J. Alarmins and immunity. Immunol. Rev. 280 (1), 41–56 (2017). Mulay, S. R., Linkermann, A. & Anders, H. J. Necroinflammation in Kidney Disease. J. Am. Soc. Nephrol. 27 (1), 27–39 (2016). Wallach, D., Kang, T. B. & Kovalenko, A. Concepts of tissue injury and cell death in inflammation: a historical perspective. Nat. Rev. Immunol. 14 (1), 51–59 (2014). Chavan, S. S., Pavlov, V. A. & Tracey, K. J. Mechanisms and Therapeutic Relevance of Neuro-immune Communication. Immunity 46 (6), 927–942 (2017). Lataro, R. M., Silva, C. A. A., Tefé-Silva, C., Prado, C. M. & Salgado, H. C. Acetylcholinesterase Inhibition Attenuates the Development of Hypertension and Inflammation in Spontaneously Hypertensive Rats. Am. J. Hypertens. 28 (10), 1201–1208 (2015). Rocha, J. A. et al. Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats. Am. J. Physiology-Regulatory Integr. Comp. Physiol. 310 (8), R697–706 (2016). Bezerra, O. C. et al. Cholinergic Stimulation Improves Oxidative Stress and Inflammation in Experimental Myocardial Infarction. Sci. Rep. 7 (1), 13687 (2017). Bandoni, R. L. et al. Cholinergic stimulation with pyridostigmine modulates a heart-spleen axis after acute myocardial infarction in spontaneous hypertensive rats. Sci. Rep. 11 (1), 9563 (2021). Rovere, M. T., La, Bigger, J. T., Marcus, F. I., Mortara, A. & Schwartz, P. J. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. Lancet 351 (9101), 478–484 (1998). Carrick, D. et al. Hypertension, Microvascular Pathology, and Prognosis After an Acute Myocardial Infarction. Hypertension 72 (3), 720–730 (2018). Kelly, M. J., Breathnach, C., Tracey, K. J. & Donnelly, S. C. Manipulation of the inflammatory reflex as a therapeutic strategy. Cell. Rep. Med. 3 (7), 100696 (2022). Soares, P. P. D. S., Da Nóbrega, A. C. L., Ushizima, M. R. & Irigoyen, M. C. C. Cholinergic stimulation with pyridostigmine increases heart rate variability and baroreflex sensitivity in rats. Autonomic Neuroscience [Internet]. Jun 30 [cited 2025 Jun 16];113(1–2):24–31. (2004). Available from: https://www.sciencedirect.com/science/article/pii/S1566070204000979?pes=vor&utm_source=wiley&getft_integrator=wiley Sayin, H. et al. Pyridostigmine enhances atrial tachyarrhythmias in aging spontaneously hypertensive rats. Clin Exp Pharmacol Physiol [Internet]. 2015 Oct 1 [cited 2025 Jun 16];42(10):1084–91. Available from: /doi/pdf/10.1111/1440-1681.12458 Macht, V. A. et al. Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness. Brain Behav. Immun. 80 , 384–393 (2019). Zoccali, C. et al. The autonomic nervous system and inflammation in chronic kidney disease. Nephrology Dialysis Transplantation [Internet]. [cited 2025 Jun 4];16(3):518–24. (2014). Available from: https://dx.doi.org/10.1093/ndt/gfaf020 Yeboah, M. M. et al. Cholinergic agonists attenuate renal ischemia–reperfusion injury in rats. Kidney Int. 74 (1), 62–69 (2008). Lekawanvijit, S. & Krum, H. Cardiorenal syndrome: acute kidney injury secondary to cardiovascular disease and role of protein-bound uraemic toxins. J. Physiol. 592 (18), 3969–3983 (2014). Ministério da Ciência T e IN de C de EA. https://www.in.gov.br/en/web/dou/-/resolucao-n-57-de-6-de-dezembro-de-2022-448572294 . RESOLUÇÃO N o 57, (2022). DE 6 DE DEZEMBRO DE 2022. McGrath, J. C. & Lilley, E. Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol [Internet]. 2015 Jul 1 [cited 2025 Sep 21];172(13):3189. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4500358/ Bayne, K. & Turner, P. V. Animal Welfare Standards and International Collaborations. ILAR J. 60 (1), 86–94 (2019). Gebbers, J. O., L࿽tscher, M., Kobel, W., Portmann, R. & Laissue, J. A. Acute toxicity of pyridostigmine in rats: histological findings. Arch. Toxicol. 58 (4), 271–275 (1986). Irigoyen, M. C. et al. Exercise Training Improves Baroreflex Sensitivity Associated With Oxidative Stress Reduction in Ovariectomized Rats. Hypertension [Internet]. 2005 Oct 1 [cited 2025 Sep 24];46(4):998–1003. Available from: /doi/pdf/10.1161/01.HYP.0000176238.90688.6b?download = true Silva, G. et al. JB,. Cardiovascular and neuroimmune adaptations to enalapril and exercise training: A comparative study in male and ovariectomized female spontaneously hypertensive rats. Autonomic Neuroscience [Internet]. Aug 1 [cited 2025 Sep 24];260:103280. (2025). Available from: https://www.sciencedirect.com/science/article/pii/S1566070225000426?via%3Dihub#s0010 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 Nov, 2025 Reviews received at journal 07 Nov, 2025 Reviews received at journal 04 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers invited by journal 30 Oct, 2025 Editor assigned by journal 30 Oct, 2025 Editor invited by journal 14 Oct, 2025 Submission checks completed at journal 26 Sep, 2025 First submitted to journal 26 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7641684","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":542225820,"identity":"3bed3a41-8c53-4a5d-ab92-d05c1139b7eb","order_by":0,"name":"Manuella Silva Teixeira","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Manuella","middleName":"Silva","lastName":"Teixeira","suffix":""},{"id":542225821,"identity":"df3c919a-4154-4aa1-95f1-994bf5f11c0b","order_by":1,"name":"Camilla Fanelli","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Camilla","middleName":"","lastName":"Fanelli","suffix":""},{"id":542225822,"identity":"f48bc2f2-4278-4752-91ee-0173cb97a77e","order_by":2,"name":"Samuel Jesus-Junior","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Jesus-Junior","suffix":""},{"id":542225823,"identity":"6a9aadb7-826e-41ee-97b2-32c597962af3","order_by":3,"name":"Flavio Teles","email":"","orcid":"","institution":"State University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Flavio","middleName":"","lastName":"Teles","suffix":""},{"id":542225824,"identity":"e8a4346b-f9f3-4129-9699-1ae83297ff74","order_by":4,"name":"Leandro Eziquiel Souza","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Leandro","middleName":"Eziquiel","lastName":"Souza","suffix":""},{"id":542225825,"identity":"3d8461a4-6161-40a1-b09b-d6872535bd9c","order_by":5,"name":"Maikon Barbosa Silva","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Maikon","middleName":"Barbosa","lastName":"Silva","suffix":""},{"id":542225826,"identity":"8fc0f266-2a92-477e-8100-b2417533cb37","order_by":6,"name":"Irene Lourdes Noronha","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"Lourdes","lastName":"Noronha","suffix":""},{"id":542225830,"identity":"cd934000-ff46-4f5b-b05d-c4311d84a552","order_by":7,"name":"Fernanda Marciano Consolim-Colombo","email":"","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"Marciano","lastName":"Consolim-Colombo","suffix":""},{"id":542225831,"identity":"8eddaf11-9c27-4dcb-8daf-768765194d6c","order_by":8,"name":"Maria Claudia Irigoyen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACA2YGhgMMBmA24wMgwcNHihZmEMXDRlALEptNAkwS0mLOznvwcEVBrZzB8d5jlV9z7GTYGJgfPrqBR4tlM1/CwTMGx40NzpxLuy27LRnoMDZj4xx8DjvMY3CwweBY4oYbOWa3JbcxA7XwsEkTp+X+G7NiyW31RGupAdrCY8b4cdthYrQA/dJgcMBY8kyOsTTjtuM8bMyE/HL+7OGPDX/q5PiOnzH8+HNbtT0/e/PDx/i0AOMORBxmUDgAjEswmxmvcriWOgb5BmCK+UFQ9SgYBaNgFIxEAADF3Uok6tLDLQAAAABJRU5ErkJggg==","orcid":"","institution":"University of São Paulo Medical School","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"Claudia","lastName":"Irigoyen","suffix":""}],"badges":[],"createdAt":"2025-09-17 15:08:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7641684/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7641684/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95663435,"identity":"80bd7a7c-cbad-4db7-b927-5ac2129c5f69","added_by":"auto","created_at":"2025-11-11 16:38:54","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3396848,"visible":true,"origin":"","legend":"","description":"","filename":"articleforsubmission2509.docx","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/be731b640bf639397b0cd1df.docx"},{"id":95663536,"identity":"d58fdb97-e3ee-454e-bb70-83f203714174","added_by":"auto","created_at":"2025-11-11 16:39:04","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9411,"visible":true,"origin":"","legend":"","description":"","filename":"e924173feb2c475190e602f46d75a7f8.json","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/38ad1ea68756b0287573f65c.json"},{"id":95663434,"identity":"f5444d98-bebf-4bf4-845f-61b17845c898","added_by":"auto","created_at":"2025-11-11 16:38:54","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89132,"visible":true,"origin":"","legend":"","description":"","filename":"e924173feb2c475190e602f46d75a7f81enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/bb3d6e6668d881f4eb833e03.xml"},{"id":95663238,"identity":"141fbe68-2530-416e-a21e-647e9b5f8647","added_by":"auto","created_at":"2025-11-11 16:38:35","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1208526,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/0a51762be9aeafa45e5284e9.png"},{"id":95663500,"identity":"18373113-29da-430e-b2eb-b1fc5aa686e8","added_by":"auto","created_at":"2025-11-11 16:39:01","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21321,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/7a6fa581916ccf02b32fcc61.png"},{"id":95663531,"identity":"7a8bece5-e6a7-4470-8799-00d8eacf453b","added_by":"auto","created_at":"2025-11-11 16:39:03","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":498871,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/736d4e52e4980c9b6c0f9c85.png"},{"id":95663530,"identity":"53bae12b-c471-4b52-9670-919a3812f5bb","added_by":"auto","created_at":"2025-11-11 16:39:03","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":201612,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/7aba1d57ad7b36527c28c095.png"},{"id":95663509,"identity":"1aed2af6-2eb5-4591-851f-ac9b78e9446e","added_by":"auto","created_at":"2025-11-11 16:39:02","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":221961,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/a9a155b240838413a09df44b.png"},{"id":95663499,"identity":"1bf43bf0-be5e-44e6-b366-1b039f981191","added_by":"auto","created_at":"2025-11-11 16:39:00","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87531,"visible":true,"origin":"","legend":"","description":"","filename":"e924173feb2c475190e602f46d75a7f81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/f007ff44cbe333ae2fb94658.xml"},{"id":95663485,"identity":"8a4c392f-81ec-4446-9385-42355ff95101","added_by":"auto","created_at":"2025-11-11 16:39:00","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96870,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/a6f881983b2ce694157b1c6e.html"},{"id":95663442,"identity":"cd84a9b4-2cd0-4fc2-853d-b0ae9395c112","added_by":"auto","created_at":"2025-11-11 16:38:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85809,"visible":true,"origin":"","legend":"\u003cp\u003eBar graphs of plasma creatinine, urea, and proteinuria concentration in the studied groups. (A) Plasma creatinine was measured 7 days post-sham surgery or AMI (±PY). (B) Normalized creatinine per 100g of body weight measured by 7 days post-sham surgery or AMI (±PY). (C) Urea was measured 7 days post-sham or AMI (±PY). (D) Proteinuria was measured 7 days post-sham or AMI (±PY). Values are presented as the mean ± standard error of the mean (SEM). * p \u0026lt; 0.05 when compared to the SHAM group. # p \u0026lt; 0.05 when compared to the AMI group. (n = 7-10, for each group).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/e099d714967bb31ca0c8511c.jpeg"},{"id":95663551,"identity":"2d83bbbf-70d8-4ba6-9ebe-8055b91b58db","added_by":"auto","created_at":"2025-11-11 16:39:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1386661,"visible":true,"origin":"","legend":"\u003cp\u003ePCNA, proliferating-cell nuclear antigen in glomerular, interstitial, and tubular cells in renal tissue. (A, E, I) Bar graphs of PCNA in glomerular, interstitial, and tubular measured 7 days post-sham surgery or AMI (±PY). (B, C, D) Representative microphotographs of PCNA glomerular. (F, G, H) Representative microphotographs of PCNA interstitial. (J, K, L) Representative microphotographs of PCNA tubular (400 × magnification). Values are presented as the mean ± standard error of the mean. Values are presented as the mean ± standard error of the mean (SEM). * p \u0026lt; 0.05 when compared to the SHAM group. # p \u0026lt; 0.05 when compared to the AMI. (n = 6-8, for each group).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/5c74a21a4ed1c826fdde9608.jpeg"},{"id":95663334,"identity":"54c0004f-6643-4dad-83eb-0742bbabf87e","added_by":"auto","created_at":"2025-11-11 16:38:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1040074,"visible":true,"origin":"","legend":"\u003cp\u003eRenal cortical Interstitial fibrosis and Periodic Acid-Schiff (PAS) in renal tissue. (A) Bar graph of the percentage of interstitial fibrosis; (B) Bar graph representation of the percentage of glomerulosclerosis; (C, D and E) Representative microphotographs of renal cortical interstitial fibrosis, evaluated in Masson’s Trichrome stained slides (200 × magnification). ; (F, G, H) Representative microphotographs of glomerulosclerosis by PAS (400 × magnification). Values are presented as the mean ± standard error of the mean (SEM). * p \u0026lt; 0.05 when compared to the SHAM group. # p \u0026lt; 0.05 when compared to the AMI. (n = 6-8, for each group).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/db4ad5bc0875c48b59334d2f.png"},{"id":95663441,"identity":"b2276d55-17e7-44d0-b1da-ea7ac9e53ade","added_by":"auto","created_at":"2025-11-11 16:38:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":705841,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of interstitial collagen in the renal tissue by Picrossirius Red staining 7 day post-sham or AMI surgery. (I) Bar graph represents the percentage of interstitial collagen. (J, K, L) Representative microphotographs of interstitial collagen in the renal tissue. Values presented as the mean ± standard error of the mean. * p \u0026lt; 0.05 when compared to the SHAM group. # p \u0026lt; 0.05 when compared to the AMI group.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/ce1a4ab1269d31c484072251.jpeg"},{"id":95797446,"identity":"e021874f-0355-4207-8674-238fc75c70ef","added_by":"auto","created_at":"2025-11-13 08:05:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4008361,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7641684/v1/8907d501-60d7-44c8-b2b8-36f060a90c55.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cholinergic stimulation after acute myocardial infarction improves hemodynamic parameters and modulates the renal injury in spontaneously hypertensive rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases remain the leading cause of mortality worldwide and comprise a heterogeneous group of disorders affecting the heart and vascular system. Among them, acute myocardial infarction (AMI) is of clinical relevance due to its strong association with atherosclerosis. Following AMI, renal function is rapidly compromised as a consequence of cardiac ischemia. Reduced oxygen delivery leads to cardiomyocyte death and local inflammatory responses, which in turn decrease cardiac output and renal perfusion, ultimately impairing kidney function. Studies correlating kidney damage from AMI have shown that the post-AMI inflammatory response is likely systemic, as evidenced by increased circulating levels of cytokines/inflammatory markers in both human and experimental models (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnother significant factor is renal interstitial fibrosis, a common feature of progressive kidney disease that leads to impaired function regardless of the nature of the initial injury (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The inflammation, post-AMI, actively participates and contributes to the progression of renal injury(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and induces cell injury and death (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Tubular cell death triggers an innate immune response through the release of damage-associated molecular patterns (DAMPs), which activate pattern recognition receptors such as Toll-like receptors on resident and recruited immune cells, leading to the secretion of pro-inflammatory cytokines and(\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) causing them to secrete pro-inflammatory cytokines and chemokines(\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This mechanism continues and forms a vicious cycle of inflammation through cell death (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecently, it has been described that the immune system is modulated by neural circuits through the inflammatory reflex, in which afferent pathways detect injury or infection, and efferent vagus nerve signals, via acetylcholine, regulate cytokine synthesis and anti-inflammatory cell recruitment, constituting the cholinergic anti-inflammatory pathway (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Experimental studies of cholinergic stimulation in myocardial infarction models demonstrated reduced pro-inflammatory cytokine production, apoptosis, and oxidative stress, along with Treg cell activation and NF-κB inhibition (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePyridostigmine (PY) is a reversible cholinesterase inhibitor that prevents acetylcholine degradation, thereby enhancing cholinergic signaling, including efferent vagus nerve activity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Its anti-inflammatory effects following acute myocardial infarction (AMI) have been previously documented (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, the renal-specific anti-inflammatory actions of PY in AMI-induced acute kidney injury (AKI) remain insufficiently explored. This knowledge gap is particularly relevant given the rising incidence of AMI-induced AKI and the limited availability of effective therapeutic options for this condition.\u003c/p\u003e\u003cp\u003eIn this study, we examined the role of pyridostigmine (PY) in modulating cardiac performance and renal inflammation following acute myocardial infarction (AMI), employing spontaneous hypertensive rats (SHRs) as a model of human-like pathophysiology. A 7-day course of PY treatment after AMI resulted in prompt cardioprotective benefits and attenuation of renal inflammation and injury. These observations underscore the relevance of PY-induced cholinergic activation as a potential strategy for future experimental and clinical investigations in AMI and kidney damage.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEffects of Pyridostigmine on Hemodynamic Indices and Cardiovascular Variability after AMI\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, hemodynamic parameters were assessed in the three groups 7 days after AMI or sham surgery. At this point, a significant decrease in systolic blood pressure (SBP) and mean blood pressure (MBP) was observed in the AMI group compared with the sham group, highlighting the early hemodynamic consequences of infarction. Interestingly, the AMI\u0026thinsp;+\u0026thinsp;PY group demonstrated a more marked reduction in SBP, DBP, and MBP relative to the sham group, as well as a greater decline in DBP compared with the AMI group, suggesting an additive effect of PY on these measures. However, the reduction in SBP in the AMI\u0026thinsp;+\u0026thinsp;PY group did not reach statistical significance when compared with the AMI group.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHemodynamic parameters of the SHAM, AMI, and AMI\u0026thinsp;+\u0026thinsp;PY groups were evaluated at 7 days of the protocol.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHemodynamic Parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSHAM\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAMI\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAMI\u0026thinsp;+\u0026thinsp;PY\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSBP (mmHg)\u003c/p\u003e\u003cp\u003eMean (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e204.0\u003c/p\u003e\u003cp\u003e(2.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e190.0 *\u003c/p\u003e\u003cp\u003e(4.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e181.6 *\u003c/p\u003e\u003cp\u003e(2.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDBP (mmHg)\u003c/p\u003e\u003cp\u003eMean (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e143.6\u003c/p\u003e\u003cp\u003e(3.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e139.5\u003c/p\u003e\u003cp\u003e(2.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e127.7 *#\u003c/p\u003e\u003cp\u003e(3.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMBP (mmHg)\u003c/p\u003e\u003cp\u003eMean (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e173.0\u003c/p\u003e\u003cp\u003e(3.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160.1 *\u003c/p\u003e\u003cp\u003e(3.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e155.0 *\u003c/p\u003e\u003cp\u003e(2.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHR (bpm)\u003c/p\u003e\u003cp\u003eMean (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e377.0\u003c/p\u003e\u003cp\u003e(9.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e372.0\u003c/p\u003e\u003cp\u003e(5.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e373,4\u003c/p\u003e\u003cp\u003e(9.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSBP\u0026thinsp;=\u0026thinsp;systolic blood pressure; DBP\u0026thinsp;=\u0026thinsp;diastolic blood pressure; MAP\u0026thinsp;=\u0026thinsp;mean arterial pressure; HR\u0026thinsp;=\u0026thinsp;heart rate; SD\u0026thinsp;=\u0026thinsp;standard deviation of the mean. * = group vs. SHAM # = group vs. AMI.\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the components of heart rate variability, along with baroreflex sensitivity, were also evaluated in the three groups 7 days post-AMI or sham surgery. Regarding the time domain, the AMI\u0026thinsp;+\u0026thinsp;PI group showed a significant increase in the RMSSD parameter compared to the AMI and SHAM groups, indicating a greater parasympathetic influence in the animals treated. Regarding the frequency domain, an increase in the high-frequency component (HF nu) was observed in the treated group, reinforcing the predominance of parasympathetic modulation compared to the AMI group. The treated group also showed a decrease in the normalized LF component, representing a sympathetic decrease compared to the AMY group.\u003c/p\u003e\u003cp\u003eFurthermore, an increase in the alpha index component was observed, demonstrating greater baroreflex parasympathetic activity, in relation to the other groups.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComponents of heart rate variability and baroreflex sensitivity of the SHAM, AMI, and AMI\u0026thinsp;+\u0026thinsp;PY groups evaluated at 7 days of the protocol.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHRV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSHAM\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAMI\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAMI\u0026thinsp;+\u0026thinsp;PY\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMSSD (ms)\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.01\u003c/p\u003e\u003cp\u003e(0.43)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.1\u003c/p\u003e\u003cp\u003e(0.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.8 *#\u003c/p\u003e\u003cp\u003e(1.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLF ab (ms\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.9\u003c/p\u003e\u003cp\u003e(0.62)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003cp\u003e(0.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.06 *\u003c/p\u003e\u003cp\u003e(0.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHF ab (ms\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.4\u003c/p\u003e\u003cp\u003e(1.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.4\u003c/p\u003e\u003cp\u003e(6.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.8 #\u003c/p\u003e\u003cp\u003e(2.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLF (nu)\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.5\u003c/p\u003e\u003cp\u003e(1.61)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.0\u003c/p\u003e\u003cp\u003e(1.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.8 #\u003c/p\u003e\u003cp\u003e(2.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHF (nu)\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e79.5\u003c/p\u003e\u003cp\u003e(1.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76.7\u003c/p\u003e\u003cp\u003e(4.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e85.1 #\u003c/p\u003e\u003cp\u003e(2.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLF/HF\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003cp\u003e(0.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003cp\u003e(0.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003cp\u003e(0.04)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlpha-index (ms\u003csup\u003e2\u003c/sup\u003e/mmHg\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003cp\u003e(0.10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003cp\u003e(0.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.98 *#\u003c/p\u003e\u003cp\u003e(0.15)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHRV\u0026thinsp;=\u0026thinsp;heart rate variability; RMSSD\u0026thinsp;=\u0026thinsp;root mean square of successive differences between normal heart beats; LF ab\u0026thinsp;=\u0026thinsp;absolute power of the low-frequency band (0.04\u0026ndash;0.15 Hz); HF ab\u0026thinsp;=\u0026thinsp;absolute power of the high-frequency band (0.15\u0026ndash;0.4 Hz); LF (nu)\u0026thinsp;=\u0026thinsp;Relative power of the low-frequency in normal units; HF (un)\u0026thinsp;=\u0026thinsp;relative power of the high-frequency band in normal units; LF/HF\u0026thinsp;=\u0026thinsp;ratio of LF-to-HF power; ALFA INDEX\u0026thinsp;=\u0026thinsp;power ratio of RR interval variability and of systolic arterial pressure series variability. * = p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 group vs. SHAM # = p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 group vs. AMI.\u003c/p\u003e\u003cp\u003eEffects of Pyridostigmine on Renal Function Markers after AMI\u003c/p\u003e\u003cp\u003ePlasma creatinine, urea, and proteinuria 24 hours concentration in the three groups of animals after 7 days post-surgery are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There is a significant difference in plasma creatinine concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) between the infarcted groups compared to the SHAM group. While no differences were observed in the urea between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, proteinuria concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) decreased in the AMI\u0026thinsp;+\u0026thinsp;PY group compared to the AMI group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffects of Pyridostigmine on cellular proliferation activity in the kidney after AMI\u003c/p\u003e\u003cp\u003eRepresentative microphotographs of renal proliferating cells are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b, c, d, f, g, h, j, k and l), and the bar graphs represent the quantitative data on cell proliferation (a, e and i), obtained by analysis of cells staining positively for PCNA. It was possible to observe that the treated group, compared to the SHAM group, presented a decrease in glomerular, interstitial, and tubular PCNA, suggesting a reduction in renal inflammation in animals undergoing treatment. While the AMI group presented less glomerular and tubular proliferation compared to the SHAM group. Moreover, no significant differences were observed between the infarcted groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffects of Pyridostigmine on renal injury after the AMI\u003c/p\u003e\u003cp\u003eRepresentative microphotographs of renal cortical interstitial fibrosis, evaluated in Masson\u0026rsquo;s Trichrome-stained slides, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (c, d and e), and the bar graph representing the quantitative data of interstitial fibrosis (a) presents the analyses of glomerular fibrosis. It was possible to observe that the AMI\u0026thinsp;+\u0026thinsp;PY group, compared to the AMI and SHAM groups, presented a decrease in the percentage of interstitial fibrosis, evidenced in the Masson trichrome staining by the positive areas, stained in blue, demonstrating the efficacy of the treatment in reduces the inflammatory process in renal tissue. Glomerular structural alterations, characterized by the development of glomerulosclerosis and by the presence of collapsed glomeruli, were accessed by PAS staining. PAS staining highlights the extracellular matrix accumulation, as well as the thickening of basement membrane, in strong pink color. Representative microphotographs of glomerulosclerosis (f, g and h), and the bar graph represents the quantitative data of percentage of glomerulosclerosis (b). No differences were observed between the groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, renal collagen content was markedly elevated in the AMI group compared with the sham group at 7 days after surgery. In contrast, AMI rats treated with PY displayed significantly lower collagen levels than both AMI and SHAM animals. These findings suggest a beneficial lasting effect of PY treatment in reducing kidney injury and collagen deposition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated the therapeutic potential of pyridostigmine (PY) in spontaneously hypertensive rats (SHRs) subjected to acute myocardial infarction (AMI). AMI triggered early renal injury, evidenced by impaired functional parameters, increased collagen deposition in the renal interstitium, and progressive histological fibrosis. These findings reinforce the concept that post-AMI renal damage develops rapidly and is multifactorial, combining impaired cardiac function with inflammatory cell recruitment. Importantly, a short-term 7-day PY treatment exerted acute cardioprotective effects and attenuated renal inflammation.\u003c/p\u003e\u003cp\u003eThe implications of AMI are strongly linked to autonomic dysfunction, characterized by worsening hemodynamic responses and reduced heart rate variability (HRV) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Nevertheless, HRV and BS are autonomic markers with independent prognostic value for mortality after myocardial infarction (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Thus, changes caused by cardiovascular autonomic dysfunction can be prevented by increasing vagal activity. Previous studies demonstrated that 7-day treatment with pyridostigmine increased the baroreflex and HRV in intact rats without altering blood pressure dynamics (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Consistent with our group\u0026rsquo;s previous protocols, PY-treated animals exhibited reduced blood pressure, improved HRV, and enhanced baroreflex sensitivity (BS) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), the study revealed important findings regarding the hemodynamic response, in which animals subjected to treatment with pyridostigmine bromide showed reduced blood pressure when compared to the other groups studied, as well as improved modulatory components of heart rate variability, evidenced by greater modulation of the parasympathetic system and reduced sympathetic system, predicting a better prognosis in hypertensive animals after an ischemic event, such as an infarction (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The present results showed that oral administration of pyridostigmine increased HRV and BS in spontaneously hypertensive rats, suggesting a parasympathetic modulating effect of the drug on cardiovascular function.\u003c/p\u003e\u003cp\u003eThe interaction between the nervous and immune systems, in which vagus nerve cholinergic signaling plays a critical role, has garnered attention for the development of novel anti-inflammatory therapies (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Pyridostigmine is a reversible acetylcholinesterase inhibitor with peripheral effects, as it does not cross the blood-brain barrier. Its main site of action is the synaptic cleft, inhibiting the hydrolysis of acetylcholine released by cholinergic neurons and has been used as a pharmacological strategy to increase vagal tone (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In the clinical setting, this drug is used to treat myasthenia gravis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In recent years, therapeutic strategies, including pharmacological ones using anticholinesterases such as pyridostigmine, have been tested to modulate the autonomic nervous system and, consequently, the inflammatory response in various pathophysiological conditions, such as kidney disease, cardiovascular disease, rheumatoid arthritis, and intestinal tract diseases (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe anti-inflammatory properties of cholinergic stimulation, through the use of α7nAChR agonists like nicotine and GTS-21, have been demonstrated in multiple preclinical studies, mainly in models of sepsis-induced AKI and renal ischemia\u0026ndash;reperfusion injury (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Moreover, a central aspect of our work was to demonstrate that pyridostigmine attenuated these renal alterations. Treated animals showed functional preservation, reduced interstitial fibrosis, and decreased inflammatory markers, such as PCNA markers, suggesting that cholinergic modulation can interfere with the course of AMI-induced renal injury.\u003c/p\u003e\u003cp\u003eRenal dysfunction following acute myocardial infarction (AMI) represents one of the main determinants of morbidity and mortality, even in patients with no prior history of kidney disease. Experimental and clinical studies demonstrate that a decrease in glomerular filtration rate, systemic inflammatory activation, and interstitial matrix deposition constitute an early manifestation of cardiorenal syndrome (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In the present study, we demonstrated that AMI induced an early renal injury, evidenced by a decrease in functional parameters and increased collagen deposition in the renal interstitium. Furthermore, histological changes consist in progressive fibrosis. These results confirm the hypothesis that post-AMI renal injury is early and multifactorial, combining cardiac function and inflammatory cell recruitment. However, it is important to emphasize that pyridostigmine did not modify the glomerulosclerosis or urea levels observed after AMI. These findings indicate that the protective action of cholinergic modulation was most evident on inflammatory and fibrosing processes in the renal interstitium, without preventing already established glomerular alterations or significantly influencing global markers of nitrogen clearance. This result is consistent with the idea that glomerulosclerosis reflects a chronic structural change that is less reversible in the short term, while interstitial fibrosis and inflammation exhibit greater plasticity when subjected to pharmacological intervention.\u003c/p\u003e\u003cp\u003eTaken together, these results demonstrate that short-term pyridostigmine treatment after AMI exerts cardioprotective and renoprotective effects by modulating autonomic function, reducing inflammation, and limiting fibrosis. Nonetheless, its actions appear more targeted to interstitial injury than to irreversible glomerular changes, supporting its role as an anti-inflammatory and antifibrotic strategy rather than a complete renoprotective agent.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eChemicals and Reagents\u003c/b\u003e. Pyridostigmine (Pyridostigmine bromide) was purchased from Sigma-Aldrich\u0026reg; (Saint Louis, MO) and dissolved in sterile, pyrogen-free PBS (Gibco\u0026reg;, Life Technologies, Grand Island, NY). Ketamine was purchased from Henry Schein Animal Health (Dublin, OH), and xylazine from Akron Animal Health (Lake Forest, IL, United States).\u003c/p\u003e\n\u003ch3\u003eBioethical statement\u003c/h3\u003e\n\u003cp\u003eAll experiments in this study were approved by the Animal Use Ethics Committee of the University of S\u0026atilde;o Paulo Medical School (FMUSP, S\u0026atilde;o Paulo, Brazil) under protocol number 1791/2022 and comply with the regulations published by the National Council for the Control of Animal Experimentation (CONCEA, Brazil) and the ARRIVE guidelines (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimals.\u003c/b\u003e Thirty male SHR rats, aged 10 to 12 weeks and weighing 250 to 300g, were obtained from the InCor animal facility at FMUSP and maintained under sanitary conditions of a conventional animal facility, with controlled humidity (50\u0026ndash;60%), temperature (22 to 24\u0026deg;C), and light control (12 hours of light and 12 hours of dark). Water and feed (Nuvilab, Nuvital brand, pelleted) were offered unrestricted, and the diet was normal protein (12% protein).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental Protocol.\u003c/b\u003e Animals were randomly assigned to one of three groups, with 6\u0026ndash;10 animals in each group: sham rats (Sham), untreated infarcted rats (AMI), and pyridostigmine-treated infarcted rats (AMI\u0026thinsp;+\u0026thinsp;PY). All animals were monitored for 7 days. The AMI\u0026thinsp;+\u0026thinsp;PY Group received pyridostigmine bromide, as described previously (40 mg/kg once a day, by gavage), started one hour after surgery and continued for seven days after this procedure. According to a prior study, the dose and period of pyridostigmine administration chosen were appropriate to inhibit approximately 40% of plasma acetylcholinesterase activity (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The echocardiogram evaluated the cardiac parameters in the five day. Catheter Implantation was done on the same day as the echocardiogram. The hemodynamic records were done in the six day to evaluate the hemodynamic, autonomic modulation, and baroreflex sensitivity. The tissues were collected for histological and biochemical analysis after euthanizing the animal by anesthetic overdose.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMyocardial infarction\u003c/b\u003e. Rats in the AMI and AMI\u0026thinsp;+\u0026thinsp;PY groups were anesthetized (80 mg/kg ketamine and 12 mg/kg xylazine intraperitoneal injected, I.P.) and underwent induction of AMI by surgical occlusion of the left coronary artery, as previously described. A left thoracotomy is performed by dissecting the third intercostal space and exposing the heart. Then, the left coronary artery was occluded with a single nylon (6.0 mm) suture 1 mm distal to the left atrial appendage. The chest was then sutured. The rats were maintained under ventilation until recovery.\u003c/p\u003e\u003cp\u003e\u003cb\u003eArterial catheterization, hemodynamic measurements, and cardiovascular variability analysis\u003c/b\u003e. On the sixth experimental day, rats were anesthetized with 2.5% isoflurane gas (1 mL/mL), and a saline-filled catheter (0.06 mL) was implanted into the femoral artery for hemodynamic measurements. The arterial catheter was connected to a pressure transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT), and arterial pressure (AP) signals as well as pulse interval\u0026ndash;derived heart rate (HR) were digitally recorded for 30 minutes in conscious, freely moving animals using a data acquisition system (WinDaq, 2 kHz; DATAQ, Springfield, OH) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). These baseline recordings were used for heart rate variability (HRV) analysis. For both time- and frequency-domain analyses, three 5-minute pulse interval (PI) segments were extracted per animal, interpolated using cubic spline (250 Hz), resampled to obtain equally spaced time points, and detrended by linear trend removal. Power spectral density was estimated using Fast Fourier Transformation, and spectral power was quantified within low-frequency (LF: 0.20\u0026ndash;0.75 Hz) and high-frequency (HF: 0.75\u0026ndash;4.0 Hz) bands by integration of the spectral density (Cardioseries software). Time-domain HRV was assessed by the root mean square of successive differences (RMSSD). The α-index in the LF band was calculated only when squared coherence between PI and systolic arterial pressure (SAP) exceeded 0.5 (range: 0\u0026ndash;1) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) .\u003c/p\u003e\u003cp\u003e\u003cb\u003eDosage of renal function markers\u003c/b\u003e. On day four of protocol, the rats were placed in individual metabolic cages, housed in a room with a controlled environment with temperatures ranging from 20 to 24\u0026deg;C, relative humidity of 50%, and a 12-hour light/12-hour dark photoperiod. They remained in cages for 24h, representing the period during which urine was collected.\u003c/p\u003e\u003cp\u003eThe measurement of urea and plasma creatinine concentration was performed according to the standardization of the Biochemistry Laboratory of the Central Laboratory of InCor - FMUSP using a commercially available Alinity c Urea Nitrogen Reagent kit and Alinity c Creatinine Reagent Kit, respectively. The proteinuria concentrations were analyzed using 24-hour urine samples using a commercially available Sensiprot kit from LabTest. Considering that rat urine is more concentrated than human urine, a 1:5 dilution was used in the samples.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry for immune cells\u003c/b\u003e. The kidney tissue sections were paraffinized and cut at a thickness of 4 \u0026micro;m and fixed to glass slides for histology. The slides were kept for 30 minutes in an oven at 60\u0026deg;C and subjected to the deparaffinization and rehydration process. To remove the paraffin and rehydrate the tissue, the slides were passed through a series of three-phase baths of xylene for 9 minutes each, followed by two baths in absolute alcohol, and two baths in 96% alcohol for 5 and 3 minutes, respectively. After removing the alcohol through a 5-minute bath in distilled water, antigen retrieval was performed by exposure to a temperature of 95\u0026deg;C for 20 minutes. The slides were then immersed in a buffer solution (sodium citrate or EDTA) according to the standardization performed for each primary antibody and kept at room temperature for 20 minutes to cool. The sections were then immersed in an endogenous peroxidase blocking solution composed of 70% methanol, 10% hydrogen peroxide, and 20% distilled water, protected from light, for 30 minutes. After washing in wash buffer, the sections were traced around the tissue using a PAP Pen.\u003c/p\u003e\u003cp\u003eTo increase the blocking of nonspecific binding, the samples were subjected to a final blocking solution composed of 6% rehydrated milk (Nestl\u0026eacute; Brasil LTDA, S\u0026atilde;o Paulo, Brazil) and 0.5% BSA (bovine serum albumin). The primary antibody (50 \u0026micro;L) was diluted in the milk and BSA solution and left overnight in a humid chamber in a refrigerator at 4\u0026deg;C. After incubation with the primary antibody, they were incubated for 20 minutes with the secondary antibody (50 \u0026micro;L). Finally, development was performed with the chromogenic substrate DAB (3,3-Diaminobenzidine) (K346811; Dako Co, Denmark), and the slides were sealed with a coverslip and counterstained with hematoxylin. The primary antibody used was DAKO Anti-PCNA, code #M0879, at a concentration of 1:1000 in 1% BSA. The secondary antibody was DAKO Envision mouse\u0026thinsp;+\u0026thinsp;rabbit, polymer-conjugated, code #K4061. Quantitative analysis of positive cells was conducted blindly, counting them under 400x microscopic magnification. The results were expressed as the number of positive cells per 10 analyzed fields, using the image analysis program Image J version 1.53d 2020 (free software, NIH, Bethesda, Maryland, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistological analysis in renal tissue\u003c/b\u003e. The kidney fragments were dehydrated and diaphanized. Subsequently, the tissues were embedded in paraffin blocks and used to prepare histological slides.\u003c/p\u003e\u003cp\u003eThe 4-\u0026micro;m-thick histological sections were cut with a microtome, mounted on previously silanized glass slides, and incubated at 60\u0026ndash;65\u0026deg;C for 2 hours to melt the paraffin and adhere the fragments to the slides.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMasson's Trichrome Reaction.\u003c/b\u003e Interstitial fibrosis percentage analyses were performed using histological Masson's Trichrome blue staining. First, conventional deparaffinization was performed, in which the slides were immersed in Weigert's iron hematoxylin solution for 10 minutes. They were then washed in running water for a few minutes to remove excess dye. Subsequently, the slides were immersed in Biebrich's scarlet solution for 10 minutes. They were washed again in running water. The slides were then placed in the differentiating solution (phosphotungstic-phosphomolybdic acid) for 5 minutes. After this step, they were washed again in running water. The slides were immersed in aniline blue solution for 10 minutes. A final wash was performed in running water. Finally, the slides were dehydrated in an ascending alcohol series, diaphanized in xylene, and coverslipped using an appropriate mounting medium.\u003c/p\u003e\u003cp\u003eThe fraction of renal interstitial fibrosis (positively stained with Masson's trichrome) was quantified using a point counting method. Thirty consecutive microscopic fields were evaluated at a final magnification of 200x, using a dotted screen containing 160 equidistant points, yielding a final value expressed as the percentage of interstitial fibrosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeriodic Acid-Schiff Reaction (PAS).\u003c/b\u003e To analyze glomerular sclerosis by counting the percentage of sclerotic glomeruli (GS%), histological slides underwent a staining protocol according to the following steps: Initially, the slides were immersed in a 1% periodic acid solution for 10 minutes. The solution was prepared with 1 g of sodium periodate (Na₂H₃IO₆) or potassium periodate (KIO₄) dissolved in 100 mL of 1 N sulfuric acid (H₂SO₄). After incubation, the slides were washed repeatedly in running water to remove excess reagent. The slides were then immersed in Schiff's relight andch was performed in a fume hood and protected from light and incubated for 30 minutes. A further wash was performed in running water until the water remained completely colorless, indicating the completion of the reaction. The slides were then immersed in Carazzi's hematoxylin solution, protected from light, and incubated for 5 minutes. Subsequently, a wash was performed in 1X TBS buffer. Finally, the slides were dehydrated in an ascending series of alcohol, cleared in xylene, and coverslipped using an appropriate mounting medium. The slides stained by the PAS reaction were submitted for analysis by a pathologist, who evaluated 50 glomeruli from each animal at a final magnification of 400x. The percentage of sclerotic glomeruli (GS%) was assessed based on the total number of glomeruli analyzed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative analysis of collagen fibers in renal tissue\u003c/b\u003e. The images for analysis will be obtained by scanning histological slides stained with picrosirius red using the Scanscope CS System (Aperio Technologies, Ic., CA, USA) with an Olympus UPlanSApo 20x objective with 40x/0.75 specifications attached to the scanner.\u003c/p\u003e\u003cp\u003eThe scanned images will be transferred to the Aperio ImageScope View digital image analysis software (Aperio Technologies, Ic., CA, USA). From the scanned images, the areas of renal tissue will be manually delimited. From this delimitation, the software will automatically quantify the total tissue area and the amount of collagen present.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e. The values of each parameter studied are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error or median\u0026thinsp;\u0026plusmn;\u0026thinsp;minimum and maximum limits, after performing the Kolmogorov-Smirnov normality test. For parametric data, one-way analysis of variance (ANOVA) was used, followed by Tukey's post-hoc test for comparison between groups. For two samples, an unpaired t-test was performed with the GraphPad Prism program, version 8.00 for Windows, from GraphPad Software, San Diego. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was jointly funded by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq): process 407871/2023-3 and process 312962/2020-7. M.S.T recevived scolarship from the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoa de N\u0026iacute;vel Superior (CAPES): process 88887.895276/2023-00.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.S.T., F.M.C.C and M.C.I contributed to the conception and design of the study. M.S.T., L.E.S., and M.B.S. performed the experiments. M.S.T., C.F., S.J.J., I.L.N. and F.T. performed the experiments and analyzed the data of figures 1D-3. M.S.T., F.M.C.C and M.C.I analyzed the data and wrote the main manuscript text and final revision. All authors reviewed the manuscrispt.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLekawanvijit, S. et al. Myocardial infarction impairs renal function, induces renal interstitial fibrosis, and increases renal KIM-1 expression: implications for cardiorenal syndrome. \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e \u003cb\u003e302\u003c/b\u003e (9), H1884\u0026ndash;H1893 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEddy, A. A. Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (6), 1273\u0026ndash;1287 (1994).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVianna, H. R., Soares, C. M. B. M., Tavares, M. S., Teixeira, M. M. \u0026amp; Silva, A. C. S. e. Inflama\u0026ccedil;\u0026atilde;o na doen\u0026ccedil;a renal cr\u0026ocirc;nica: papel de citocinas. Jornal Brasileiro de Nefrologia. ;33(3):351\u0026ndash;64. (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChawla, L. S., Eggers, P. W., Star, R. A. \u0026amp; Kimmel, P. L. Acute Kidney Injury and Chronic Kidney Disease as Interconnected Syndromes. \u003cem\u003eN. Engl. J. Med.\u003c/em\u003e \u003cb\u003e371\u003c/b\u003e (1), 58\u0026ndash;66 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMolitoris, B. A. Therapeutic translation in acute kidney injury: the epithelial/endothelial axis. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e124\u003c/b\u003e (6), 2355\u0026ndash;2363 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKurts, C., Panzer, U., Anders, H. J. \u0026amp; Rees, A. J. The immune system and kidney disease: basic concepts and clinical implications. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (10), 738\u0026ndash;753 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeaf, I. A. et al. Pericyte MyD88 and IRAK4 control inflammatory and fibrotic responses to tissue injury. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e (1), 321\u0026ndash;334 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, H. et al. TLR4 activation mediates kidney ischemia/reperfusion injury. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e (10), 2847\u0026ndash;2859 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, D., Han, Z. \u0026amp; Oppenheim, J. J. Alarmins and immunity. \u003cem\u003eImmunol. Rev.\u003c/em\u003e \u003cb\u003e280\u003c/b\u003e (1), 41\u0026ndash;56 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulay, S. R., Linkermann, A. \u0026amp; Anders, H. J. Necroinflammation in Kidney Disease. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e (1), 27\u0026ndash;39 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWallach, D., Kang, T. B. \u0026amp; Kovalenko, A. Concepts of tissue injury and cell death in inflammation: a historical perspective. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 51\u0026ndash;59 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChavan, S. S., Pavlov, V. A. \u0026amp; Tracey, K. J. Mechanisms and Therapeutic Relevance of Neuro-immune Communication. \u003cem\u003eImmunity\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e (6), 927\u0026ndash;942 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLataro, R. M., Silva, C. A. A., Tef\u0026eacute;-Silva, C., Prado, C. M. \u0026amp; Salgado, H. C. Acetylcholinesterase Inhibition Attenuates the Development of Hypertension and Inflammation in Spontaneously Hypertensive Rats. \u003cem\u003eAm. J. Hypertens.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (10), 1201\u0026ndash;1208 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRocha, J. A. et al. Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats. \u003cem\u003eAm. J. Physiology-Regulatory Integr. Comp. Physiol.\u003c/em\u003e \u003cb\u003e310\u003c/b\u003e (8), R697\u0026ndash;706 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBezerra, O. C. et al. Cholinergic Stimulation Improves Oxidative Stress and Inflammation in Experimental Myocardial Infarction. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (1), 13687 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBandoni, R. L. et al. Cholinergic stimulation with pyridostigmine modulates a heart-spleen axis after acute myocardial infarction in spontaneous hypertensive rats. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (1), 9563 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRovere, M. T., La, Bigger, J. T., Marcus, F. I., Mortara, A. \u0026amp; Schwartz, P. J. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e351\u003c/b\u003e (9101), 478\u0026ndash;484 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarrick, D. et al. Hypertension, Microvascular Pathology, and Prognosis After an Acute Myocardial Infarction. \u003cem\u003eHypertension\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e (3), 720\u0026ndash;730 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKelly, M. J., Breathnach, C., Tracey, K. J. \u0026amp; Donnelly, S. C. Manipulation of the inflammatory reflex as a therapeutic strategy. \u003cem\u003eCell. Rep. Med.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (7), 100696 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoares, P. P. D. S., Da N\u0026oacute;brega, A. C. L., Ushizima, M. R. \u0026amp; Irigoyen, M. C. C. Cholinergic stimulation with pyridostigmine increases heart rate variability and baroreflex sensitivity in rats. Autonomic Neuroscience [Internet]. Jun 30 [cited 2025 Jun 16];113(1\u0026ndash;2):24\u0026ndash;31. (2004). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sciencedirect.com/science/article/pii/S1566070204000979?pes=vor\u0026amp;utm_source=wiley\u0026amp;getft_integrator=wiley\u003c/span\u003e\u003cspan address=\"https://www.sciencedirect.com/science/article/pii/S1566070204000979?pes=vor\u0026amp;utm_source=wiley\u0026amp;getft_integrator=wiley\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSayin, H. et al. Pyridostigmine enhances atrial tachyarrhythmias in aging spontaneously hypertensive rats. Clin Exp Pharmacol Physiol [Internet]. 2015 Oct 1 [cited 2025 Jun 16];42(10):1084\u0026ndash;91. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/doi/pdf/10.1111/1440-1681.12458\u003c/span\u003e\u003cspan address=\"/doi/pdf/10.1111/1440-1681.12458\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMacht, V. A. et al. Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness. \u003cem\u003eBrain Behav. Immun.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 384\u0026ndash;393 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZoccali, C. et al. The autonomic nervous system and inflammation in chronic kidney disease. Nephrology Dialysis Transplantation [Internet]. [cited 2025 Jun 4];16(3):518\u0026ndash;24. (2014). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dx.doi.org/10.1093/ndt/gfaf020\u003c/span\u003e\u003cspan address=\"10.1093/ndt/gfaf020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYeboah, M. M. et al. Cholinergic agonists attenuate renal ischemia\u0026ndash;reperfusion injury in rats. \u003cem\u003eKidney Int.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e (1), 62\u0026ndash;69 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLekawanvijit, S. \u0026amp; Krum, H. Cardiorenal syndrome: acute kidney injury secondary to cardiovascular disease and role of protein-bound uraemic toxins. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cb\u003e592\u003c/b\u003e (18), 3969\u0026ndash;3983 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMinist\u0026eacute;rio da Ci\u0026ecirc;ncia T e IN de C de EA. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.in.gov.br/en/web/dou/-/resolucao-n-57-de-6-de-dezembro-de-2022-448572294\u003c/span\u003e\u003cspan address=\"https://www.in.gov.br/en/web/dou/-/resolucao-n-57-de-6-de-dezembro-de-2022-448572294\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. RESOLU\u0026Ccedil;\u0026Atilde;O N\u003csup\u003eo\u003c/sup\u003e 57, (2022). DE 6 DE DEZEMBRO DE 2022.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcGrath, J. C. \u0026amp; Lilley, E. Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol [Internet]. 2015 Jul 1 [cited 2025 Sep 21];172(13):3189. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pmc.ncbi.nlm.nih.gov/articles/PMC4500358/\u003c/span\u003e\u003cspan address=\"https://pmc.ncbi.nlm.nih.gov/articles/PMC4500358/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBayne, K. \u0026amp; Turner, P. V. Animal Welfare Standards and International Collaborations. \u003cem\u003eILAR J.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e (1), 86\u0026ndash;94 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGebbers, J. O., L࿽tscher, M., Kobel, W., Portmann, R. \u0026amp; Laissue, J. A. Acute toxicity of pyridostigmine in rats: histological findings. \u003cem\u003eArch. Toxicol.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e (4), 271\u0026ndash;275 (1986).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIrigoyen, M. C. et al. Exercise Training Improves Baroreflex Sensitivity Associated With Oxidative Stress Reduction in Ovariectomized Rats. Hypertension [Internet]. 2005 Oct 1 [cited 2025 Sep 24];46(4):998\u0026ndash;1003. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/doi/pdf/10.1161/01.HYP.0000176238.90688.6b?download\u0026thinsp;=\u0026thinsp;true\u003c/span\u003e\u003cspan address=\"/doi/pdf/10.1161/01.HYP.0000176238.90688.6b?download\u0026thinsp;=\u0026thinsp;true\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva, G. et al. JB,. Cardiovascular and neuroimmune adaptations to enalapril and exercise training: A comparative study in male and ovariectomized female spontaneously hypertensive rats. Autonomic Neuroscience [Internet]. Aug 1 [cited 2025 Sep 24];260:103280. (2025). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sciencedirect.com/science/article/pii/S1566070225000426?via%3Dihub#s0010\u003c/span\u003e\u003cspan address=\"https://www.sciencedirect.com/science/article/pii/S1566070225000426?via%3Dihub#s0010\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acute myocardial infarction, renal inflammation, autonomic nervous system, cholinergic stimulation, bromide of pyridostigmine, renal injury","lastPublishedDoi":"10.21203/rs.3.rs-7641684/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7641684/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study examined the impact of pharmacological cholinergic activation on cardiac function and renal inflammatory responses in spontaneously hypertensive rats (SHRs) subjected to acute myocardial infarction (AMI). Adult male SHRs were allocated into three groups: sham-operated, AMI (infarcted), and AMI\u0026thinsp;+\u0026thinsp;PY (infarcted and treated with the cholinesterase inhibitor pyridostigmine bromide [PY] at 40 mg/kg, administered once daily for seven days). Animals were euthanized seven days after surgery by anesthetic overdose, and clinical parameters were evaluated the day prior to euthanasia. Following euthanasia, blood samples were collected and kidney tissues were processed for histological analysis to assess inflammation and injury. At seven days post-surgery, the AMI\u0026thinsp;+\u0026thinsp;PY group showed improvements in blood pressure regulation and autonomic dysfunction. In addition, treatment reduced plasma creatinine, proteinuria, cell proliferation, and collagen accumulation compared with both AMI and sham groups. These findings indicate that cholinergic stimulation with PY provides cardiac and renal protection by mitigating post-AMI injury and inflammation.\u003c/p\u003e","manuscriptTitle":"Cholinergic stimulation after acute myocardial infarction improves hemodynamic parameters and modulates the renal injury in spontaneously hypertensive rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 16:31:08","doi":"10.21203/rs.3.rs-7641684/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-12T10:51:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-07T11:52:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T08:04:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123776333552948711904185294183816451416","date":"2025-10-31T06:09:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186753741365259232962144636130993354655","date":"2025-10-31T06:02:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T16:13:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-30T16:04:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-14T06:12:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-26T14:27:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-26T14:24:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3bcf6a7a-f288-49fd-93d1-780fd0f977f8","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57681411,"name":"Health sciences/Cardiology"},{"id":57681412,"name":"Health sciences/Diseases"},{"id":57681413,"name":"Biological sciences/Drug discovery"},{"id":57681414,"name":"Health sciences/Medical research"},{"id":57681415,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-09T04:25:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 16:31:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7641684","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7641684","identity":"rs-7641684","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}