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1Research Results in Pharmacology
Research Results in Pharmacology 9(2): 85-97 UDC: 615.225.3: 616.13.002.2-004.6 DOI 10.18413/rrpharmacology.9.10032
Correction of renal ischemia/reperfusion injury with the Combination of Infliximab and the erythropoietin-derived peptide mimetic pHBSP
Aleksandr S. Netrebenko1
1 Belgorod State National Research University, 85 Pobecly St., Belgorod. 308015. Russia Corresponding author: Aleksandr S. Netrebenko (AlexNetrebenkoMmail.ru)
Academic editor: Oleg Gudyrev ♦ Received 22 February 2023 ♦ Accepted 25 May 2023 ♦ Published 19 June 2023
Citation: Netrebenko AS (2023) Correction of renal ischemia/reperfusion injury with the combination of Infliximab and the erythropoietin-derived peptide mimetic pHBSP. Research Results in Pharmacology 9(2): 85-97. https://doi.org/10.18413/ rrpharmacology. 9.10032
Introduction: Due to the high social and economic value of acute kidney injury, the scientific community is focused on methods of diagnosis and treatment of this pathology. A number of studies have already revealed cytoprotective effects of the helix B-derived erythropoietin peptide and infliximab in simulated ischemia/ reperfusion injury of liver, myocardium, and nervous tissue. The aim of this research was to study the renoprotective effects of the combination of pHBSP and infliximab on the renal ischemia/reperfusion injury.
Materials and Methods: The experiment was performed in 230 white male Wistar rats. The animals were treated with pHBSP and infliximab. Under anesthesia, a unilateral right nephrectomy was performed and the contralateral renal pedicle was clamped. Functional tests were performed and tissue samples were taken for laboratory studies 5 minutes, 24 hours and 72 hours after reperfusion.
Results and Discussion: The results obtained confirm the dose-dependent renoprotective activity of the helix B-derived erythropoietin peptide and infliximab. The nephroprotective activity of the combination of pHBSP at a dose of 25 mcg/kg and infliximab at a dose of 10 mg/kg significantly exceeded the effect of a single-drug therapy. This is evidenced by the normalization of renal tubule function, a significant increase in the microcirculation level, the absence of rough lesion during pathomorphological examination, as well as a decrease in the expression of TNF-a by 54% and IL-ip by 65% in comparison with the ischemia/reperfusion group according to immunohistochemistry examination. The important role of ATP-sensitive potassium channel in the renoprotective activity of pHBSP has been confirmed.
Conclusion: The renoprotective activity of the helix B-derived erythropoietin peptide and infliximab has been confirmed, and the advantage of their combined administration for the correction of morphofunctional disorders in simulated renal ischemia/reperfusion injury due to the multimodal effect on pathogenetic processes has been established.
Abstract
Copyright Netrebenko AS et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Keywords
pHBSP, infliximab, ischemia/reperfusion, TNF-a, preconditioning, inflammation
Introduction
Oncourological morbidity in Russia remains extremely high. According to current recommendations, the optimal method of treatment at the early stages is a preserving surgery (kidney resection) under WIT conditions (Forbes et al. 2016; Ragulina et al. 2017; Jiang et al. 2019). The most threatening complication of the devascularized kidney is the acute kidney injury (Basile et al. 2014), the main pathogenetic mechanism of which is ischemia/ reperfusion kidney injury (Hwang 2013).
The clinical outcome of acute kidney injury in many cases also remains unsatisfactory. In a mixed population of patients being treated in hospital, mortality can reach 72.6% (Gobe et al. 2015), which exceeds the total mortality from breast cancer and prostate cancer (Sabbisetti et al. 2014).
One of the promising methods for the prevention of ischemia/reperfusion injury is pharmacological preconditioning (Skachilova et al. 2015). In a number of major studies, a preconditioning activity of the glycoprotein hormone erythropoietin has been proved (Brookset al. 2015).
The biological effects of erythropoietin are realized by binding to specific receptors (Yakovlev et al. 2016). There are two types of receptors: homodimeric (EPOR) and heterodimeric (EPOR/pcR). In an adult, erythropoietin binding to the homodimer receptor results in apoptosis inhibition and erythropoiesis activation (Xiaoet al. 2012). Cytoprotective effects of erythropoietin are caused by activation of the heterodimer receptor (Netrebenko et al. 2021). The realization of these effects is mediated by JAK-2, STAT5, and PI3K (Grebien et al. 2008).
Being expressed in several non-hematopoietic tissues, erythropoietin plays an important role in protection against apoptosis and inflammation, and also has proliferative activity. Erythropoietin significantly reduces damage in stroke (Thériault et al. 2016; Jia et al. 2016), myocardial infarction (Arthuret al. 2014) and ischemia/ reperfusion kidney injury (Golmohammadi et al. 2020). However, erythropoietin has dangerous side effects: arterial hypertension, thrombosis and stimulation of the growth and progression of malignant neoplasms (Lund et al. 2014; Yakovlev et al. 2016). Therefore, a small erythropoietin-derived peptide mimetic(pHBSP), capable of selectively binding to EPOR/pcR, was developed and synthesized (Zhang et al. 2017). In a series of experiments, pHBSP has already demonstrated a number of positive effects in simulated ischemia/reperfusion injury to the liver (Tan et al. 2018); it positively influences on the course of connective tissue diseases (Huang et al. 2018) and relieves acute lung injury (Bi et al. 2020).
Pro-inflammatory cytokines play an equally important role in the development of acute kidney injury (Netrebenko et al. 2021). One of the most significant cytokineis tumor necrosis factor alpha (Bethesda et al. 2017), which is released by macrophages and monocytes in damaged tissues and triggers pathological inflammatory reactions, stimulating the production of IL-IB, IL-6, IL-8, enhancing this process (Netrebenko et al. 2021). Infliximab is one of the known drugs that reduce the activity of TNF-a. It has a high affinity for TNF-a and is able to effectively block it (Netrebenko et al. 2022). Infliximab is used as essential therapy in patients with Crohn's disease (Ponsioen et al. 2017) and various forms of sarcoidosis (Bakker et al. 2021). Modern
science makes steps to study the nephroprotective properties of infliximab in the renal ischemia/reperfusion injury, but the results are contradictory (Tasdemir et al. 2012).
Based on the features of pathogenetic processes in renal ischemia/reperfusion, a set of receptor mechanisms and cascades of pathological inflammatory reactions, it can be suggested that one of the promising ways of nephroprotection may be the combined administration of infliximab and pHBSP, taking into account the multimodal mechanisms of their effects and the theoretical possibility of their mutual potentiating.
The aim of the study: to make an experimental confirmation of the prospectivity of renal ischemia/ reperfusion injury correction with the combination of infliximab and the erythropoietin-derived peptide mimetic.
Material and Methods
Compliance with ethical and regulatory requirements
The study was conducted at the Research Institute of Pharmacology of Living Systems of Belgorod National Research University in accordance with regulatory legal acts and guidelines governing the conduct of experimental research in the Russian Federation. The ethical principles of the treatment of laboratory animals meet requirements of the European Convention for the Protection of Vertical Animals Used for Experimental and Other Scientific Purposes. CETSN170.
Experimental animals
The experiment was performed in 230 white male Wistar rats weighing 280-320g, which met all the necessary criteria and were kept in accordance with the current regulations. The experimental protocols were approved by the local independent Ethical committee of Belgorod State National Research University (Minutes No. 3.10 of 28.10.2019).
Study design
After randomization of animals by weight, the following experimental groups were formed:
1 group - Intact animals
2 group - Sham-operated animals (24 hours)
3 group - Sham-operated animals (72 hours)
4 group - Ischemia/reperfusion (24 hours)
5 group - Ischemia/reperfusion (72 hours)
6 group - Ischemia/reperfusion + pHBSP 5 mcg/kg (24 hours)
7 group - Ischemia/reperfusion + pHBSP 5 mcg/kg (72 hours)
8 group- Ischemia/reperfusion + pHBSP 25 mcg/kg (24 hours)
9 group- Ischemia/reperfusion + pHBSP 25 mcg/kg (72 hours)
10 group - Ischemia/reperfusion + infliximab 2 mg/kg (24 hours)
11 group - Ischemia/reperfusion + infliximab 2 mg/kg (72 hours)
12 group - Ischemia/reperfusion + infliximab 10 mg/kg (24 hours)
13 group - Ischemia/reperfusion + infliximab 10 mg/kg (72 hours)
14 group - Ischemia/reperfusion + EPO (24 hours)
15 group - Ischemia/reperfusion + EPO (72 hours)
16 group - Ischemia/reperfusion + pHBSP + infliximab (24 hours)
17 group - Ischemia/reperfusion + pHBSP + infliximab (72 hours)
18 group- Ischemia/reperfusion + glibenclamide (24 hours)
19 group - Sham-operated animals + glibenclamide (72 hours)
20 group - Ischemia/reperfusion + pHBSP + glibenclamide (24 hours)
21 group - Ischemia/reperfusion + pHBSP + glibenclamide (72 hours)
22 group - Ischemia/reperfusion + infliximab + glibenclamide (24 hours)
23 group - Ischemia/reperfusion + infliximab + glibenclamide (72 hours)
The activity of the erythropoietin mimetic peptide (pHBSP) (provided by a pharmaceutical company PHARMAPARK LLC) was studied at the doses of 5 mcg/ kg and 25 mcg/kg; the activity of infliximab (Remicade, MSD) was studied at the doses of 2 mg/kg and 10 mg/kg; the activity of recombinant erythropoietin (Epocrine, Research Institute of Highly Pure Biopreparations, Russia) was studied at the dose of 50 IU/kg; the activity of glibenclamide (Maninil, Berlin-Chemie AG, Germany) was studied at the dose of 5 mg/kg. These doses were selected based on previously identified protective effects on ischemia/reperfusion models or were calculated taking into account the recommended human doses using conversion factors (Nagata et al. 2016; Kostina et al. 2021; Firsova et al. 2022). The dose schedule is based on the pharmacokinetic profile of the drug.
Simulation of the renal ischemia/reperfusion injury
The animals were anesthetized by intraperitoneal injection of chloral hydrate at the dose of 300 mg/kg (Bratchikov et al. 2018). After surgical field preparation, median laparotomy was performed, and a renal body with elements of a renal pedicle was pushed out on both sides (Bratchikov et al. 2018). Microcirculation in the renal parenchyma was measured according to a generally accepted method (Elagin and Bratchikov 2018). Left renal pedicle clamping for 40 minutes followed by a right nephrectomy was performed. Urine was collected during reperfusion. Twenty-four or 72 hours after reperfusion, the rats were anesthetized again; relaparotomy was performed; microcirculation parameters in the renal parenchyma were measured; blood was taken from the right ventricle for biochemical studies, and tissue samples were taken.
Measurement of biochemical and functional parameters
Serum creatinine and urea levels were measured using a biochemical analyzer URIT800 Vet (URIT Medical Electronic Co. Ltd., China). The concentrations of potassium and sodium ions in the blood serum were detected according to the standard procedure using the kits for the automatic analyzer K/N "Ionomer ETS-59". Endogenous creatinine clearance (glomerular filtration rate) and fractional sodium excretion were calculated using standard formulas.
Morphological examination
The kidney samples were fixed in 10% formalin. The slides were stained with hematoxylin and eosin. All
studies were performed using a Leica DM4000B microscope.
Immunohistochemistry
The immunohistochemical study was performed in serial paraffin sections with a thickness of 2-3 microns placed on adhesive glasses coated with poly-L-lysine (Super Frost Plus, "Mainzel Glazer, Germany). Antibodies to IL-1 beta (ThermoFisher, 1:100), IL-4 (ThermoFisher, 1:100), IL-6 (ThermoFisher, 1:100), IL-10 (ThermoFisher, 1:100), CD68 (514H12; LeicaRTU) were used as primary antibodies. All immunohistochemical reactions were performed manually, and reaction on CD68 was performed in automatic mode (Bond-Max immunohistosteiner "Leica", Germany). The primary antibodies were anti-rat. Secondary antibodies were a universal two-component detection system HiDef Detection™ HRP Polymer system, ("Cell Marque", USA), mouse/rabbit anti-IGG, horseradish peroxidase (HRP) and DAB substrate. The cell nuclei were stained with Mayer's hematoxylin. The evaluation of immunohistochemical reactions was based on the intensity of staining and separation of immunopositive cells according to the recommendations of D.J. Dabbs "Diagnostic immunohistochemistry" (4rd Edition, 2014).
Statistical data processing
Descriptive statistics were applied to all the data: the data were tested for the normality of distribution. The type of distribution was determined using the Shapiro-Wilk's test. The mean value (M) and the standard error of the mean (m) were calculated in a normal distribution. Taking into account the normal distribution of the results, a parametric method (Student's t-test) was used to analyze the intergroup differences. All calculations were made using the Microsoft Excel 10.0 statistical software package.
Results and Discussion
Assessment of morphofunctional disorders in the renal ischemia/reperfusion injury
Pathology simulation by the opening of the retroperitoneal space during laparotomy and applying of the atraugrip on the left vascular renal pedicle for 40 minutes leads to a complex of changes corresponding to the modern criteria for acute renal injury KDIGO 201224 and 72 hours after reperfusion. After 24 hours in the ischemia/reperfusion group, the serum creatinine level was 102.6±3.6 mmol/L, glomerular filtration rate was 0.09±0.01 ml/min, and fractional sodium excretion was 2.77±0.1%; in the group of the sham-operated animals creatinine was 45.9±0.8 mmol/L, glomerular filtration rate was 0.75±0.02 ml/min, and fractional sodium excretion was 0.37± 0.01%. Seventy-two hours after reperfusion in the ischemia/ reperfusion group, serum creatinine level was 125.6±6.4 mmol/L, glomerular filtration rate was 0.06±0.01 ml/min, and fractional sodium excretion was 6.83±0.29%, in the group of the sham-operated animals creatinine was 47.0±2.3 mmol/L glomerular filtration rate was 0.77±0.04 ml/min, and fractional sodium excretion was 0.37±0.04% (Fig. 1).
The dynamics of the microcirculation index in the renal parenchyma was as follows: the microcirculation level was 900±42 PU5 minutes, 881±38 PU24 hours and 890±36 PU72 hours after reperfusion in the group of sham-operated animals. Simulation of the acute kidney injury was accompanied by a statistically significant decrease in the microcirculation level 5 minutes after reperfusion to 219±12 PU with a moderate improvement in this index 24 and 72 hours after reperfusion to 430±20 PU and 410±20 PU, respectively.
Figure 1. The values of serum creatinine (A), urea (B), glomerular filtration rate (C) and fractional sodium excretion (D) 24 and 72 hours after reperfusion in the simulated renal ischemia/reperfusion injury. Note: control - intact animals; sham - sham-operated animals; IR - renal ischemia/ reperfusion; x - p<0.05 in comparison with the group of sham-operated animals.
Microscopic examination of kidney sections 24 and 72 hours after reperfusion revealed the presence of destructive changes consisting in a large number of oxyphilic masses, the predominance of shrunken glomeruli in most fields, which was confirmed by a decrease in the cross-sectional area of the glomerular vascular pole by 1.2 times compared with the group of sham-operated animals, a decrease in the height of epithelial cells of the proximal tubules by 1.4 times, most likely due to necrosis (Table 1).
Table 1. Morphometric characteristics of the structural elements of the nephron in the simulated ischemia/reperfusion (M±m)
Group
Cross-sectional area of the
renal corpuscle, pm2
Height of epithelial cells of the proximal tubules, pm
Cross-sectional area of the
renal corpuscle, pm2
Height of epithelial cells of the proximal tubules, pm
24 hours
72 hours
Sham 10496±123 11.9±0.7 10345±118 11.8±0.6 IR 8973±241x 8.3±0.3x 8293±227x 6.4±0.5x
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; x - р<0.05 in comparison with the group of sham-operated animals.
Immunohistochemical examination of the kidneys for pro-inflammatory and anti-inflammatory cytokines revealed that in the glomeruli and tubules of nephrons of the sham-operated animals the number of cells expressing both pro-inflammatory and anti-inflammatory cytokines varies on the average from 3.0% to 6.0% (Table 2). Investigation of the intensity of the macrophages and monocytes infiltration of the kidney structural elements in sham-operated animals showed that the relative level of CD68-positive cells in interstitial tissue was 20%.
Twenty-four hours after ischemia/reperfusion surgery of the kidney, a statistically significant increase in the number of cells expressing both pro-inflammatory and anti-inflammatory cytokines was observed in all its structural elements, and the level of CD68-positive cells in interstitial tissue reached 61.8±0.42%. Seventy-two hours after ischemic reperfusion injury of the kidney, a decrease in the number of cells expressing both pro-inflammatory cytokines and anti-inflammatory cytokines was observed in all its structural elements compared to those after 24 hours (Table 2).
Thus, the proposed method of renal ischemia simulation with a subsequent reperfusion period of 24 or 72 hours is an adequate experimental model for acute renal injury and can be used to evaluate the effectiveness of new drugs.
Renoprotective effects of the erythropoietin-derived peptide mimetic in ischemia/reperfusion injury of the kidney
Administration of pHBSP at the doses of 5 mcg/kg and 25 mcg/kg restored the glomerular filtration rate to 0.27±0.01 ml/min and 0.29±0.01 ml/min respectively 24 hours after and 0.27±0.02 ml/min and 0.38±0.02 ml/min, respectively, 72 hours afterwards, which was accompanied by a decrease in serum creatinine and urea concentrations. Twenty-four hours later, a significant decrease in fractional sodium excretion by more than 2 times was revealed compared with the ischemia/reperfusion group (Fig. 2).
pHBSP administration at the doses of 5 mcg/kg and 25 mcg/kg led to a dose-dependent improvement in kidney function 72 hours after the clamps were removed from the renal pedicle manifested in a decrease in serum creatinine concentration to 88.3±3.9 mmol/L and 62.2±3.3 mmol/L and urea to 16.2±1.1 mmol/L and 9.7±0.9 mmol/L, respectively, and the fractional sodium excretion index was 2.7±0.17% and 2.1±0.16%, respectively. The renoprotective effects of pHBSP significantly exceeded the effects of a single therapy with recombinant human erythropoietin at a dose of 50 lU/kg.
Table 2. The levels of expression of pro-inflammatory and anti-inflammatory cytokines in the kidney structures (M±m)
Tissue Group IL-1ß, % TNF-a, % IL-4, % IL-10, %
24 hours
Sham 5.8±0.3 4.9±0.3 4.2±0.2 7.8±0.4
Glomerulus
IR 49.9±1.1x 69.7±1.3x 15.5±0.8x 12.4±0.4x
Sham 5.9±0.3 5.7±0.3 4.0±0.2 5.8±0.2
Nephrontubules
IR 56.5±1.5x 71.1±1.2x 16.2±0.9x 13.7±0.3x
72 hours
Sham 5.6±0.3 5.5±0.3 4.9±0.2 8.3±0.4
Glomerulus
IR 45.0±0.9x 63.6±1.2x 11.5±0.6x 20.2±0.6x
Nephrontubules
Sham IR
4.7±0.3 49.4±1.3x
5.7±0.3 62.2±1.3x
4.2±0.2 15.0±0.9x
6.0±0.2 10.5±0.3x
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; x - р<0.05 in comparison with the group of sham-operated animals.
Figure 2. The effect of the erythropoietin-derived peptide mimetic (pHBSP) on the concentration of serum creatinine (A), urea (B), glomerular filtration rate (C) and fractional sodium excretion (D) 24 hours after reperfusion. Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; EPO - erythropoietin (at the dose of 50 IU/kg); pHBSP 5 - erythropoietin-derived peptide mimetic (at the dose of 5 mcg/kg); pHBSP 25 -erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group.
A single administration of pHBSP at the doses of 5 mcg/ kg and 25 mcg/kg led to restoration of the microcirculation level in all control time periods, significantly exceeding the indicators of the EPO group (Table 3).
Table 3. The effect of the erythropoietin-derived peptide mimetic on the renal microcirculation (M±m)
Microcircula-Experimental tion index group 5 minutes,
PU
Microcircula- Microcirculation index tion index 24 hours, PU 72 hours, PU
Sham IR
IR + EPO IR + pHBSP 5 IR + pHBSP 25
900±42у 219±12x 637±27xy 492±21xy 693±28xy
881±38у 430±20x 733±31xy 607±28xy 771±27xy
890±36у 410±20x 539±39xy 584±32xy 625±36xy
Table 4. Morphometric characteristics of the structural elements of the nephron against the background of nephroprotection with pHBSP (M±m)
C Height of C Height of
Cr°SS~, epithelial C™SS~, epithelial
sectional „ ? sectional „ ?
cells of cells of
area oi the area oi the
the the
renal renal
„ . proximal . proximal
Group corpuscle, . , , corpuscle, . , ,
1 , tubules, 1 , ' tubules,
am2 am2
^ am ^ am
Sham
IR
24 hours
72 hours
10496±123у 11.9±0.7у 10345±118у 11.8±0.6у
8973±241x 8.3±0.3xy 8293±227x 6.4±0.5x
IR + EPO 8938±102x 8.9±0.1xy 8974±98xy 6.9±0.2x
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; EPO - erythropoietin (at the dose of 50 IU/kg); pHBSP 5 -erythropoietin-derived peptide mimetic (at the dose of 5 mcg/kg); pHBSP 25 - erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group.
Microscopic evaluation of the kidneys slices of the animal groups administered with pHBSP for nephroprotection revealed a significant dose-dependent improvement in the histological pattern. The number of edematous or shrunken renal corpuscles was insignificant, and the cross-sectional area of the renal corpuscles and the height of the epithelial cells increased, which indicates a reduction of the ischemia/reperfusion injury (Table 4).
IR +
pHBSP 5
IR +
pHBSP 25
8894±85x 9.1±0.1xy 9126±85xy 7.1±0.1xy
9029±98x
9.6±0.1xy
9344±88xy
;.1±0.2xy
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; EPO - erythropoietin (at the dose of 50 IU/kg); pHBSP 5 -erythropoietin-derived peptide mimetic (at the dose of 5 mcg/kg); pHBSP 25 - erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group.
Twenty-four hours after reperfusion, pHBSP had a dose-dependent effect, consisting in a smaller increase in the cells expressing pro-inflammatory cytokines (Fig. 3), and an increase in cells expressing IL-10 in all parts of the nephron compared to the group of control untreated animals (Table 5). The dose-dependent effect of pHBSP on the intensity of macrophages and monocytes infiltration of the kidney tissues was also noted. pHBSP administration led to a less pronounced increase in CD68-positive cells in the interstitial tissue of the renal parenchyma. This indicator was 37.88±0.5% and 31.98±0.45%, respectively, in the groups of animals treated with pHBSP at the doses of 5 mcg/kg and 25 mcg/ kg. Seventy-two hours after the clamps removal from the renal pedicle and reperfusion, the maintaining of dose-dependent effect like the one 24 hours after reperfusion was observed in the animals that had been injected with pHBSP (Table 5). The number of CD68-positive cells in the interstitial tissue was 34.94±0.47% and 32.39±0.43%.
Table 5. The effect of pHBSP on the expression of pro-inflammatory and anti-inflammatory cytokines in the kidney (M±m)
Tissue Group IL-1ß, % TNF-a, % IL-4, % IL-10, %
24 hours
Sham 5.8±0.3 4.9±0.3 4.2±0.2 7.8±0.4
IR 49.9±1.1x 69.7±1.3x 15.5±0.8x 12.4±0.4x
Glomerulus IR + EPO 40.6±0.9xy 62.2±1.3xy 15.9±0.9x 31.2±1.0xy
IR + pHBSP 5 40.6±0.9xy 61.8±1.3xy 14.3±0.9x 33.7±1.0xy
IR + pHBSP 25 32.7±0.8xy 49.4±1.1xy 17.1±0.9x 46.9±1.3xy
Nephrontubules Sham 5.9±0.3 5.7±0.3 4.0±0.2 5.8±0.2
IR 56.5±1.5x 71.1±1.2x 16.2±0.9x 13.7±0.3x
IR + EPO 47.8±0.9xy 62.9±1.1xy 18.7±1.1x 23.3±0.8xy
IR + pHBSP 5 50.9±0.9xy 61.4±1.0xy 20.7±1.2x 22.3±0.7xy
IR + pHBSP 25 36.9±0.8xy 51.7±0.7xy 18.6±1.2x 46.8±0.7xy
72 hours
Sham 5.6±0.3 5.5±0.3 4.9±0.2 8.3±0.4
IR 45.0±0.9x 63.6±1.2X 11.5±0.6x 20.2±0.6x
Glomerulus IR + EPO 38.3±0.7xy 56.8±1.3xy 11.6±0.5x 28.4±0.8xy
IR + pHBSP 5 37.6±0.8xy 57.0±1.4xy 13.1±0.7x 25.5±0.7xy
IR + pHBSP 25 29.3±0.6xy 42.4±1.0xy 16.4±0.9xy 40.3±1.2xy
Sham 4.7±0.3 5.7±0.3 4.2±0.2 6.0±0.2
IR 49.4±1.3x 62.2±1.3x 15.0±0.9x 10.5±0.3x
Nephrontubules IR + EPO 46.3±0.9xy 55.7±0.9xy 14.4±0.9x 15.7±0.6xy
IR + pHBSP 5 43.6±0.9xy 60.4±0.6у 16.1±0.9x 16.8±0.5xy
IR + pHBSP 25 34.1±0.7xy 39.3±0.8xy 16.8±1.0x 37.5±0.7xy
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; EPO - erythropoietin (at the dose of 50 IU/kg); pHBSP 5 - erythropoietin-derived peptide mimetic (at the dose of 5 mcg/kg); pHBSP 25 - erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group.
Б D
Figure 3. The effect of pHBSP 25 mcg/kg on the expression of IL-1ß (A, B) and TNFa (C, D) in the renal cells 24 hours after reperfusion. Note: immunohistochemical reaction with antibodies to IL-1ß and TNFa; light microscopy, magnification x400.
Thus, the obtained results indicate the dose-dependent renoprotective properties of the erythropoietin-derived peptide mimetic: pHBSP administration led to a decrease in the concentration of nitrogen metabolism products in blood plasma, normalization of the glomerular filtration rate and fractional sodium excretion. The level of renal parenchymal perfusion significantly increased. Also, morphological and immunohistochemical studies revealed greater protective capabilities compared to recombinant human erythropoietin.
Renoprotective effects of infliximab in ischemia/ reperfusion kidney injury
The injection of infliximab at the dose of 10 mcg/kg intraperitoneally one hour before ischemia contributed to a significant decrease in serum creatinine to 63.2±2.5 mmol/L 24 hours later, and also led to an increase in glomerular filtration rate to 0.22±0.01 ml/min, which significantly differed from the ischemia/reperfusion group. Seventy-two hours after the clamps removal from the renal pedicle, a decrease in serum creatinine concentration to 108.4±5 mmol/L and 69.3±2.9 mmol/L, and an increase in glomerular filtration rate to 0.19±0.02 ml/min and 0.33±0.02 ml/min were revealed in the groups treated with infliximab at the doses of 2 mg/kg and 10 mg/kg, respectively. The concentration of urea in the blood also decreased under the influence of infliximab, reaching the levels of 20.4±1.4 mmol/L and 13.6±1.3 mmol/L24 hours later, and 15.6±1.1 mmol/L and 9.0±1.0 mmol/L 72 hours later, respectively. The obtained values in the group of infliximab 10 mg/kg significantly differed from the ischemia/reperfusion group and came close to the group of sham operated animals. Twenty-four hours after the restoration of renal blood supply, infliximab administration in both doses led to a pronounced decrease in fractional sodium excretion by more than 2 times compared with the ischemia/ reperfusion group. On the 3rdday of the experiment, protection with infliximab had a positive effect on the fractional sodium excretion index, which was 2.34±0.19% and 1.5±0.14% for the doses of 2 mg/kg and 10 mg/kg, respectively, which significantly differs from the values in the ischemia/reperfusion group.
The administration of infliximab at the dose of 10 mg/ kg contributed to the improvement of microcirculation in all control time periods, significantly exceeding the indicators of the ischemia/reperfusion group (Table 6).
Table 6. The influence of infliximab on the renal microcirculation (M±m)
Experimental group Microcirculation index 5 minutes, PU Microcirculation index 24 hours, PU Microcirculation index 72 hours, PU
Sham 900±42у 881±38у 890±36у
IR 219±12x 430±20x 410±20x
IR + INF 2 418±17xy 448±20x 522±43xy
IR+ INF 10 679±31xy 743±34xy 631±30xy
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; INF 2 - infliximab (at the dose of 2 mg/kg); INF 10 - infliximab (at the dose of 10 mg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/ reperfusion group.
Histological examination revealed the dose-dependent nephroprotective effect of infliximab. Infliximab at the dose of 2 mg/kg scarcely led to an improvement in the microscopic pattern and morphometry indicators in comparison with the pathology simulated group, while in the course of the treatment with infliximab at the dose of 10 mg/kg, a moderate number of shrunken renal corpuscles were noted in the kidney sections, subcapsular spaces were slightly dilated 24 and 72 hours after reperfusion. This is consistent with the morphometry, according to which, an increase in the cross-sectional area of the glomerular vascular pole, as well as in the height of the epithelial cells of the proximal and distal tubules of the nephron, was revealed (Table 7).
Table 7. Morphometric characteristics of the structural elements of the nephron against the background of nephroprotection with infliximab (M±m)
Group Cross-sectional area of the renal corpuscle, pm2 Height of epithelial cells of the proximal tubules, pm Cross-sectional area of the renal corpuscle, pm2 Height of epithelial cells of the proximal tubules, pm
24 hours 72 hours
Sham 10496±123у 11.9±0.7у 10345±118у 11.8±0.6у
IR 8973±241x 8.3±0.3xy 8293±227x 6.4±0.5x
IR + INF 2 8716±113x 8.9±0.1xy 9092±92xy 7.4±0.1xy
IR+ INF 10 8994±75xy 9.7±0.1xy 9208±106xy 8.5±0.2xy
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; INF 2 - infliximab (at the dose of 2 mg/kg); INF 10 - infliximab (at the dose of 10 mg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/ reperfusion group.
Twenty-four hours after the restoration of blood supply of the renal parenchyma, a dose-dependent effect of infliximab was observed, consisting in a smaller increase in the cells expressing pro-inflammatory cytokines and an increase in the number of cells expressing IL-10 in all elements of the nephron compared to the group of control untreated animals (Table 8). There was also a less pronounced increase in the CD68-positive cells, compared with the group of control untreated animals; their level in the interstitial tissue reached 35.33±0.49% and 33.5±0.42%, respectively (Fig. 4). Immunohistochemical methods of examination of the expression of pro-inflammatory and anti-inflammatory cytokines, CD68-positive cells in kidney structures showed the maintaining of dose-dependent effect 72 hours after reperfusion like the one 24 hours after reperfusion in the animals that had been injected with infliximab (Table 8).
The obtained results confirm the renoprotective activity of infliximab: infliximab administration decreased the concentration of nitrogen metabolism indicators in blood plasma, normalized the glomerular
Table 8. The effect of infliximab on the expression of pro-inflammatory and anti-inflammatory cytokines in the kidney (M±m)
Tissue Group IL-1p, % TNF-a, % IL-4, % IL-10, %
24 hours
Sham 5.8±0.3 4.9±0.3 4.2±0.2 7.8±0.4
IR 49.9±1.1x 69.7±1.3X 15.5±0.8x 12.4±0.4x
Glomerulus
IR + INF 2 30.4±0.6xy 50.1±1.1xy 22.0±1.1xy 32.2±1.0xy
IR+ INF 10 25.0±0.6xy 40.6±0.9xy 18.4±0.9xy 49.7±1.1xy
Sham 5.9±0.3 5.7±0.3 4.0±0.2 5.8±0.2
IR 56.5±1.5X 71.1±1.2x 16.2±0.9x 13.7±0.3x
Nephrontubules IR + INF 2 36.6±0.7xy 49.8±0.9xy 18.9±1.1x 27.9±0.8xy
IR+ INF 10 29.9±0.6xy 43.6±0.7xy 18.8±1.1x 50.8±0.9xy
72 hours
Sham 5.6±0.3 5.5±0.3 4.9±0.2 8.3±0.4
IR 45.0±0.9* 63.6±1.2x 11.5±0.6* 20.2±0.6x
Glomerulus
IR + INF 2 25.0±0.6xy 42.6±1.1xy 13.1±0.9x 26.2±0.7xy
IR+ INF 10 22.9±0.4xy 33.9±0.9xy 18.4±0.9xy 41.1±1.1xy
Sham 4.7±0.3 5.7±0.3 4.2±0.2 6.0±0.2
IR 49.4±1.3x 62.2±1.3x 15.0±0.9x 10.5±0.3x
Nephrontubules IR + INF 2 31.6±0.7xy 41.5±0.9xy 17.4±0.9x 22.1±0.7xy
IR+ INF 10 27.9±0.7xy 36.2±0.9xy 18.5±1.1xy 42.1±0.7xy
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; INF 2 - infliximab (at the dose of 2 mg/kg); INF 10 - infliximab (at the dose of 10 mg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group.
Renoprotective properties of the combination of the helix B-derived erythropoietin peptide and infliximab
The combined administration of pHBSP at the dose of 25 mcg/kg and infliximab at the dose of 10 mg/kg in the ischemia/reperfusion kidney injury had a positive effect on the filtration function of the kidneys; the effect of the combination significantly exceeded the effect of these drugs in a single-drug therapy. So, 24 hours after reperfusion, the glomerular filtration rate reached 0.42±0.02 ml/min and was as close as possible to the group of sham operated animals. The same trend is observed for the nitrogen metabolism indicators: creatinine and serum urea, as well as fractional sodium excretion (Fig. 5). Seventy-two hours after the clamps were removed from the renal pedicle, the combined therapy with pHBSP and infliximab slightly exceeded the effectiveness of the single-drug therapy, which was reflected in a decrease in plasma creatinine and urea levels, an increase in glomerular filtration rate and a decrease in fractional sodium excretion.
A single administration of the combination of pHBSP and infliximab restored the level of microcirculation in all control time periods, significantly exceeding the values for these drugs in a single-drug therapy (Table 9).
filtration rate and fractional sodium excretion. The level of renal parenchymal perfusion significantly increased. Also, a pathomorphological study with morphometry revealed an improvement in the histological pattern of renal tissue.
The obtained immunohistochemistry results clearly demonstrate the significant role of pro-inflammatory cytokines that negatively affect the renal functions after the episode of ischemia-reperfusion. In turn, infliximab, blocking them, had a significant renoprotective effect.
Figure 4.The effect of infliximab at the doses of 2 mg/kg (A) and 10 mg/kg (B) on the macrophage infiltration of kidney tissues 24 hours after reperfusion. Note: immunohistochemical reaction with antibodies to CD68; light microscopy, magnification x400
Figure 5. The effect of the erythropoietin-derived peptide mimetic (pHBSP) on the concentration of serum creatinine (A), urea (B), glomerular filtration rate (C) and fractional sodium excretion (D) 24 hours after reperfusion. Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; EPO - erythropoietin (at the dose of 50 IU/kg); pHBSP 5 - erythropoietin-derived peptide mimetic (at the dose of 5 mcg/kg); pHBSP 25 -erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group.
Histological examination of the animal kidneys treated with the combination of pHBSP 25 mcg/kg + Infliximab 10 mg/kg showed glomeruli without any signs of destruction, which is confirmed by an increase in the cross-sectional area of the renal corpuscle, renal glomerulus and subcapsular space in comparison with the single-drug therapy groups of animals (Table 10).
Table 9. The influence of the combination of pHBSP and infliximab on the renal microcirculation (M±m)
Experimental group
Microcircula- Microcircula- Microcircula-
tion index
tion index
Sham IR
900±42yz 219±12xz
881±38yz 430±20xz
tion index
5 minutes, PU 24 hours, PU 72 hours, PU
890±36yz 410±20xz
in all structural elements of the kidney. Their level was on average 3 times lower than in the group of untreated animals.
Table 10. Morphometric characteristics of the structural elements of the nephron against the background of nephroprotection with the combination of pHBSP and infliximab (M±m)
Group
Sham
Cross-sectional area of the
renal corpuscle, pm2
Height of epithelial cells of the proximal tubules, pm
Cross-sectional area of the
renal corpuscle, pm2
Height of epithelial cells of
the proximal tubules, pm
24 hours
72 hours
10496±123у
11.9±0.7у
10345±118у 11.8±0.6у
IR + pHBSP 25
IR + INF 10
IR + pHBSP + INF
693±28xy 678±23xy 809±41yz
771±27xy 743±34ху 802±10у
625±36ху 631±30xy 762±41xyz
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; pHBSP 25 - erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); INF 10 - infliximab (at the dose of 10 mg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group; z - p<0.05 in comparison with the pHBSP 25 group and p<0.05 in comparison with the infliximab group (10 mg/kg).
Twenty-two hours after reperfusion, the combined therapy with pHBSP and infliximab resulted in the minimal increase in the cells expressing pro-inflammatory cytokines
IR
IR +
pHBSP
25
IR + INF 10
IR +
pHBSP + INF
8973±241x
9029±98x
8994±75xy
8.3±0.3xy
9.6±0.1xy
9.7±0.1xy
8293±227x
9344±88xy
9208±106xy
6.4±0.5x
8.5±0.2xy
8.5±0.2xy
9724±122xyz 10.3±0.2xyz 9854±115xyz 9.9±0.2xyz
Note: sham - sham-operated animals; IR - renal ischemia-reperfusion; pHBSP 25 - erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); INF 10 - infliximab (at the dose of 10 mg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group; z - p<0.05 in comparison with the pHBSP 25 group and p<0.05 in comparison with the infliximab group (10 mg/kg).
On the other hand, there was an increase in the number of IHC-positive cells expressing antiinflammatory cytokines in the kidneys (Table 11). The minimal increase in CD68-positive cells in the interstitial kidney tissue (26.51±0.38%) was noted against the background of the combined therapy with pHBSP and infliximab 24 hours after reperfusion (Fig. 6). There was a decrease in cells expressing both pro-inflammatory and anti-inflammatory cytokines in all structural elements of the kidney 72 hours after reperfusion compared to those 24 hours later (Table 11). It was revealed that only combined therapy with pHBSP and infliximab leads to a significant increase in the expression of the anti-inflammatory cytokine IL-4, which reduces the severity of pathomorphological changes after ischemia/ reperfusion kidney injury and reduces the risk of delayed fibrotic changes.
Table 11. The effect of the combination of pHBSP and infliximab on the expression of pro-inflammatory and anti-inflammatory cytokines in the kidney (M±m)
Tissue Group IL-1ß, % TNF-a, % IL-4, % IL-10, %
24 hours
Sham 5.8±0.3 4.9±0.3 4.2±0.2 7.8±0.4
IR 49.9±1.1x 69.7±1.3x 15.5±0.8x 12.4±0.4x
Glomerulus IR + pHBSP 25 32.7±0.8xy 49.4±1.1xy 17.1±0.9x 46.9±1.3xy
IR + INF 10 25.0±0.6xy 40.6±0.9xy 18.4±0.9xy 49.7±1.1xy
IR + pHBSP + INF 17.6±0.6xyz 32.9±0.8xyz 20.0±1.2xy 57.9±1.1xyz
Sham 5.9±0.3 5.7±0.3 4.0±0.2 5.8±0.2
IR 56.5±1.5x 71.1±1.2x 16.2±0.9x 13.7±0.3x
Nephrontubules IR + pHBSP 25 36.9±0.8xy 51.7±0.7xy 18.6±1.2x 46.8±0.7xy
IR + INF 10 29.9±0.6xy 43.6±0.7xy 18.8±1.1x 50.8±0.9xy
IR + pHBSP + INF 19.5±0.6xyz 35.9±0.9xyz 21.5±1.3xy 63.6±0.9xyz
72 hours
Sham 5.6±0.3 5.5±0.3 4.9±0.2 8.3±0.4
IR 45.0±0.9x 63.6±1.2x 11.5±0.6x 20.2±0.6x
Glomerulus IR + pHBSP 25 29.3±0.6xy 42.4±1.0xy 16.4±0.9xy 40.3±1.2xy
IR + INF 10 22.9±0.4xy 33.9±0.9xy 18.4±0.9xy 41.1±1.1xy
IR + pHBSP + INF 15.4±0.6xyz 21.6±0.7xyz 18.1±0.9xy 51.3±0.9xyz
Sham 4.7±0.3 5.7±0.3 4.2±0.2 6.0±0.2
IR 49.4±1.3x 62.2±1.3x 15.0±0.9x 10.5±0.3x
Nephron tubules IR + pHBSP 25 34.1±0.7xy 39.3±0.8xy 16.8±1.0x 37.5±0.7xy
IR + INF 10 27.9±0.7xy 36.2±0.9xy 18.5±1.1xy 42.1±0.7xy
IR + pHBSP + INF 20.1±0.6xyz 24.4±0.9xyz 20.9±1.2xy 58.2±0.7xyz
Note: sham - sham-operated animals; IR - renal ischemia/reperfusion; pHBSP 25 - erythropoietin-derived peptide mimetic (at the dose of 25 mcg/kg); INF 10 - infliximab (at the dose of 10 mg/kg); x - p<0.05 in comparison with the group of sham-operated animals; y - p<0.05 in comparison with the ischemia/reperfusion group; z - р<0.05 in comparison with the pHBSP 25 group and р<0.05 in comparison with the infliximab group (10 mg/kg).
Figure 6. The effect of infliximab at the doses of 2 mg/kg (A) and 10 mg/kg (B) on the macrophage infiltration of kidney tissues 24 hours after reperfusion. Note: immunohistochemical reaction with antibodies to CD68; light microscopy, magnification x400.
The obtained results evidence the advantage of the combined administration of pHBSP and infliximab for the nephroprotection in simulated ischemia/reperfusion kidney injury surpassing in effectiveness the protective effects of pHBSP and infliximab in a single-drug therapy, due to the multimodal effect on pathogenetic processes involving in ischemia/reperfusion kidney injury.
The immunohistochemistry results confirmed the mechanism of renoprotective activity of infliximab and pHBSP: these substances block the macrophage and monocyte infiltration of kidney tissues, which leads to a significant decrease in the expression of pro-inflammatory cytokines in the structural elements of the nephron and contribute to the retention of the renal structure and function after simulated ischemia/ reperfusion injury.
Determination of the role of ATP-sensitive potassium channels in the nephroprotective effect of the helix B-derived erythropoietin peptide and infliximab in simulated renal ischemia/reperfusion
The inhibition of ATP-sensitive potassium channels with glibenclamide led to a pronounced subsidence of the nephroprotective effects of pHBS , which was confirmed by an increase in plasma creatinine levels to 91.9±4.1 mmol/L and 109.8±5.6 mmol/L, and urea to 19.4±1.6 mmol/L and 17.8±1.9 mmol/L 24 hours and 72 hours after reperfusion, respectively. Similar dynamics were noted for glomerular filtration rate, which was 0.14±0.01 ml/min and 0.13±0.01 ml/min, and fractional sodium excretion, which was 2.25±0.1% and 5.82±0.42% 24 hours and 72 hours after reperfusion, respectively. The level of microcirculation at all time points in the groups of animals treated with glibenclamide together with pHBSP was comparable to that of the ischemia/ reperfusion group. The administration of glibenclamide together with pHBSP significantly worsened the histological pattern and the results of morphometry: microscopic examination revealed pronounced destructive changes, multiple local deposits of oxyphilic masses between the renal cortex and renal medulla, as well as in the tubule lumen and collector tubules. Shrunken glomeruli were found in most fields. The tubule
lumen is dilated, and severe intracellular edema was noted. There was no significant dependence of the protective activity of infliximab on ATP-sensitive potassium channels.
It should be concluded that ATP-sensitive potassium channels play an important role in the realization of renoprotective effect of pHBSP in the simulated ischemia/reperfusion kidney injury, unlike infliximab, which showed nephroprotective activity through other ways independent of ATP-sensitive potassium channels.
Conclusion
The results of the performed study reliably confirm the renoprotective properties of pHBSP and infliximab, and also verify the advantage of their combined administration for correction of morphofunctional disorders in simulated ischemia/reperfusion kidney injury. The results of immunohistochemical study confirmed the mechanism of the renoprotective effect of infliximab and pHBSP: these substances reduce the macrophage and monocyteinfiltration of kidney tissues, which leads to a significant decrease in the expression of pro-inflammatory cytokines in the structural elements of the nephron and contributes to the retention of the renal structure and function after simulated ischemia/ reperfusion injury.
Funding
The author has received no funding to report.
Conflict of Interests
The author declares no conflict of interests.
Acknowledgements
The author thanks M.A. Zatolokina for her help and methodological support in conducting and evaluating the results of the pathomorphological study.
References
■ Bakker ALM, Mathijssen H, Azzahhafi J, Swaans MJ, Veltkamp M, Keijsers RGM, Akdim F, Post MC, Grutters JC (2021) Effectiveness and safety of infliximab in cardiac Sarcoidosis. International Journal of Cardiology 330: 179-185. https://doi.org/10.1016/ j.ijcard.2021.02.022 [PubMed]
■ Basile DP, Yoder MC (2014) Renal endothelial dysfunction in acute kidney ischemia reperfusion injury. Cardiovascular &Hematological Disorders-Drug Targets 14(1): 3-14. https://doi.org/ 10.2174/1871529x1401140724093505 [PubMed] [PMC] Bethesda MD (2017) LiverTox: Clinical and Research Information on Drug-Induced Liver Injury. National Institute of Diabetes and Digestive and Kidney Diseases. [PubMed]
■ Bi XG. Li ML. Xu W. You JY Xie D. Yuan XF. Xiang Y (2020) Helix B surface peptide protects against acute lung injury through reducing oxidative stress and endoplasmic reticulum stress via activation of Nrf2/HO-1 signaling pathway. European Review for Medical and Pharmacological Sciences 24(12): 6919-6930. https:// doi.org/10.26355/eurrev_202006_21683 [PubMed]
■ Bratchikov OI. Pokrovskiy MV. Elagin VV. Kostina DA (2018) The correction of endothelial dysfunction with the help of distant ischemic and pharmacological preconditioning with thermal local asphyxia of kidney. Urology Herald 6(2): 4-12.
https://doi.org/10.21886/2308-6424-2018-6-2-4-12
■ Elagin VV, Bratchikov OI (2018) Correction of microcirculatory damages in ischemic and reperfusive kidney injury in experiment. SmolenskyMedical Almanac [SmolenskiyMeditsinskiyAlmanakh] 4: 95-97. [in Russian]
■ Elagin VV, Bratchikov OI, UlyanovaAA (2018) Approaches to correction of ischemic and reperfusion kidney injuries in experiment. Research Results in Biomedicine 4(3): 63-69. https://doi.org/ 10.18413/2313-8955-2018-4-3-0-6
Forbes CM, Rendon RA, Finelli A, Kapoor A, Moore RB, Breau RH, Lacombe L, Kawakami J, Drachenberg DE, Pautler SE, Jewett MMA, Saarela O, Liu Z, Tanguay S, Black PC (2016) Disease progression and kidney function after partial vs. radical nephrectomy for T1 renal cancer. Urologic Oncology 34(11): 486-486. https:// doi.org/10.1016/j.urolonc.2016.05.034 [PubMed]
■ Gobe GC. Coombes JS. Fassett RG Endre ZH (2015) Biomarkers of drug-induced acute kidney injury in the adult. Expert Opinion on Drug Metabolism &Toxicology 11(11): 1683-1694. https://doi.org/ 10.1517/17425255.2015.1083011 [PubMed]
■ Golmohammadi MG, Banaei S, Nejati K, Chinifroush-Asl MM (2020) Vitamin D3 and erythropoietin protect against renal ischemia-reperfusion injury via heat shock protein 70 and microRNA-21
expression. Scientific Reports 10(1): 20906. https://doi.org/10.1038/ s41598-020-78045-3 [PubMed] [PMC]
Grebien F, Kerenyi MA, Kovacic B, Kolbe T, Becker V, Dolznig H, Pfeffer K, Klingmüller U, Müller M, Beug H, Müllner EW, Moriggl R (2008) Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2. Blood 111(9): 4511-4522. https://doi.org/10.1182/ blood-2007-07-102848 [PubMed] [PMC]
Jiang YL, Peng CX, Wang HZ, Qian LJ (2019) Comparison of the
long-term follow-up and perioperative outcomes of partial nephrectomy
and radical nephrectomy for 4 cm to 7 cm renal cell carcinoma: a
systematic review and meta-analysis. BMC Urology 19(1): 48. https://
doi.org/10.1186/s12894-019-0480-6 [PubMed] [PMC]
Khvan MA (2013) Mediators of inflammation in acute kidney
injury.Nephrology and Dialysis 15(2): 106-115.
Kostina DA, Pokrovskaya TG, Poltev VY (2021) Renoprotective
effect of carbamylated darbepoetin and udenafil in ischemia-
reperfusion of rat kidney due to the effect of preconditioning and
inhibition of nuclear factor kappa B. Research Results in
Pharmacology 7(1): 1-19. https://doi.org/10.3 89 7/
rrpharmacology.7.63059
Li X, Tang Y, Ding Y, Chen Y, Hou M, Sun L, Qian G, Qin L, Lv H (2021) Higher efficacy of infliximab than immunoglobulin on Kawasaki disease, a meta-analysis. European Journal of Pharmacology 899: 173985. https://doi.org/10.1016/ j.ejphar.2021.173985 [PubMed]
Lou J, Zhang H, Qi J, Xu Y, Wang X, Jiang J, Hu X, Ni L, Cai Y, Wang X, Gao W, Xiao J, Zhou K (2022) Cyclic helix B peptide promotes random-pattern skin flap survival via TFE3-mediated enhancement of autophagy and reduction of ROS levels. British Journal of Pharmacology 179(2): 301-321. https://doi.org/10.1111/ bph.15702 [PubMed]
NagataY, Fujimoto M, NakamuraK, IsoyamaN, MatsumuraM, FujikawaK, UchiyamaK, TakakiE, TakiiR, Nakai A, MatsuyamaH (2016) Anti-TNF-aagent infliximab and splenectomy are protective against renal ischemia-reperfusion injury. Transplantation 100(8): 1675-1682. https://doi.org/10.1097/TP.0000000000001222 [PubMed]
Netrebenko AS, Gureev VV, Pokrovskii MV, Gureeva AV, Tsuverkalova YM, Rozhkov IS (2021) Assessment of the nephroprotective properties of the erythropoietin mimetic peptide and infliximab in kidney ischemia-reperfusion injury in rats. Archives of Razi Institute 76(4): 995-1004. https://doi.org/10.22092/ ari.2021.355849.1728 [PubMed] [PMC]
NetrebenkoAS, GureevVV, PokrovskiiMV, YakushevVI, AvdeevaEV, GureevaAV, ZatolokinaMA (2022) Research of the renoprotective effect of a combination of the peptide mimicking the spatial structure of the b erythropoietin chain and infliximab in a renal ischemia-reperfusion injury model. Journal of Volgograd State Medical University [Vestnik Volgogradskogo Meditsinskogo Universiteta] 19(1): 167-172. https://doi.org/10.19163/1994-9480-2022-19-1-167-172 [in
Ponsioen CY, de Groof EJ, Eshuis EJ, Gardenbroek TJ, Bossuyt PMM, Hart A, Warusavitarne J, Buskens CJ, van Bodegraven AA, Brink MA, Consten ECJ, van Wagensveld BA, Rijk MCM, Crolla RMPH, Noomen CG, Houdijk APJ, Mallant RC, Boom M, Marsman WA, Stockmann HB, Mol B, de Groof AJ, Stokkers PC, D'Haens GR, Bemelman WA (2017) Laparoscopic ileocaecal resection versus infliximab for terminal ileitis in Crohn's disease: a randomised controlled, open-label, multicentre trial. The Lancet Gastroenterology and Hepatology 2(11): 785-792. https://doi.org/ 10.1016/S2468-1253(17)30248-0 [PubMed]
Ragulina VA, Kostina DA, Dovgan AP, Burda YE, Nadezhdin SV (2017) Nuclear factor kappa B as a potential target for pharmacological correction endothelium-associated pathology. Research Results in Pharmacology 3(1): 114-124. https://doi.org/ 10.18413/2500-235X-2017-3-1-114-124
Sabbisetti VS, Waikar SS, Antoine DJ, Smiles A, Wang C, Ravisankar A, Ito K, Sharma S, Ramadesikan S, Lee M, Briskin R, De Jager PL, Ngo TT, Radlinski M, Dear JW, Park KB, Betensky R, Krolewski AS, Bonventre JV (2014) Blood kidney injury molecule-1 is a biomarker of acute and chronic kidney injury and predicts progression to ESRD in type I diabetes. Journal of the American Society of Nephrology 25(10): 2177-2186. https://doi.org/10.1681/ ASN.2013070758 [PubMed] [PMC]
Skachilova S, Danilenko L, Kesarev O, Kochkarova I (2015) Pharmacological protection of the ischemic myocardium by derivatives of 3-(2,2,2-trimethylhydrazinium) propionate and evaluation of their antioxidant activity. Research Results in Pharmacology 1 ( 1 ): 23-27. https://doi.org/ 10.18413/2500-235X-2015-1-4-25-31
Tan R, Tian H, Yang B, Zhang B, Dai C, Han Z, Wang M, Li Y, Wei L, Chen D, Wang G, Yang H, He F, Chen Z (2018) Autophagy and Akt in the protective effect of erythropoietin helix B surface peptide against hepatic ischaemia/reperfusion injury in mice. Scientific Reports 8(1): 14703. https://doi.org/10.1038/s41598-018-33028-3 [PubMed] [PMC]
Tasdemir C, Tasdemir S, Vardi N, Ates B, Parlakpinar H, Kati B, Karaaslan MG, Acet A (2012) Protective effect of infliximab on ischemia/reperfusion-induced damage in rat kidney. Renal Failure 34(9): 1144-1149. https://dpo.org/10.3109/0886022X.2012.717490 [PubMed]
Wang W (2004) Renal potassium channels: recent developments. Current Opinion in Nephrology and Hypertension 13(5): 549-555. https://doi.org/10.1097/00041552-200409000-00011 [PubMed] Yakovlev AK, L.A. GayderovaLA, AlpatovaNA, (2016) Studying of the standardization principles of pharmacological activity of recombinant erythropoietin preparations. Reference Materials 1: 820. https://doi.org/10.20915/2077-1177-2016-0-1-8-20 Zhang C, Yang C, Zhu T (2017) From erythropoietin to its peptide derivatives: Smaller but stronger. Current Protein &Peptide Science 18(12): 1191-1194. https://doi.org/ 10.2174/1389203717666160909130006 [PubMed]
Author Contributions
■ Aleksandr S. Netrebenko, postgraduate student, Department of Pharmacology and Clinical Pharmacology of Belgorod State National Research University, e-mail: AlexNetrebenko@mail.com; ORCID ID https://orcid.org/ 0000-0003-2212-0508.
