RU2737802C2 - Method for preventing and treating acute postischemic renal insufficiency - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to regenerative medicine, and can be used for the purpose of prevention and treatment of acute renal insufficiency. Complex of biological active peptides and proteins with molecular weight from 10 to 250 kDa, which is an active component of Cellex pharmacopoeial preparation, chromatographically isolated from tissues of embryos of animals or brain embryos of pigs, subcutaneously before operation in dose of 0.015–0.1 ml/kg, followed by daily introduction for 10 days.

EFFECT: use of the declared method enables more rapid and more complete recovery of the function of injured kidneys as compared to using stem cell cultivation, eliminates expensive and prolonged stage of their cultivation.

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Description

The method relates to the field of medicine, namely urology, surgery and regenerative medicine. Many operations on the kidney require temporary clamping of the renal vessels (nephrolithotomy, resection of the kidney, reconstruction of the renal artery in vasorenal hypertension), which can lead to a violation of the functional state of the organ. With such operations on a single kidney in the postoperative period, it is possible to develop acute renal failure (ARF), which poses a significant threat to the patient's life. In addition, with the development of severe infectious complications in a number of urological diseases (obstructive pyelonephritis, acute abscess prostatitis) or after surgery, it can lead to the development of infectious toxic shock and / or sepsis, which is also fraught with the development of acute renal failure. In these cases, the ischemic factor also plays an important pathogenetic role in connection with a decrease in blood pressure and impaired renal microcirculation.

Treatment of patients with developed acute renal failure involves the implementation of a set of measures in the framework of intensive care up to hemodialysis. In connection with the development of modern trends in medicine, based on cellular technologies and stimulation of the regeneration of cellular structures of damaged organs (regenerative medicine), the possibilities of prevention and treatment of acute renal failure using stem cells or their metabolic products are being intensively studied. At the same time, the possibility of using embryonic, fetal stem or progenitor cells, as well as stem cells of an adult organism is being studied. In this case, stem / progenitor cells are obtained from different sources - bone marrow, organs of embryos or fetuses, as well as subcutaneous adipose tissue (Maeshima A, Nakasatomi M, Nojima Y. Regenerative medicine for the kidney: Renotropic factors, renal stem / progenitor cells, and stem cell therapy. Biomed Res Int 2014; 2014: 595493. Doi: 10.1155 / 2014/595493)

Closest to the claimed method (prototype) is a method for treating acute or chronic renal failure using stem cells of the bone marrow or pod kidney. In accordance with this method, previously isolated and then cultured in a nutrient medium, fetal stem cells are injected directly into the damaged kidney (Kirpatovsky V.I., Kazachenko A.V., Plotnikov E.Yu. and others. Functional consequences of intraparenchymal administration of fetal stem and progenitor human cells in chronic and acute renal failure in rats. Cell technologies in biology and medicine. 2006. No. 2. P. 70-76) or intravenously (Kirpatovskii VI, Kazachenko AV, Plotnikov EY et al. Experimental intravenous cell therapy of acute and chronic renal failure Bulletin of Experimental Biology and Medicine 2007 Vol. 143, No. 1. P. 160-165).

The disadvantages of this method are the need for long-term cultivation of fetal cells to obtain a sufficient amount of cellular material, the need for expensive equipment and reagents, as well as specially trained personnel. In addition, the injection of cells directly into the kidney is an invasive intervention that requires anesthesia and surgical intervention, which creates additional risks in ARF conditions. An important factor is the ethical and legal problems of using fetal material to obtain stem cells.

The inventive method avoids these disadvantages. Since it is based on the fact that, according to many authors, the effect of cell therapy is achieved not due to the inclusion of transplanted cells into damaged organ structures, but due to the action of humoral factors secreted by these cells (cytokines, chemokines, growth factors, angiogenic factors) that stimulate regeneration, which are united by the term "stem cell secretion" (Bi B, Schmitt R, Israilova M. et al. Stromal cells protect against acute tubular injury via an endocrine effect. J Am Soc Nephrol. 2007; 18 (9): 2486-96). Using the products of secretion or metabolism of stem cells allows achieving the same results as using the cells themselves (van Koppen A, Joles JA, van Balkom BW et al. Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease.PLoS One 2012; 7 (6): e38746.doi: 10.1371 / journal.pone.0038746, Lavoie JR, Rosu-Myles M. Uncovering the secretes of mesenchymal stem cells.Biochimie 2013; 95 (12): 2212- 21. Doi: 10.1016 / j.biochi.2013.06.017).

The technical result of the claimed method is to increase the efficiency and safety of prevention and treatment of acute renal failure due to stimulation of regenerative processes in the kidney using a secretome of animal embryonic tissues (products of secretion of stem cells of embryonic tissues).

The specified technical result in the implementation of the invention is achieved due to the fact that, as in the known method, enter stem cells or products of their vital activity.

The peculiarity of the proposed method is that a complex of biologically active peptides and proteins with a molecular weight of 10 to 250 kDa, chromatographically isolated from the tissues of animal embryos or the brain of pig embryos, is injected subcutaneously the day before or on the day of the operation at a dose of 0.015-0.1 ml / kg, followed by daily administration for 10 days.

The claimed method uses a protein-peptide complex isolated from the tissues of animal embryos, in particular the brain of pig embryos (the secretion of stem cells of embryonic tissues), which is an active component of the pharmacopoeial drug "Cellex". This complex contains biologically active peptides and proteins with a molecular weight of 10 to 250 kDa, chromatographically isolated from the tissues of animal embryos or the brain of pig embryos, belonging to the groups of growth factors and cell differentiation and angiogenic factors produced by stem cells of the embryonic tissue, which it is rich in ... Their concentration in the extract is significantly higher than that which is achieved in the tissue around the implanted cells, since the complex of active metabolites produced by them is in a concentrated form. It should also be borne in mind that during cell transplantation, not all signaling protein or peptide molecules capable of stimulating the regeneration of cellular structures can be released into the surrounding tissues due to the high molecular weight and selective permeability of cell membranes, whereas in the protein-peptide complex isolated from the embryonic fabrics they are present in the full spectrum. Due to the high concentration of biologically active substances and their high solubility during parenteral administration, including with the help of subcutaneous or intramuscular injections, the components of the drug can reach the target organ through the circulatory system in a biologically significant concentration, which avoids the need to inject the drug directly into the kidney , that is, to exclude the invasive nature of therapy. An important point for achieving a significant therapeutic effect is the duration of the therapeutic effect. In stem cell therapy, the duration of their influence is determined by the period of preservation of cell viability after transplantation, which is relatively short due to the elimination of genetically introduced foreign cells by the body's immune system. There is evidence that 7 days after the introduction of stem cells into the kidney, only a few cells remain viable (Kirpatovsky VI, Kazachenko AV, Nadtochiy ON Prospects for the use of stem cells in the treatment of acute and chronic renal failure. Urology. 2007. No. 6. S. 82-87). When using the proposed method, there is the possibility of repeated repeated injections of the drug, that is, course therapy of the required duration.

The invention is illustrated by a detailed description, tables, experimental examples and illustration, which shows: a) a hypertrophied glomerulus in the kidney of rats treated with protein-peptide secretion of embryonic tissue; b) a shriveled glomerulus in a rat kidney, a control group; c) dividing cells of the tubular epithelium (indicated by the arrow).

The method is carried out as follows.

In order to prevent the development of acute renal failure in cases where there is a risk of its development, the preparation of the protein-lipid complex of the embryonic tissue of animals, in particular the brain, is injected subcutaneously on the eve or on the day of surgery at a dose of 0.015-0.1 ml / kg (0.02 -0.1 mg of active substances), followed by daily subcutaneous administration of the drug in the same dose for 10 days. In the case of already developed acute renal failure, the drug is administered immediately after the diagnosis is made subcutaneously at a dose of 0.015-0.1 ml / kg and then daily at the same dose for 10 days. If necessary, repeat the course of therapy.

The effectiveness of the proposed method is illustrated by the following examples.

Example 1.

Stimulation of kidney regeneration in ARF modeling using a protein-peptide secretome of embryonic pig brain tissue.

ARF was modeled in 10 rats by unilateral nephrectomy and clamping of the vascular pedicle of the remaining kidney for 60 minutes, followed by restoration of its blood supply. In the control series (5 rats), no therapy was carried out, and in the experimental series (5 rats), a protein-peptide complex isolated from the brain of pig embryos was injected subcutaneously from the day of operation for 10 days at a dose of 0.1 ml / kg ( 0.03 ml per rat). After 14 days, the remaining kidney was removed and its weight was determined by weighing and a histological examination of the removed organ was performed.

Determination of the degree of hypertrophy of the ischemic kidney showed that its weight by the 14th day of the postischemic period in the control series (without therapy) increased from 1.56 ± 0.07 g (average weight of one intact kidney) to 1.80 ± 0.05 g (by 13.4%), while during therapy with protein-peptide secretion, the mass of the kidney increased to 2.55 ± 0.06 g (by 48.2%). The differences between the groups were statistically highly significant at p <0.001. A histological study of the removed kidney preparations showed that in experiments with therapy with a protein-peptide secretion more than half of the glomeruli were hypertrophied, while in the control series more than 1/3 of the glomeruli were in a collapsed state with signs of incipient glomerulosclerosis (See Fig. A, b) ... In separate renal tubules of rats treated with protein-peptide secretion of embryonic tissue, dividing epithelial cells of renal tubules were detected (see Fig. C), while in the control series this was not observed.

This example confirms that therapy with protein-peptide secretion of the brain tissue of porcine embryos stimulates the proliferation of renal glomeruli cells and the regeneration of tubular epithelium in the kidney damaged by ischemia and promotes more pronounced hypertrophy of the organ under conditions of increased functional load.

Example 2.

Stimulation of renal function restoration in rats with simulated ARF using protein-peptide secretome isolated from the brain tissue of pig embryos.

ARF was modeled in 24 male rats by unilateral nephrectomy and clamping of the vascular pedicle of the remaining kidney for 60 or 90 minutes. In the 1st and 2nd groups (6 rats in each) with ischemia of 60 or 90 minutes, respectively, no therapy was performed. In the 3rd and 4th series (6 rats in each group) with ischemia for 60 or 90 minutes, respectively, daily from the day of the operation, a protein-peptide secretion isolated from the brain of pig embryos was injected subcutaneously for 10 days at a dose of 0.03 ml per rat. 3, 7, and 14 days after ARF simulation, the animals were placed for a day in exchange cages to collect daily urine, and blood samples were taken from the tail vein. A biochemical analysis of blood and urine was carried out with the determination of the concentration of urea, creatinine, sodium and calcium, on the basis of which the indicators characterizing the functional state of the ischemic kidney were calculated.

The analysis showed that all animals developed ARF, which was manifested in a pronounced decrease in glomerular filtration, a significant decrease in tubular reabsorption of sodium and calcium, as well as daily excretion of creatinine (table 1). The most pronounced changes were found in rats of the 2nd group. In the experimental series (series 3 and 4, therapy with a protein-peptide secret), these parameters were restored more quickly. Glomerular filtration already by the 7th day approached normal values, and after 14 days even in the 3rd group it exceeded them, while in the control groups this indicator did not normalize. The values of tubular reabsorption of sodium and calcium were also higher in the rats treated with protein-peptide secretion compared with the control groups.

Thus, the therapy of rats with ARF with protein-peptide secretion isolated from the embryonic pig brain, in accordance with the claimed method, helps to accelerate the recovery of impaired functional parameters of the kidney state both in ARF of moderate severity (60-minute ischemia) and in severe form ARF (90 minute ischemia). Both the filtration function of the kidneys and the reabsorbing function of the renal tubules improved more rapidly.

Example 3.

Comparison of the effectiveness of prevention of acute renal failure using cultured fetal mesenchymal bone marrow stem cells (prototype method) and protein-peptide secretome of embryonic tissues (the claimed method).

ARF was induced in 18 rats by removing the right kidney and clamping the vascular pedicle of the left kidney for 90 minutes. In series 1, no therapy was performed. In the second series, immediately after the clamp was removed and the blood supply to the kidney was resumed, 10 ⁶ cultured mesenchymal cells of the human fetal bone marrow were injected into it with a puncture insulin syringe. In the 3rd series, 10 ⁶ cultured fetal kidney cells were similarly injected. In series 4, immediately after the restoration of blood supply in the kidney, a protein-peptide secretion isolated from the brain of pig embryos was injected subcutaneously at a dose of 0.1 ml / kg, followed by daily injections of the drug for 10 days. After 3 days and 2 weeks, the animals were planted in exchange cages to collect daily urine and blood samples were taken from the tail vein, followed by biochemical analysis of blood and urine and calculation of indicators of the functional state of the kidney, as described in example 2.

When assessing the indicators of the functional state of the kidneys, it was revealed that in all 3 experimental series, the functional indicators changed less significantly than in the series without therapy (Table 2). However, in experiments using the proposed method (4th series), the restoration of functional parameters occurred more quickly than in experiments using mesenchymal cells of the bone marrow or fetal kidney (2nd and 3rd series). The most pronounced differences were revealed on the 3rd day after ischemic exposure, when in all functional parameters (diuresis, concentration of urea and creatinine in the blood, glomerular filtration and tubular reabsorption of sodium and calcium) in the series with the therapy of rats with protein-peptide secretion, significantly better results than when using both variants of the prototype method.

When compared with the use of bone marrow mesenchymal cells by 14 days after ischemia, the differences with the claimed method in most parameters disappeared, with the exception of daily urine output and glomerular filtration, which were significantly closer to normal in experiments using the protein-peptide secretome of embryonic tissues.

The functional results of using fetal kidney cells turned out to be worse than when using mesenchymal bone marrow cells, and all the more worse than when using protein-peptide secretion of embryonic tissues.

This example illustrates the advantages of the proposed method in comparison with the prototype method in relation to the speed and usefulness of the restoration of the functional parameters of the kidney with the development of postischemic ARF.

Example 4.

Proof of the possibility of treating postischemic acute renal failure using the proposed method.

Postischemic ARF was modeled in 12 rats as described in previous examples. In 6 of them no therapy was performed (group 1), and in the other 6 animals, starting from the 3rd day, after establishing the biochemical parameters characteristic of ARF, a protein-peptide secretion isolated from the brain of pig embryos at a dose of 0.1 ml / kg (group 2). Determined biochemical indicators of the functional state of the kidneys on the 3rd day (start of therapy) and 14 days later (end of therapy). The results showed that, with the initially identical degree of deterioration of the kidney function on the 3rd day after ARF modeling, against the background of therapy with the protein-peptide secretion of the tissues of pig embryos, by the 14th day there is a significantly more complete recovery of all functional parameters compared to the control series (without therapy). ) (Table 3).

The presented results indicate that therapy with the protein-peptide secretion of pig embryonic tissues after the development of ARF, as well as with the prophylactic administration of this drug, contributes to a more complete restoration of the functional state of the kidney.

Thus, all the presented examples testify to the high efficiency of the proposed method in the prevention and treatment of postischemic ARF by stimulating the regeneration of damaged renal structures (both glomeruli and tubules) and accelerating the restoration of the functional usefulness of the organ. The inventive method has significant advantages over others, in particular, the introduction of cultured stem cells into a damaged kidney or intravenously, since it provides higher functional results, does not require long-term cultivation of stem cells with the possibility of immediate use of the finished product.

The presented method provides the prevention of the development of acute renal failure by therapy with a protein-peptide complex of embryonic tissues, which is started in advance with the threat of acute renal failure. Moreover, in order to treat the developed acute renal failure, therapy with protein-peptide complexes of embryonic tissues of pigs begins immediately after the diagnosis of acute renal failure is established and is carried out for 10 days.

The use of the proposed method makes it possible to achieve a faster and more complete restoration of the function of damaged kidneys, eliminates the costly and lengthy stage of their cultivation, and also allows eliminating existing ethical and legal restrictions.

Claims (1)

A method for the prevention and treatment of acute post-ischemic renal failure in kidney surgery, including the introduction of stem cells or their metabolic products, characterized in that a complex of biologically active peptides and proteins with a molecular weight of 10 to 250 kDa, which is an active component of the pharmacopoeial drug "Cellex" , chromatographically isolated from the tissues of animal embryos or the brain of pig embryos, is injected subcutaneously before surgery at a dose of 0.015-0.1 ml / kg, followed by daily administration for 10 days.

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