Application of peptides in kidney pathology: clinical and experimental studies

Relevance. Pathological processes affecting the main organs of the urinary system are one of the most common causes of a decrease in the quality of life of people of working age. Kidney disease is a global problem of modern healthcare around the world, which is of great medical and social importance. The prevalence of kidney disease ranges from 10 to 20%. Screening and identification of risk factors are inseparable from measures for the primary prevention and comprehensive treatment of kidney diseases. Timely prevention and comprehensive treatment of kidney diseases in many cases make it possible to avoid disability and death, and maintain a high quality of life in middle-aged and older people.

For the prevention and complex therapy of kidney pathology, the following peptide and non-peptide bioregulators can be recommended: Pielotax, Renefort and Ovagen.

Pielotax is a complex of polypeptides obtained from the kidney parenchyma of young animals. Pielotax has a selective effect on kidney cells, normalizes their metabolism and functions, and regulates the functioning of the urinary system. Pielotax is available in the form of a biologically active food supplement (dietary supplement) — capsules and drops under the tongue (lingual form). When studying acute toxicity in outbred male mice, it was shown that a single administration of Pielotax to animals at a dose exceeding the therapeutic dose (recommended for clinical use) by more than 5000 times does not cause toxic reactions. A study of the subacute and chronic toxicity of the drug on outbred male rats and guinea pigs indicates the absence of side effects with long-term use of Pielotax in doses exceeding the therapeutic dose by 300 — 3000 times. Pielotax does not change the morphological composition and biochemical parameters of the peripheral blood of guinea pigs 3 and 6 months after the start of administration [Khavinson V.Kh. et al., 2007].

A study was conducted on the effectiveness of Pielotax in gouty nephropathy. The study involved patients with gouty nephropathy aged 43-57 years. The control group included 10 people who received standard treatment and the study group included 15 people who used, in addition to standard therapy, 5 mg of Pielotax in 2 ml of saline, intramuscularly, daily for 10 days. In 78% of cases, under the influence of Pielotax, an improvement in well-being, normalization of sleep, a decrease in the frequency and intensity of joint pain, and an improvement in laboratory parameters for blood and urine tests were revealed. Activation of the metabolism of renal tissues with increased secretory function of the kidneys was observed [Khavinson V.Kh. et al., 2007].

Renefort is a complex drug, a nephro- and oncoprotector that improves the solubility of stones and has an anti-inflammatory effect. Renefort contains: rosehip extract and dihydroquercetin, horsetail, hibiscus, rosemary, lingonberry leaves, hesperdin, coenzyme Q10.

Pielotax and Ovagen (also known as a regulator of liver functions) have proven themselves to be regulators of cellular renewal in kidney tissue. The influence of Pielotax and Ovagen was assessed in an organotypic culture of kidney tissue from old (24 months) Wistar rats. Study groups: control — without the addition of peptides, 2 — addition of Pielotax at a dosage of 20 ng/ml, 3 — addition of Ovagen at a dosage of 0.05 ng/ml. In kidney cell cultures, the synthesis of proliferation proteins Ki67 (cell division) and apoptosis p53 (programmed cell death) was assessed using immunocytochemistry. Pielotax increases Ki67 protein expression and also decreases p53 protein expression in kidney explants obtained from young and old animals. Ovagen also stimulates proliferation and reduces apoptosis of kidney cells, but to a lesser extent than Pielotax [Chalisova N.I. et al., 2015].

Further, the effect of Ovagen on the aging of kidney cells in culture was studied, and data on the cell growth curve were obtained. The study was conducted on a primary dissociated cell culture from kidney tissue obtained from young Wistar rats. To construct a cell growth curve, cells of the 14th passage were seeded onto a 24-well plate at an initial concentration of 10,000 cells per 1 ml of medium. Then the number of cells was counted in Goryaev’s chamber on the 3rd, 5th, 7th, 10th, 13th day. Cell growth curve is a curve describing the dependence of the logarithm of the number of living cells on the cultivation time. In control cell cultures, an increase in the number of cells was observed on the 3rd and 5th days, on the 7th day their number stabilized and by the 10th — 13th days the number of cells decreased sharply. Ovagen had an effect on the course of the cell growth curve, significantly increasing the number of kidney cell subpopulations from the 3rd to the 13th day. At the same time, the maximum effect of Ovagen was observed on the 10th and 13th days, which indicates a slowdown in the process of cell death [Khavinson V.Kh. et al., 2014].

A study of the effect of Ovagen on the aging of kidney cells in culture (assessment of the synthesis of proteins p16, p21, p53 and sirtuin-6) was carried out on a primary culture of kidney cells of young (3-month-old) Wistar rats. To assess the synthesis of the «youth» proteins Sirtuin-6, «aging» and apoptosis (programmed cell death) p16, p21 and p53 in kidney cells, the immunocytochemistry method was used. Control cells were grown without the addition of peptide. Ovagen was added to the experimental cultures at each subculture at a concentration of 20 ng/ml. Ovagen increased the synthesis of Sirtuin-6 by 1.6 and 3.3 times in «young» and «old» cell cultures, respectively, and decreased the expression of the following markers: p16 — by 2.8 and 1.8 times, p21 — by 2 .9 and 1.6 times and p53 by 1.7 and 3.2 times [Khavinson V.Kh. et al., 2014].

The nephroprotective effect of Ovagen was studied in models of ischemic reperfusion injury of the kidneys and toxic gentamicin nephropathy in rats. The study was conducted on 42 outbred white rats. In the first part of the study, the animals were divided into 3 groups: 1 — control — falsely operated animals (surgical intervention is performed without further kidney damage), 2 — modeling of ischemic reperfusion kidney damage, 3 — modeling of ischemic reperfusion kidney damage and administration of Ovagen at a dose of 3 µg/kg for 3 days before pathology modeling. Ischemia was modeled under aseptic conditions under general anesthesia (sodium ethaminal, 40 mg/kg) by applying a clamp to each renal pedicle for 60 minutes followed by 24-hour reperfusion, after which urine was collected for 2 hours under conditions of induced aqueous diuresis (enteral intragastric administration of 5% drinking water heated to 37 degrees of body weight). In the second part of the study, the animals were divided into 3 groups: 1 — control, 2 — modeling of gentamicin nephropathy (administration of a 4% solution of gentamicin sulfate at a dose of 80 mg/kg once for 6 days), 3 — modeling of gentamicin nephropathy and administration of Ovagen at a dose of 3 µg/kg 40 minutes after each injection of gentamicin. Ovagen has been found to have a protective effect on renal tissue in a model of ischemic reperfusion injury and toxic gentamicin nephropathy. This is evidenced by the restoration of excretory kidney function, improvement of nephron energy supply, restriction of free radical processes and increased activity of antioxidant defense enzymes [Zamorsky I.I. et al., 2017].

In addition, studies were conducted to study the effect of Pielotax and Ovagen on the morphofunctional state of the kidneys in old rats. The study was carried out on 35 non-linear white old (20–24 months) rats. The animals were divided into 3 groups: 1 — control; 2 — administration of Pielotax at a dose of 300 μg/kg for 10 days intraperitoneally in the form of an aqueous solution; 3 — administration of Ovagen at a dose of 3 μg/kg intraperitoneally for 10 days in the form of an aqueous solution. Ovagen increased diuresis by 1.3 times compared to the control, did not affect the concentration of creatinine in the blood plasma and glomerular filtration rate, but led to a decrease in water reabsorption by 2.9%, which indicates a tubular mechanism of increased diuresis. Under the influence of Ovagen, an increase in the excretion of sodium ions by 1.3 times and an increase in the distal transport of sodium in the kidneys were established. Pielotax significantly increased diuresis by 1.2 times compared to the control against the background of a tendency to increase glomerular filtration rate and decrease water reabsorption. A hypoazotemic effect of Pielotax was detected, as indicated by a decrease in the concentration of creatinine in the blood plasma by 1.4 times compared to the control. Pielotax significantly reduced the protein content in urine by 1.8 times and its excretion by 1.5 times compared to the control. In terms of the acid-regulating function of the kidneys, under the influence of Pielotax, an increase in urine pH by 5% was detected against the background of a significant increase in the excretion of ammonium ions by 1.3 times. Under the influence of Pielotax, a 1.2-fold increase in the distal transport of sodium ions was observed. Thus, Pielotax and Ovagen affect the processes of tubular transport of water, protein and ions in the nephrons of the kidneys, and also exhibit a weak diuretic effect [Zamorsky I.I. et al., 2019].

It was found that Pielotax and Ovagen restore the functional state of the kidneys in a model of cisplatin acute renal failure in rats. The study included 42 outbred young (3 month old) rats, which were divided into the following groups: 1 — control (intact rats), 2 — rats with cisplatin-induced acute renal failure (ARF), 3 — ARF and administration of Pielotax at a dose of 300 µg/ kg intraperitoneally for 4 days before and 3 days after the administration of cisplatin once a day in the morning, 4 — acute renal failure and administration of Ovagen at a dose of 3 mcg/kg intraperitoneally for 4 days before and 3 days after the administration of cisplatin once a day in the morning hours. An experimental model of cisplatin-induced acute renal failure in rats was induced by a single intraperitoneal injection of cisplatin at a dose of 6 mg/kg 72 hours before the animals were removed from the experiment. It has been established that the administration of Pielotax and Ovagen has a restorative effect on the functional state of the kidneys of rats with acute renal failure. Also, these peptides normalize the excretory and ion-regulating functions of the kidneys. The greatest nephroprotective effect in this study was shown by Ovagen [Zamorsky I.I. et al., 2015].

Conclusions

1. Pielotax showed high efficacy in a clinical study in middle-aged patients with gouty nephropathy. In 78% of cases, under the influence of Pielotax, an improvement in well-being, normalization of sleep, a decrease in the frequency and intensity of joint pain, and an improvement in laboratory parameters for blood and urine tests were revealed. Under the influence of Pielotax, activation of the metabolism of kidney tissue and an increase in their secretory function were observed. Pielotax increases the synthesis of the kidney cell division protein (Ki67) by 2.2 times and reduces the synthesis of the apoptosis protein p53 (cell death) in these cells by 20%. Thus, Pielotax stimulates cell renewal in kidney tissue.
2. Ovagen, also known as a hepatoprotector, has shown its effectiveness as a nephroprotector in aging and kidney pathologies of various origins in experiments. The molecular mechanisms of the nephroprotective action of Ovagen during aging of kidney cells were studied. It has been established that Ovagen activates cell growth by reducing the expression of the «aging» and apoptosis proteins p16, p21, p53 and increasing the expression of the «youth» protein Sirtuin-6.
3. The effectiveness of Ovagen and Pielotax in a model of cisplatin-induced renal failure in animals has been demonstrated. Pielotax and Ovagen normalize diuresis, the concentration of creatinine in the urine and its excretion, the glomerular filtration rate, the absolute reabsorption of sodium ions, reduced the concentration of protein in the urine and its excretion, the concentration of sodium and potassium ions in the urine and other indicators in this kidney pathology.
4. Ovagen has a nephroprotective effect in experimental models of gentamicin nephropathy and ischemia-reperfusion kidney injury in rats. The nephroprotective effect of Ovagen is manifested in preventing the development of oliguria and azotemia, reducing proteinuria and sodium excretion, antioxidant effect and normalizing the energy supply of kidney cells.

Scheme for using peptide and non-peptide bioregulators for the prevention of accelerated aging and kidney diseases:

Scheme of application of peptide and non-peptide bioregulators in the complex treatment of kidney diseases:

Literature

1. Zamorsky I.I., Shchudrova T.S., Zelenyuk V.G., Linkova N.S., Nichik T.E., Khavinson V.H. Nephroprotective effect of EDL peptide in acute kidney injury of various genesis. Bulletin of Experimental Biology and Medicine. 2017. Vol. 163, No. 3. P. 389-393. https://khavinson.info/assets/files/skan/2017-zamorskii.pdf
2. Zamorsky I.I., Shchudrova T.S., Zelenyuk V.G., Linkova N.S., Nichik T.E., Khavinson V.H. The effect of peptides on the morphofunctional state of the kidneys in old rats. Advances in Gerontology. 2019. No. 9. P. 75-80. https://link.springer.com/article/10.1134/S207905701901017X3.
3. Zamorsky I.I., Shchudrova T.S., Zelenyuk V.G., Linkova N.S., Nichik T.E., Khavinson V.Kh. Peptides restore the functional state of the kidneys in cisplatin acute renal failure. Bulletin of Experimental Biology and Medicine. 2015. Vol. 159. N. 6. P. 736-739. https://link.springer.com/article/10.1007/s10517-015-3062-y
4. Khavinson V.Kh., Malinin V.V., Ryzhak G.A. Means, normalizing kidney function, and a method of its obtaining. 2007. Patent EA200700621A. https://patents.google.com/patent/EA200700621A1/en
5. Khavinson V.Kh., Tarnovskaya S.I., Linkova N.S., Polyakov O.O., Durnova A.O., Nichik T.E., Kvetnoy I.M., Dyakonov M.M., Yakutseni P.P. Tripeptides slow down the aging process in kidney cell cultures. Advances in Gerontology. 2014. Vol. 27. No. 4. pp. 651-656. (in Russian) https://pubmed.ncbi.nlm.nih.gov/25946838/
6. Chalisova N.I., Linkova N.S., Nichik T.E., Ryzhak A.P., Dudkov A.V., Ryzhak G.A. Peptide regulation of cellular renewal processes in kidney tissue cultures of young and old animals. Bulletin of Experimental Biology and Medicine. 2015. Vol. 159. P. 124-127. https://link.springer.com/article/10.1007/s10517-015-2906-9