RU2373942C2 - Method for treating chronic and acute renal failure by foetal stem and progenitor cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to cell medicine and nephrology, and concerns treatment of renal failure. That is ensured by preparing donor foetal material, except human, containing monoculture or mixed culture of cell population including culture of stem cells consisting of haemopoetic and mesenchymal cells. Said cells are recovered as a suspension from foetal bone marrow of tubular bones and/or as a culture of progenitor cells, from epithelial and mesenchymal cells from foetal kidneys. Further, primary cell suspension of said cells is cultivated to monolayer condition in nutritious growth medium DMEM/F-12 in the ratio 1:1 with 10% veal serum and 0.02% gentamycin added. Then the prepared cell donor material is introduced to an object of treatment as suspended in physiologic saline with concentration of cell elements 5 to 20 million cells per 1 kg of weight of object of treatment.

EFFECT: owing to use of mixed culture of marrow and mesenchymal stem cells of foetal kidneys, the method provides effective normalisation of renal function indicators in acute chronic or chronic renal failure during 4-11 days after intraparenchymal or intravenous introduction of specified suspended cells.

11 cl, 4 tbl, 4 ex

Description

The invention relates to medicine and can be used to treat renal failure, and especially for the treatment of chronic and acute renal failure using cell donor material, and specifically with the help of fetal stem and progenitor cells.

Acute and chronic renal dysfunction is a common condition that threatens the patient's life. In chronic renal failure (CRF), there is a need to maintain or completely replace the function of the damaged organ by dialysis or kidney transplantation.

Known methods of treating patients suffering from renal failure by filtering the blood. Blood is sequentially passed through a hemofiltration element and a hemodialysis element, and ultrafiltrate is passed through an outlet connector of the hemofiltration element and a filter, preferably with an uncoated activated carbon, is introduced as a regenerated ultrafiltrate into the channel. Thus, the purified ultrafiltrate is used as a reinfusion solution for repeated administration to the patient (RU 2086264, IPC 6 A61M 1/38, A61M 1/34, published on 08/10/1997). Or through the combined effects of bicarbonate hemodialysis and hemodiafiltration and with isolated blood ultrafiltration, drug nephroprotective therapy and automation of the treatment process through the use of a hemodialysis database, computer processing and quick calculation of the source data, organization and use of graphic graphics, tables, speech comments and implementation urological objects about patients, time, quantity, doses of delivery-reception and results of actions vii prescribed drugs with the diagnosis and names of patients (RU 2154499, IPC 7 A61M 1/34, published on 08/20/2000).

However, during flow hemodialysis in one session, up to 200 l of dialysis solution is passed through the dialyzer, which is drained, which requires a large consumption of dialysate concentrate and water. In addition, along with toxins, a number of useful substances are removed from the blood, which leads to various complications and requires subsequent additional procedures to adjust the blood composition.

The problems of detoxification of the body are solved in "artificial kidney" apparatuses by a method in which during the dialysis fluid circulation the contact time of the dialysis fluid with the platinum filter carbon is set for at least 10 s, the concentration of sodium hypochlorite in front of the filter is measured, maintaining it at a level of 50 ± 10 mg / l by adjusting the density of the power supply current of the electrolyzer, setting at the beginning of the session its maximum value, and then reducing it so that the concentration of sodium hypochlorite remains within the specified tolerance Mykh limits. In addition, to adjust the pH, an additional buffer additive is included in the initial composition of the dialysing solution of the treatment loop, for example, sodium bicarbonate in concentrations from 3 to 5 mg / L (RU 2110283, IPC 6 A61M 1/14, published on 05/10/1998).

Known methods of hemodialysis and hemodiafiltration. Firstly, the need for a sterile pyrogen-free substitute solution or normotonic solution, balanced by electrolyte composition in a container of 4-5 liters, and secondly, special equipment in the form of an “artificial kidney” apparatus with volumetric ultrafiltration control, capable of carrying out the latter at a speed not less than 3000 ml / h, thirdly, for safe hemofiltration, it is necessary to have a special system that carries out dosed infusion of a replacement solution (perfusion pump), and control SPS by weighing containers with the solution throughout the procedure. It is also necessary to constantly monitor the compliance of the infusion and ultrafiltration rates, taking into account the volume of fluid accumulated during the interdialysis period. Moreover, errors of 1.5-2 liters can lead to serious complications associated with hyper- and hypovolemia.

However, the needs for these types of treatment are far ahead of the possibilities of modern medicine both in Russia and in other developed countries. After kidney transplantation, patients with end-stage CRF often develop acute functional transplant failure, which requires long-term auxiliary hemodialysis, complicates the postoperative period, worsens the conditions for diagnosis of transplant rejection crises, and ultimately negatively affects the immediate and long-term results of the operation. Methods for treating this condition have not yet been developed. In acute renal failure (ARF) of both ischemic and toxic genesis, despite the identification of new effective medications in experimental studies in the last decade, there has been no significant progress in the effectiveness of treatment of this difficult situation. In this regard, studies are being conducted on alternative methods of adjuvant or renal replacement therapy, in particular, the possibility of cell therapy using various types of stem cells is being studied.

A known method of therapeutic transplantation of fetal tissue of the kidney and liver of a person on the 49-56th day of gestation or pigs on the 28-29th days of gestation under the capsule of the kidney, in the kidney tissue, in the anterior chamber of the eye, in the omentum, in the intestinal loop, in the abdominal cavity without or with the introduction of peripheral blood monocytes and immunosuppressants (US application publication No. 2005226854, dated October 13, 2005, “Therapeutic transplantation using developing, human or porcine, renal or hepatic, grafts”). It was shown that during transplantation into mice (immunocompetent and immunodeficient), kidney tissue is formed from the transplanted cells with zones containing glomeruli and tubules, with the germination of blood vessels in it and with the formation of cysts containing primary urine. However, the newly formed kidney tissue does not integrate with the urinary system of the recipient's kidney, therefore the functional effect of this method is doubtful. The description of the patent on the functional consequences of using this method is not indicated. For transplantation, uncultivated fetal tissue of the kidney or liver is used and, due to its small size, it is unlikely to be able to improve the kidney function of an adult, or tissue from several sources must be used at once, which is also problematic. In addition, this method involves the use of immunosuppressive drugs that have significant side effects and can cause serious complications that are life-threatening to the patient.

There is also known a method of treating acute and chronic heart failure, impaired liver, kidney, endocrine organs, which involves obtaining stem cells from monocytes of peripheral blood of an adult by dedifferentiating them when cultured in a nutrient medium containing macrophage colony stimulating factor (M-CSF) and interleukin 3 (IL-3) and their transformation into mature cells of various tissues (myocytes, adipocytes, neurons and neuroglia, endothelium, insulin-producing cells, keratinocytes) during cultivation testing with the addition of a nutrient medium in which one or another type of mature cells from the same individual was cultured (target cells) (US application publication No. 2005233477, 10/20/2005, “Dedifferentiated, programmable stem cells of monocytic orgin, and their production and use "). The disadvantages of the method are the need for long-term cultivation of isolated stem cells, multi-stage cultivation, as well as the need to obtain autologous mature somatic cells of the patient by biopsy of the corresponding tissue or organ.

There is also known a method for the treatment of acute and chronic renal failure, as well as multiple organ failure and accelerate wound healing by introducing allogeneic or autologous adult bone marrow stem cells (a combination or separate administration of hematopoietic and mesenchymal stem cells), introducing differentiated by culturing mesenchymal bone marrow cells ( specific progenitor cells, for example endothelial, glomerular, tubular) or hemangioblasts, also the introduction of stem cell stimulants (granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage KSM, stem cell stimulation factor, interleukins-1,2,3,4,6,7,8,11,12, erythropoietin, etc.) (International application W02004090112. A61K 38/00, "STEM CELL, PRECURSOR CELL, OR TARGET CELL-BASED TREATMENT OF MULTI-ORGAN FAILURE AND RENAL DYSFUNCTION RELATED APPLICATIONS", published October 21, 2004). The disadvantages of this method is the need for puncture of the bone marrow of a sick person to obtain their own bone marrow hematopoietic and mesenchymal cells. A long cultivation period is also necessary to obtain a sufficient number of stem cells that can provide the necessary functional effect, and in acute renal or acute liver failure such a period of time, as a rule, is not. When using foreign (allogeneic) hematopoietic stem cells, the likelihood of a graft versus host reaction is high and this requires the use of immunosuppressants, which, as mentioned above, can themselves cause serious complications. In particular, when the dose is exceeded, the most common immunosuppressant cyclosporin has pronounced nephrotoxicity, which makes it extremely dangerous for patients with already impaired renal function. Stem cell pre-cultivation to produce more differentiated progenitor cells requires a multi-stage cultivation, a long period of time and is difficult to implement.

An object of the invention is the development of a method for the treatment of chronic and acute renal failure using fetal stem and progenitor cells, devoid of the above disadvantages of the prototype methods.

The stated technical problem is achieved by the fact that in the method of treating chronic and acute renal failure, including obtaining donor material and introducing it to the treatment object, donor material is obtained containing a mono- or mixed culture of a cell population including a stem cell culture consisting of hematopoietic and mesenchymal cells isolated as a suspension from the fetal bone marrow of the tubular bones, and / or a culture of progenitor cells consisting of epithelial and mesenchymal cells, ELENITE in suspension from fetal kidney, followed by culturing the primary cell suspension of the cells to the monolayer state in a nutrient growth medium DMEM / F-12 in a ratio of 1: 1 supplemented with 10% calf serum and 0.02% gentamycin; the obtained cellular donor material is administered to the treatment object in the form of a suspension in physiological solution with a concentration of cellular elements from 5 to 20 million cells per 1 kg of the weight of the treatment object.

By the method of the invention, cultured cells (cellular donor material) are administered directly to a damaged kidney or renal artery, or systemically (intravenously), to a treatment object with chronic renal failure.

According to the method of the invention, bone marrow stem cells (from fetal bone marrow of the tubular bones) are administered in an amount of 1 million cells per 1 kg of weight to each kidney, and progenitor cells from fetal kidneys are administered in an amount of 1 million cells per 1 kg of weight in each kidney.

So, the method according to the invention involves the implementation of several stages, namely, the stage of obtaining donor material, the stage of cultivation and the stage of transplantation of cultured cells to the object of treatment.

Fetal kidneys and fetal bone marrow of tubular bones, for example, pigs, are used as donor material. From fetal kidneys of different gestation periods, a suspension of epithelial and mesenchymal cells is obtained, which are subsequently cultivated under special conditions. Cells were cultured on DMEM and F-12 (1: 1 ratio) supplemented with 10% calf serum and 0.02% gentamicin. The resulting total kidney cell culture was grown to a monolayer state in culture bottles (25 cm ² , Coming, USA) at 37 ° C in a humidified incubator containing 5% CO ₂ .

A suspension of hematopoietic and mesenchymal cells is obtained from the bone marrow of the tubular bones by washing them out with DMEM containing 2 mM EDTA as an anticoagulant. Then the cell suspension is layered on a solution of ficoll-urografin (density 1.077 g / ml) and centrifuged for 30 minutes at 2000 rpm. A fraction of mononuclear cells was collected at the phase boundary, resuspended in the medium and centrifuged again for 5 minutes at 1500 rpm. The resulting pellet was resuspended in complete culture medium (DMEM and F-12 (1: 1 ratio) with the addition of 10% calf serum and 0.02% gentamicin) and planted on culture bottles (25 cm ² , Coming, USA). After the mesenchymal cells were attached to the plastic, the non-attached cells were washed, and the adhesive culture was grown to a monolayer state. Thus, a culture of bone marrow mesenchymal cells (KM-MSCs) was obtained. The cells were subsequently cultured on DMEM and F-12 medium (in a 1: 1 ratio) with the addition of 10% calf serum and 0.02% gentamicin to the state of a monolayer in culture bottles (25 cm ² , Coming, USA) at 37 ° C in a humidified incubator containing 5% CO ₂ .

The resulting cultures of fetal stem (KM-MSC) and / or progenitor (kidney cells) cells are used for administration to a recipient with chronic or acute renal failure using various methods.

The method according to the invention is illustrated by the following specific example, not limiting the invention.

EXAMPLE 1

From fetal kidneys, for example, pigs, a suspension of epithelial and mesenchymal cells is obtained by mechanical disaggregation, which are subsequently cultured on DMEM and F-12 medium (in a 1: 1 ratio) with the addition of 10% calf serum and 0.02% gentamicin to a monolayer state in culture bottles (25 cm ² , Corning, USA) at 37 ° C in a humidified incubator containing 5% CO ₂ . Hematopoietic and mesenchymal cells are washed out of the tubular bones with DMEM medium containing 2 mM EDTA as an anticoagulant. The resulting cell suspension is layered on a solution of ficoll-urographin (density 1.077 g / ml) and centrifuged for 30 minutes at 2000 rpm. A fraction of mononuclear cells was collected at the phase boundary, resuspended in the medium and centrifuged again for 5 minutes at 1500 rpm. The resulting pellet was resuspended in complete culture medium (DMEM and F-12 (1: 1 ratio) with the addition of 10% calf serum and 0.02% gentamicin) and planted on culture bottles (25 cm ² , Coming, USA). After the mesenchymal cells are attached to the plastic, the non-attached cells are washed away, and the adhesive culture is grown to a monolayer state. For cultivation, cells with viability above 90% are used, which is determined using vital dyes of trypan blue and propidium iodide.

For administration to the recipient, a primary non-passaged cell culture is used both in the case of renal cells and in the case of KM-MSCs.

For the introduction of the adhesive culture cells dissociate with a solution of 0.25% trypsin-EDTA. The resulting suspension is washed from the dissociating agents by centrifugation in DMEM for 5 minutes at 2000 rpm. The resulting cell pellet was resuspended in DMEM and the number of viable cells was counted (cell viability should be at least 98%). The resulting suspension is again centrifuged for 5 min at 2000 rpm and diluted with saline to the concentration necessary for administration to the treatment object.

Depending on the type of pathology (chronic renal failure or acute renal failure) and its severity (stage of chronic renal failure, severity of acute renal failure) monoculture of one type of cell or total culture with mesenchymal and epithelial cells is used for administration.

The introduction of cell suspension to the object of treatment can be done in different ways. The most optimal route of administration is intraparenchymal injection of cells directly into the damaged organ. This can be done during open surgery on the kidney or endoscopically by puncture of the kidney under ultrasound or x-ray control. Perhaps intravenous administration of a suspension of cells into a peripheral or central vein, as well as intra-arterial administration directly to the renal artery.

The therapeutic range of the number of introduced cells is from 5 to 20 million cells. The optimal dose is - 10-15 million cells (for renal cells and KM-MSCs).

EXAMPLE 2

The effectiveness of the treatment of chronic renal failure with intraparenchymal administration of cultured KM-MSCs and renal fetal cells

In rats with a sharply reduced volume of the functioning kidney parenchyma, signs of functional insufficiency of this organ were revealed, which persisted throughout the entire 1.5 months of observation (Table 1). There was a significant increase in the concentration of creatinine, urea and potassium in the blood with a decrease in clearance of endogenous creatinine and sodium reabsorption in the renal tubules.

Table 1
Indicators of kidney function in rats before and after modeling CRF Indicators Intact rats 2 weeks after modeling 6 weeks after modeling Blood creatinine (μmol / L) 46 ± 2 106 ± 8 *** 76 ± 5 ** Blood Urea (mmol / L) 4.4 ± 0.1 5.3 ± 0.4 * 5.9 ± 0.4 * Blood Sodium (mol / L) 141 ± 1 141 ± 2 142 ± 1 Blood potassium (mol / L) 6.5 ± 0.2 7.8 ± 0.4 * 8.2 ± 0.5 * Creatinine clearance (ml / min / kg) 3.32 ± 0.12 1.77 ± 0.05 *** 1.93 ± 0.07 *** Sodium Reabsorption (%) 99.64 ± 0.05 99.35 ± 0.12 * 99.24 ± 10 *

In the control series of experiments, the introduction of physiological saline into the damaged kidney led to a significant deterioration in all functional parameters by 4 days after injection, the function improved slightly by 11 days, but subsequently all indicators remained impaired, indicating the preservation of chronic renal failure (Table 2).

In a series of experiments with the introduction of a culture of fetal renal cells, after 4 days there was a decrease in the concentration of creatinine and blood urea, an increase in creatinine clearance and normalization of sodium reabsorption. By 11-12 days, all indicators of kidney function are normalized and maintained at normal or subnormal values until 39-40 days (Table 2). In a more distant period (61-72 days), 3 out of 4 rats maintained normal values, and 1 recurred chronic renal failure.

With the introduction of KM-MSCs after 4 days, there was a slight increase in blood creatinine and blood urea levels despite an increase in creatinine clearance and sodium reabsorption. Moreover, the growth of these indicators was less than in the series with the introduction of a kidney cell culture by this observation period. In the future, indicators of kidney function gradually improved with the normalization of most indicators by 11-12 days and the maintenance of normal or subnormal values until the 39-40th day (Table 2). In the long-term period (on the 72nd day), 1 out of 2 rats showed a recurrence of chronic renal failure, while the other retained normal renal function.

Noteworthy is the steady growth of diuresis in rats that were injected with fetal stem and progenitor cells, which correlated with an increase in glomerular filtration, estimated by the clearance of endogenous creatinine.

Although the sodium concentration in the blood as a whole was stable, reliable hyponatremia was observed in the series with the introduction of stem cells on the 11-12th days, despite the fact that sodium reabsorption was almost normal by this time. Apparently, this dynamics is associated with a sharp increase in the filtered sodium fraction due to increased diuresis, which leads to increased sodium excretion despite normal percent reabsorption. Thus, the filtered sodium fraction in the series with the introduction of fetal renal cells increased from 267 ± 16 to 417 ± 22 μmol / min (p <0.01), and in the series with the introduction of KM-MSC - from 284 ± 18 to 677 ± 35 μmol / min (p <0.001). The excreted sodium fraction increased from 0.52 ± 0.04 to 0.69 ± 0.05 μmol / min (p <0.05) in the series with the introduction of fetal renal cells and from 0.78 ± 0.05 to 1 , 01 ± 0.06 μmol / min (p <0.05), i.e. by 33% and 29%, respectively.

A rapid increase in creatinine clearance, reflecting an increase in glomerular filtration, after administration of stem and progenitor cells in combination with a diuretic effect, is possibly associated with hemodynamic reconstruction of the intraorgan vascular bed, primarily glomerular vessels, apparently under the influence of humoral factors secreted by the introduced cells. It is difficult to associate such rapid changes with any structural changes in the kidney tissue.

When comparing the dynamics of the main indicators characterizing the excretory function of the kidney, it is clear that the restoration of renal function after transplantation of a culture of fetal kidney cells occurs somewhat faster than after transplantation of a culture of mesenchymal stem cells, but in the future there was no significant difference between these groups.

table 2
The effect of fetal cell transplant on renal function in chronic renal failure Indicator Series Before cell injection After 4 days After 11-12 days After 20-21 days After 39-40 days After 61-72 days Blood creatinine (μmol / L) Fiz. solution 72 ± 4 164 ± 7 64 ± 3 112 ± 5 86 ± 3 - Fetal cells of the kidney 80 ± 4 72 ± 3 55 ± 2 60 ± 2 63 ± 2 149 ± 45 KM-MSC 74 ± 3 107 ± 4 68 ± 2 50 ± 1 56 ± 2 80 ± 7 Blood Urea (mmol / L) Fiz. solution 8.0 ± 0.2 9.4 ± 0.3 5.7 ± 0.2 8.1 ± 0.3 7.2 ± 0.1 - Fetal cells of the kidney 6.7 ± 0.2 5.0 ± 0.1 3.9 ± 0.1 4.6 ± 0.1 4.6 ± 0.1 8.8 ± 2.3 KM-MSC 5.2 ± 0.1 5.7 ± 0.1 5.0 ± 0.1 3.6 ± 0.1 4.2 ± 0.1 6.0 ± 0.9 Blood Sodium (mmol / L) Fiz. solution 142 ± 1 143 ± 2 136 ± 1 142 ± 1 142 ± 1 - Fetal cells of the kidney 140 ± 1 141 ± 2 123 ± 8 142 ± 2 141 ± 1 143 ± 1 KM-MSC 142 ± 1 142 ± 1 134 ± 5 143 ± 1 142 ± 2 142 ± 1 Blood potassium (mmol / L) Fiz. solution 8.6 ± 0.2 8.2 ± 0.2 9.6 ± 0.3 8.5 ± 0.2 9.2 ± 0.3 - Fetal cells of the kidney 8.3 ± 0.2 8.5 ± 0.3 8,00,2 8.5 ± 0.3 9.3 ± 0.3 8.7 ± 0.2 KM-MSC 8.0 ± 0.4 8.3 ± 0.3 7.9 ± 0.2 8.4 ± 0.3 8.2 ± 0.2 9.1 ± 0.1 Diuresis (ml) Fiz. solution 22.1 ± 4.2 28.2 ± 3.6 43.5 ± 7.4 45.3 ± 4.2 25.4 ± 3.2 - Fetal cells of the kidney 15.4 ± 3.1 15.5 ± 3.9 20.5 ± 3.1 16.9 ± 2.2 27.5 ± 3.3 20.4 ± 1.4 KM-MSC 18.6 ± 3.7 18.5 ± 5.1 25.1 ± 5.9 28.3 ± 2.8 27.3 ± 4.1 10.5 ± 2.1 Creatinine clearance (ml / min) Fiz. solution 1.74 ± 0.06 0.96 ± 0.23 3.92 ± 0.36 1.66 ± 0.19 1.54 ± 0.16 - Fetal cells of the kidney 1.88 ± 0.08 2.45 ± 0.37 3.39 ± 0.55 2.63 ± 0.11 2.76 ± 0.17 3.09 ± 0.51 KM-MSC 2.09 ± 0.12 2.54 ± 0.71 5.05 ± 0.43 2.78 ± 0.36 3.37 ± 0.32 1.77 ± 0.69 Sodium Reabsorption (%) Fiz. solution 99.41 ± 0.03 99.54 ± 0.02 99.50 ± 99.23 ± 99.07 ± - Fetal cells of the kidney 99.37 ± 0.04 99.76 ± 0.05 99.63 ± 0.04 99.60 ± 0.02 99.36 ± 0.04 98.96 ± 0.42 KM-MSC 99.11 ± 0.21 99.31 ± 0.22 99.78 ± 0.01 99.51 ± 0.02 99.57 ± 0.02 99.40 ± 0.08 Creatinine Excretion (μmol / day) Fiz. solution 232 ± 27 274 ± 34 277 ± 31 188 ± 16 192 ± 13 - Fetal cells of the kidney 197 ± 54 249 ± 25 269 ± 23 201 ± 10 249 ± 11 318 ± 32 KM-MSC 204 ± 31 242 ± 23 286 ± 39 269 ± 35 270 ± 24 188 ± 51

EXAMPLE 3

The efficacy of acute renal failure therapy with intraparenchymal administration of cultured fetal CM-MSCs

In the control series of experiments, in which rats after 90-minute heat ischemia of a single kidney were injected intraparenchymally with physiological saline, all rats died with uremia on 2–3 days after modeling of acute renal failure. In a series of experiments with the introduction of a fetal kidney cell culture, 5 out of 6 operated rats died, and the death of the animals occurred on 1-2 days. After transplantation of fetal KM-MSCs, none of the 5 rats died of uremia. One rat died on the 9th day for an unknown reason with improving renal function.

The indicators of the functional state of the ischemic kidney in the only surviving rat in a series with intraparenchymal administration of a fetal kidney cell culture remained impaired until the 39th day of observation. At this time, creatinine clearance and sodium reabsorption remained at subnormal values. By the 80th day, kidney function was completely normal.

In the series with the introduction of mesenchymal stem cells, reliably better indicators of the functional state of the ischemic kidney were noted already in the early stages (from the 4th day of the post-ischemic period) (Table 3). In a more distant period, the function of ischemic kidney in 1 out of 3 long-surviving rats returned to normal after 20-21 days, and in the remaining 2 by the 39th day. Normal values remained until the 80th day (maximum observation period).

As in experiments with chronic renal failure, a decrease in the concentration of sodium in the blood was noted on the 11-12th day of the postischemic period, but in this series this was associated with a decrease in sodium reabsorption in the renal tubules during polyuria.

Table 3
The effect of fetal stem cell transplant on the dynamics of kidney function in rats with postischemic acute renal failure Indicator Series 4 days 11-12 days 20-21 days 39 days 80 days Blood creatinine (μmol / L) The control 600 - - - - Fetal cells of the kidney 490 460 70 60 fifty KM-MSC 266 ± 64 135 ± 8 60 ± 3 55 ± 2 40 Blood Urea (mmol / L) The control 24.2 - - - - Fetal cells of the kidney 20.1 18,4 6.3 4,5 4.0 KM-MSC 14.0 ± 2.1 10.4 ± 1.1 4.6 ± 0.5 3.6 ± 0.1 3,1 Blood Sodium (mmol / L) The control 137 - - - - Fetal cells of the kidney 139 99 141 141 144 KM-MSC 140 ± 1 125 ± 3 * 142 ± 1 141 ± 1 145 Blood potassium (mmol / L) The control 9.8 - - - - Fetal cells of the kidney 7.8 - 9.5 9.9 9.0 KM-MSC 8.6 ± 0.3 7.6 ± 0.3 6.1 ± 0.4 7.2 ± 0.1 9.2 Diuresis (ml) The control 21 - - - - Fetal cells of the kidney 22.5 23.5 22.5 17.0 12.5 KM-MSC 26.1 ± 3.7 29.2 ± 0.9 29.5 ± 2.6 15.3 ± 1.1 14.0 Creatinine clearance (ml / min) The control 0.02 - - - - Fetal cells of the kidney 0.35 0.54 2.65 2.66 4.10 KM-MSC 0.84 ± 0.11 1.93 ± 0.09 2.60 ± 0.18 3.39 ± 0.05 5.22 Sodium Reabsorption (%) The control 75.34 - - - - Fetal cells of the kidney - 97.87 99.05 99.35 99.76 KM-MSC 97.06 ± 0.88 99.72 ± 0.02 99.32 ± 0.04 99.56 ± 0.01 99.79 Creatinine Excretion (μmol / kg / day) The control 17 - - - - Fetal cells of the kidney 248 345 249 230 276 KM-MSC 297 ± 33 350 ± 39 208 ± 27 269 ± 16 288

EXAMPLE 4

The efficacy of CRF therapy with intravenous administration of cultured KM-MSCs and renal fetal cells

The initial functional state of the kidneys (before the start of cell therapy) in the 1st and 2nd series of experiments is presented in the table. More pronounced changes in all indicators are noted in the 1st series. They corresponded to the compensated stage of chronic renal failure according to N.A. classification Lopatkina. In the 2nd series, all indices were also significantly worse than in intact rats, and their condition was evaluated as a latent stage of chronic renal failure (Table 4).

Table 4
The functional state of the kidneys with chronic renal failure in the 1st and 2nd series of experiments Blood creatinine (μmol / L) Blood Urea (mol / L) Creatinine clearance (ml / min / kg) Sodium Reabsorption (%) Intact rats 46 ± 2 4.4 ± 0.1 3.32 ± 0.12 99.64 ± 0.05 1st series 85 ± 3 *** 6.7 ± 0.4 ** 1.74 ± 0.05 *** 99.09 ± 0.8 * 2nd series 75 ± 4 ** 5.8 ± 0.5 * 2.33 ± 0.07 ** 99.24 ± 0.10 *

The introduction of rats of the 1st series of both cultured KM-MSCs and cultured renal cells (PC) led to a gradual improvement of all functional parameters. Recovery dynamics were faster in PC experiments. In these experiments, after 2 weeks, all functional parameters returned to normal, and they remained within the normal range for more than 2 months. In a more distant period, there was a slight increase in the concentration of blood creatinine, a decrease in creatinine clearance and sodium reabsorption, however, these indicators remained at subnormal values. In experiments with the introduction of KM-MSC, the improvement of the functional state of the kidneys was slower: it took more than 1 month to reach normal or subnormal values. However, in the future, all indicators except sodium reabsorption were kept within normal limits for the entire observation period. The latter indicator was maintained at a subnormal level.

In the 2nd series of experiments, in which rats with less significant impaired functional state of the kidney corresponding to the latent stage of chronic renal failure were used, exacerbation of renal failure was caused by an additional 40-minute ischemic effect, which imitated a potential exacerbation of chronic renal failure after kidney surgery with compression of the renal vessels, for example with nephrolithotomy. A suspension of stem cells was injected several minutes after the restoration of blood flow in the kidney.

In the control series of experiments, where the rats were injected with saline, all animals died from uremia within 5 days. In both experimental series, rat survival was 100%.

Assessing the dynamics of the functional parameters of the kidney after intravenous administration of stem cells, we noted the beneficial effect of both cell therapy options. The main functional indicators gradually recovered after a 2-week latent period, during which there was even a slight increase in the concentration of creatinine and urea in the blood. Their dynamics (with the exception of tubular reabsorption of sodium) in experiments with the introduction of PC and KM-MSC was approximately the same. After 1-1.5 months, these indicators returned to normal, but after 2-2.5 months, a temporary increase in blood creatinine and urea levels was noted with a decrease in creatinine clearance. In a more distant period of 3-4 months, renal function again improved with the normalization of these parameters. Sodium reabsorption after administration of PC normalized after 4 days, but later on there was a tendency to a gradual deterioration of this indicator and in the long term it persisted at the lower limit of the norm or at subnormal values. In experiments with the introduction of KM-MSCs, sodium reabsorption in the renal tubules remained reduced within 2 weeks, but later normalized and remained at a normal level for 2-2.5 months. In a more distant period, a steady decrease in this indicator was noted.

When comparing the results of experiments of the 1st and 2nd series, it is seen that the introduction of PC turned out to be more effective in terms of the onset of the therapeutic effect in experiments with stable CRF (series 1) compared with experiments where they caused exacerbation of CRF with additional ischemic effect ( 2nd series). The dynamics of functional indicators with the introduction of KM-MSC in rats in the 1st and 2nd series was similar. The therapeutic effect occurred only after 2 weeks.

In both series, a transient deterioration in functional indicators was noted after 2-2.5 months, the cause of which is still unclear.

It is noteworthy that the filtration function of the renal glomeruli during cell therapy is restored more fully than the reabsorption function of the renal tubules both after the introduction of PC and after the introduction of KM-MSC. Reabsorption of sodium after a period of normalization in the long term is again reduced to subnormal figures.

Claims (11)

1. A method for the treatment of chronic and acute renal failure, including obtaining donor material and administering it to a treatment object, characterized in that donor fetal material other than human is obtained, containing a monoculture or a mixed culture of a cell population including a stem cell culture consisting of hematopoietic and mesenchymal cells isolated as a suspension from the fetal bone marrow of the tubular bones and / or a culture of progenitor cells, consisting of epithelial and mesenchymal cells ok, isolated in the form of a suspension from fetal kidneys, followed by cultivation of the primary cell suspension of these cells to a monolayer in the growth medium DMEM / F-12 in the ratio 1: 1 with the addition of 10% calf serum and 0.02% gentamicin, and the obtained cell donor material is administered to the treatment object in the form of a suspension in physiological solution with a concentration of cellular elements from 5 to 20 million cells per 1 kg of the weight of the treatment object. 2. The method according to claim 1, characterized in that the cultured cells are administered to the treatment object with chronic renal failure. 3. The method according to claim 2, characterized in that the cultured cells are injected directly into the damaged kidney. 4. The method according to claim 2, characterized in that the cultured cells are introduced into the renal artery. 5. The method according to claim 2, characterized in that the cultured cells are administered intravenously. 6. The method according to claim 1, characterized in that the cultured cells are administered to the treatment object with acute renal failure. 7. The method according to claim 6, characterized in that the cultured cells are injected directly into the damaged kidney. 8. The method according to claim 6, characterized in that the cultured cells are introduced into the renal artery. 9. The method according to claim 6, characterized in that the cultured cells are administered intravenously. 10. The method according to claim 1, characterized in that the stem marrow cells are administered in an amount of 1 million cells per 1 kg of weight in each kidney. 11. The method according to claim 1, characterized in that the progenitor renal cells are administered in an amount of 1 million cells per 1 kg of weight in each kidney.