In-vitro kidney perfusion preserving fluid and preparation method thereof

Abstract

The invention discloses an in-vitro kidney perfusion preserving fluid and a preparation method thereof, and belongs to the technical field of organ preservation. The preserving fluid consists of the following components: polyethylene glycol, adenosine, sodium sulfate, magnesium sulfate, tetramethylpyrazine, potassium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, mannitol and sterile water. The method can relieve cell edema caused by low-temperature storage, prevent cell acidification, prevent cell gap swelling and prevent oxygen free radical damage in the reperfusion process, and provides a regenerated high-energy phosphate compound substrate: the high-energy phosphate compound substrate, such as adenosine, is added to keep the intracellular environment stable, so that the regeneration of the high-energy phosphate compound substrate is realized. The purposes of complete localization, cost saving and better kidney perfusion effect are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of organ preservation, in particular to an isolated kidney perfusion preservation solution and a preparation method thereof. Background Art

[0002] Kidney transplantation is recognized as the most effective treatment for end-stage renal disease. Currently, the shortage of transplantable organs is still a global problem that restricts the progress of transplantation. According to statistics, only 25% of patients can undergo kidney transplantation within 5 years of waiting, while a large number of patients are left on dialysis and experience a long wait in the registration list. The acquisition, preservation and ischemia-reperfusion injury of donor kidneys after transplantation are important factors affecting the prognosis of transplantation. Since the development of organ transplantation technology in the 20th century, preservation strategies have always been a research hotspot in the field of transplantation in order to extend the preservation time, ensure the quality of donor kidneys and improve the survival rate of donor kidneys. Organ preservation solutions corresponding to the static cold storage (SCS) technology, such as Collins solution, the University of Wisconsin solution (UW solution), histidine-tryptophan-ketoglu tarate solution (HTK solution), and hypertonic citrateadenine solution (HC-A solution) and HCA-Ⅱ solution independently developed by Shanghai Changzheng Hospital and Shanghai Central Blood Station, have been launched one after another.

[0003] To date, SCS still dominates the preservation of kidneys for kidney transplantation compared to mechanical perfusion organ preservation technology due to its advantages such as convenience, economy and effectiveness. Among them, UW solution has been proven to have excellent preservation effects and can effectively preserve kidneys for 72 hours. It is considered the "gold standard" of organ preservation fluids, but it still has its drawbacks. First, as a preservation fluid designed for multi-organ preservation, the formula cannot be specific for kidney preservation, and there are useless components for kidney preservation and room for improvement; second, the high price of UW fluid has hindered its clinical promotion. Correspondingly, my country's domestically produced HCA-Ⅱ fluid has been widely used in China due to its high cost-effectiveness, but there is still a gap in the preservation effect compared to UW fluid. In summary, a kidney preservation fluid with high performance and low price is an urgent need in the current clinical practice. We have invented a set of formulas for in vitro kidney preservation fluids. Existing experiments have proved that it can be a safe, effective and economic kidney preservation fluid, and it is expected to further get rid of the constraints of relying on imported preservation fluids, saving a lot of clinical costs, which is of great significance. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present application provides an isolated kidney perfusion preservation solution, characterized in that it is composed of the following components:

[0005] Polyethylene glycol, adenosine, sodium sulfate, magnesium sulfate, ligandrazine, potassium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, mannitol and sterile water.

[0006] Furthermore, the concentration of polyethylene glycol is 1-1.5 mmol / L; the concentration of adenosine is 10-12 mmol / L; the concentration of sodium sulfate is 9.8563 mmol / L; the concentration of magnesium sulfate is 5 mmol / L; the concentration of ligustrazine is 2-

[0007] 4mmol / L; the concentration of potassium citrate is 40-45mmol / L; the concentration of disodium hydrogen phosphate is 0.09578mmol / L; the concentration of sodium dihydrogen phosphate is 0.07836mmol / L; the concentration of mannitol is 135-

[0008] 150mmol / L.

[0009] Further, the concentration of polyethylene glycol is 1.5mmol / L; the concentration of adenosine is 10mmol / L; the concentration of sodium sulfate is 9.8563mmol / L; the concentration of magnesium sulfate is 5mmol / L; the concentration of ligustrazine is 2mmol / L; the concentration of potassium citrate is 40mmol / L; the concentration of disodium hydrogen phosphate is

[0010] 0.09578mmol / L; the concentration of sodium dihydrogen phosphate is 0.07836mmol / L; the concentration of mannitol is 135mmol / L.

[0011] Furthermore, the pH of the preservation solution is 7.0-7.4.

[0012] Furthermore, the osmotic pressure of the preservation solution is 345 mOsm / L.

[0013] The beneficial effects produced by the present invention are: 1. Reducing cell edema caused by low-temperature preservation: adding non-permeable substances to reduce cell edema. Generally speaking, the larger the molecular weight of the added substance, the better the effect. 2. Preventing cell acidification: adding hydrogen ion buffers can effectively prevent cell acidification. 3. Preventing swelling of the intercellular space: the colloidal components are believed to be able to effectively prevent swelling of the intercellular space. 4. Preventing damage by oxygen free radicals during reperfusion: adding oxygen free radical scavengers to reduce ischemia-reperfusion injury. 5. Providing regenerative high-energy phosphate substrates: adding high-energy phosphate substrates, such as adenosine. 6. Maintaining the stability of the intracellular environment: It was previously believed that the potassium and sodium ion concentrations and osmotic pressure of the preservation solution were crucial to maintaining the stability of the intracellular environment, which directly affected the effect of organ preservation. The purpose of complete localization, cost savings, and better kidney perfusion effect has been achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of light microscopy observations of UW, HCA and the three preservation solutions of the present invention at different time periods and the comparison charts;

[0015] Figure 2 The results of TUNEL staining at different time periods for UW, HCA and the three preservation solutions of the present invention;

[0016] Figure 3 The survival rates of in vitro rat donor kidney cells at different time periods were measured by UW, HCA and the three preservation solutions of the present invention;

[0017] Figure 4 This is the formula solution and placement result diagram of Comparative Example 3;

[0018] Figure 5 This is the formula solution and placement result diagram of Comparative Example 4;

[0019] Figure 6 This is the formula solution and placement result diagram of Comparative Example 5;

[0020] Figure 7 This is the formula solution and placement result diagram of Comparative Example 6;

[0021] Figure 8 This is the formula solution and placement result diagram of Comparative Example 7;

[0022] Fig. 9 This is the formula solution and placement result diagram of Comparative Example 8;

[0023] Fig.10 This is a diagram showing the placement results after each component is dissolved and sterilized separately;

[0024] Fig.11 Different concentrations of phosphate buffer were added to the five groups;

[0025] Fig.12 It is the solution of the formula of Comparative Example 5;

[0026] Fig.13 is the osmotic pressure of different concentrations of PEG;

[0027] Fig.14 is the osmotic pressure of mannitol at different concentrations;

[0028] Fig.15 It is the pH value of the formula corresponding to different concentrations of mannitol in the formula. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0030] Example 1

[0031] An isolated kidney perfusion preservation solution, comprising the following components:

[0032] Sterile water: Add 800 ml of double distilled water to a 1000 ml container.

[0033] Magnesium sulfate: Add 0.6 g of magnesium sulfate and stir to dissolve.

[0034] Adenine nucleoside: Add 2.68 g of adenine nucleoside and stir to dissolve.

[0035] Chuanxiongzine: Add 0.27 g of Chuanxiongzine and stir to dissolve.

[0036] Sodium sulfate: Add 1.4 g of sodium sulfate and stir to dissolve.

[0037] Potassium citrate: Add 9.25 g potassium citrate and stir to dissolve.

[0038] Disodium hydrogen phosphate: Add 0.0136 g of disodium hydrogen phosphate and stir to dissolve.

[0039] Sodium dihydrogen phosphate: Add 0.0094 g of sodium dihydrogen phosphate and stir to dissolve.

[0040] Mannitol: Add 24.58 g mannitol and stir to dissolve.

[0041] Polyethylene glycol 700: Add 1.05 g of polyethylene glycol 700 and stir to dissolve.

[0042] Volume adjustment: Add sterile water until the total volume of the solution is 1000 ml, stir to make the solution uniform, colorless, clear liquid, and obtain the organ preservation solution of the present invention. The pH of the organ preservation solution is 7.0-7.4, and the osmotic pressure is 345 mOsm / L.

[0043] The concentrations of the components in the organ preservation solution are shown in Table 1 : polyethylene glycol was polyethylene glycol 700;

[0044] Table 1

[0045]

[0046] The organ preservation solution prepared in Example 1 was used to perform perfusion repair of isolated rat donor kidneys for 72 hours;

[0047] When the isolated rat donor kidneys were perfused with organ preservation solution at 0, 6, 12, 24, 48, and 72 hours, the renal tissues were observed under an optical microscope, and the apoptosis of renal cells was observed by TUNEL staining at 0, 6, 12, and 24 hours.

[0048] In order to further illustrate the beneficial effects of the present invention, the following comparative examples are constructed:

[0049] Comparative Example 1: UW liquid, composed of the following components:

[0050] Table 2 UW solution (Clinical Nephrology, 2006)

[0051]

[0052]

[0053] The organ preservation solution of Comparative Example 1 was used to perform perfusion repair of isolated rat donor kidneys for 72 hours. The other conditions and test methods were the same as those of Example 1.

[0054] Comparative Example 2: HCA-II liquid, composed of the following components:

[0055] Table 3 HCA-II

[0056]

[0057] The organ preservation solution of Comparative Example 2 was used to perform perfusion repair of isolated rat donor kidneys for 72 hours. The remaining conditions and test methods were the same as those of Example 1.

[0058] The results of optical microscopy observations are as follows Figure 1 As shown, when 0, 6, and 12 hours were preserved, the renal tissue structures of the three groups were intact, the renal tubules were almost not damaged, there was no significant difference, and they were well preserved; at 24h, the renal tubules were slightly damaged, the glomerular structure was basically intact, there was a small amount of degenerated renal tubular epithelial cells, and the interstitial was slightly edematous; at 48h, the damage was aggravated, the degeneration of renal tubular epithelial cells was aggravated, multiple severe vacuolar changes, small focus tubular necrosis increased, the outline of glomerular capillaries was blurred, the interstitium was obviously edematous, and the brush border part fell off; at 72h, large-area points and flake necrosis appeared in the renal tubular epithelium, the renal tubular capillaries were blurred, and the curved tubule structure was unclear. The preservation time was extended, and the renal injury of each group was aggravated, and there was no significant difference in the renal tissue injury of the three groups preserved for 24h; the renal tissue injury of the homemade preservation liquid group (embodiment 1) preserved for 48h and 72h was equivalent to the HCA liquid group, and there was no significant difference (P>0.05); the renal tissue injury of the UW liquid group at 48h and 72h was better than that of the other two groups.

[0059] TUNEL staining results Figure 2 As shown in the figure, in a static cold storage environment at 0-4℃, at different time points (0 hours, 6 hours, 12 hours, 24 hours), the effects of the three storage solutions (UW, A, HCA) on renal tissue cell apoptosis were significantly different. Figure 3 As shown, with the extension of storage time, the apoptosis rate of renal cortical cells in the three groups showed an upward trend. Within 12 hours of storage, there was no significant difference in the number of apoptotic cells in the UW liquid group, HCA liquid group and homemade storage liquid group (Example 1); at 24 hours, the number of apoptotic cells in the HCA liquid group was significantly higher than that in the UW liquid group and the homemade storage liquid group, and there was a significant difference compared with the homemade storage liquid group (Example 1) (P<0.05); at the same time, the number of apoptotic cells in the UW liquid group and the homemade storage liquid group (Example 1) was similar, and there was no significant difference between the two (P>0.05). This shows that the effect of the homemade storage liquid (Example 1) in inhibiting cell apoptosis is comparable to that of the UW liquid, and is better than that of the HCA liquid. The homemade storage liquid (Example 1) shows a better cell protection effect in long-term storage (24 hours).

[0060] As mentioned above, the UW solution of Comparative Example 1 is a preservation solution designed for multi-organ preservation. The formula cannot be specific for kidney preservation. There are useless components for kidney preservation and room for improvement. Secondly, the price of UW solution is expensive, which hinders its clinical promotion. In addition, it is generally reflected in clinical work that the UW solution is viscous due to the addition of macromolecular solutes such as hydroxyethyl starch, and there is insufficient perfusion during kidney perfusion. As mentioned above, the HCA-II of Comparative Example 2 still has a gap in kidney preservation efficiency compared to the preservation effect of UW solution, and there is still room for cost reduction.

[0061] Comparative Example 3

[0062] In order to reduce the cost of HCA-II and improve the perfusion preservation effect, the formula of Comparative Example 2 and Comparative Example 1 was combined to prepare Comparative Example 3.

[0063] Table 4 First preparation ingredients

[0064]

[0065]

[0066] After the preservation solution of Comparative Example 3 was not sterilized and stood for 7 days, flocculent precipitation appeared in the solution and the solution turned yellow; it may be that some substance in the solution was denatured and precipitated.

[0067] like Figure 4 As shown, the preservation solution formula in Table 4 was improved, and after removing ligustrazine, after being placed without sterilization for 7 days, flocculent precipitation appeared in the solution and the solution turned yellow;

[0068] The preservation solution of Comparative Example 3 was sterilized and left for 1 day, and the color of the solution became darker.

[0069] Comparative Example 4:

[0070] Table 5 First preparation ingredients

[0071] Element Content (mol / l) 100ml(g) PEG700 1 0.07g Reduced Glutathione 3 0.092g Histidine 180 3.072g Potassium chloride 25 0.186g Magnesium sulfate 5 0.06g Adenosine 5 0.134g Ligustrazine 4 83.48mg pH 7.65

[0072] Compared with the storage solution in Table 4, polyethylene glycol 700 (PEG700) was used instead of raffinose as the non-permeable substance, and ligustrazine was not added. After being placed without sterilization for 7 days, Figure 5 The solution is transparent, but contains small white impurities;

[0073] After adding ligustrazine for sterilization and leaving it for another day, the solution turned slightly yellow;

[0074] Refrigerate for another day and let it darken in color.

[0075] Comparative Example 5:

[0076] Table 6 Second preparation ingredients

[0077]

[0078]

[0079] like Figure 6 As shown, based on Comparative Example 3, without adding reduced glutathione, the color still changes after sterilization, and the color still becomes darker when glutathione solution is added after sterilization.

[0080] Comparative Example 6:

[0081] Table 7 Second preparation composition

[0082] Element Content (mol / l) 100ml(g) PEG700 1 0.07g Histidine 180 3.072g Potassium chloride 25 0.186g Magnesium sulfate 5 0.06g Adenosine 5 0.134g Ligustrazine 4 83.46mg

[0083] like Figure 7 As shown, based on Comparative Example 4, without adding reduced glutathione, the color still changes after sterilization, and the color still becomes darker when glutathione solution is added after sterilization.

[0084] Comparative Example 7:

[0085] Table 8 Third preparation composition

[0086] Element Content (mol / l) 100ml(g) Raffinose 30 1.513g Histidine 180 3.072g Potassium chloride 25 0.186g Adenosine 5 0.134g

[0087] like Figure 8 As shown, based on Comparative Example 3, no reduced glutathione, magnesium sulfate, or ligustrazine was added, and the solution still changed color after sterilization.

[0088] Comparative Example 8:

[0089] Table 9 Third preparation composition

[0090]

[0091]

[0092] like Fig. 9 As shown, based on Comparative Example 4, no reduced glutathione, magnesium sulfate, or ligustrazine was added, and the solution still changed color after sterilization.

[0093] like Fig.10 As shown, the components in the formulations of Comparative Examples 7 and 8 were dissolved and sterilized separately, and all of them were colorless solutions; and when they were mixed together after sterilization, the solutions had no precipitation and no color.

[0094] This indicates that histidine causes the color change.

[0095] Comparative Example 8:

[0096] like Fig.11 As shown, based on Comparative Example 4, the formula was modified and a phosphate buffer was added; the intracellular fluid was divided into the following 5 groups.

[0097] Table 10

[0098]

[0099] After one day of sterilization, the solutions in each group changed as follows Fig.12 As shown: Fig.12 After sterilization, only the solutions of Group 2 and Group 3 did not turn yellow, indicating that raffinose caused the solutions to turn yellow.

[0100] The pH changes of the 6 experimental groups were as follows: Group 1, pH = 7.11; Group 2, pH = 6.63, pH was low; Group 3, pH = 7, pH was low; Group 4, pH = 6.9, pH was low; Group 5, pH = 6.81, pH was low;

[0101] The pH value of 7.2-7.4 was the standard. The osmotic pressure effect of group 1 was better in groups 1-5. The ion concentration test results of groups 1-5 are shown in Table 11.

[0102] Table 11

[0103]

[0104] In Group 1, the pH value could not be adjusted to the standard by changing the ratios of the buffer pairs: KH2PO4:Na2HPO4 (25mmol / l:22mmol / l), KH2PO4:Na2HPO4 (25mmol / l:37.5mmol / l), KH2PO4:Na2HPO4+NaOH (25mmol / l:25mmol / l), KH2PO4+NaOH (25mmol / l+0.2mmol / l), KH2PO4+NaOH (50mmol / l+0.1mmol / l), and KH2PO4+NaOH (25mmol / l+0.1mmol / l).

[0105] Comparative Example 9:

[0106] To get the pH right, measure the acidity or alkalinity of each ingredient:

[0107] Acidity: reduced glutathione, ligustrazine, PEG700; reduced glutathione is the most acidic;

[0108] Alkaline: adenine nucleoside, potassium citrate;

[0109] Adjust the ratio of buffer pair KH2PO4 to K2HPO4.

[0110] By deleting reduced glutathione, ligustrazine was reduced from 4 mol / l to 2 mol / l; adenosine was increased from the original 5 mol / l to 10 mol / l, and the ideal ratio of KH2PO4 to K2HPO4 was calculated to be 72%:28%, but it was later changed to 54%:45% based on continuous experiments.

[0111] According to the modified formula, the osmotic pressure cannot reach 330mOsm / L, as shown in Table 12

[0112] Table 12

[0113]

[0114] like Fig.13 As shown in the figure, the content of PEG700 was first increased to increase the osmotic pressure, and it was found that 1.5 mol / l was the highest osmotic pressure, but it still did not meet the requirements. Fig.14 As shown in Figure 2, the modified formula lacks non-osmotic substances, so a gradient of mannitol is added to adjust the osmotic pressure, such as Fig.15 When the concentration of mannitol is 135-150, the pH of the preservation solution is 7-7.18, and the formulation of Example 1 is finally obtained.

[0115] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An isolated kidney perfusion preservation solution, characterized in that: It is composed of the following components: Polyethylene glycol, adenosine, sodium sulfate, magnesium sulfate, ligandrazine, potassium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, mannitol and sterile water.

2. The isolated kidney perfusion preservation solution according to claim 1, characterized in that The concentration of polyethylene glycol is 1-1.5mmol / L; the concentration of adenosine is 10-12mmol / L; the concentration of sodium sulfate is 9.8563mmol / L; the concentration of magnesium sulfate is 5mmol / L; the concentration of chuanxiongzine is 2-4mmol / L; the concentration of potassium citrate is 40-45mmol / L; the concentration of disodium hydrogen phosphate is 0.09578mmol / L; the concentration of sodium dihydrogen phosphate is 0.07836mmol / L; and the concentration of mannitol is 135-150mmol / L.

3. The isolated kidney perfusion preservation solution according to claim 1, characterized in that The concentration of polyethylene glycol is 1.5mmol / L; the concentration of adenosine is 10mmol / L; the concentration of sodium sulfate is 9.8563mmol / L; the concentration of magnesium sulfate is 5mmol / L; the concentration of chuanxiongzine is 2mmol / L; the concentration of potassium citrate is 40mmol / L; the concentration of disodium hydrogen phosphate is 0.09578mmol / L; the concentration of sodium dihydrogen phosphate is 0.07836mmol / L; and the concentration of mannitol is 135mmol / L.

4. The isolated kidney perfusion preservation solution according to claim 1, characterized in that The pH of the preservation solution is 7.0-7.

4.

5. The isolated kidney perfusion preservation solution according to claim 1, characterized in that: The osmotic pressure of the preservation solution is 345 mOsm / L.

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