FGF23 mutant and medicine for preventing or treating acute kidney injury

Abstract

The invention relates to the technical field of biological medicine manufacturing, in particular to an FGF23 mutant capable of being used for preventing or treating acute kidney injury and a medicine. The FGF23 mutant is a novel FGF23 partial agonist designed by mutating the amino acid site of wild type FGF23. The FGF23 mutant disclosed by the invention can keep the AKI treatment activity and avoid adverse reaction on phosphate metabolism at the same time. Experiments prove that the FGF23 mutant disclosed by the invention shows excellent prevention and treatment effects on acute kidney injury in vivo and in vitro, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical manufacturing technology, and in particular to an FGF23 mutant and a drug that can be used to prevent or treat acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a sudden and rapid deterioration of kidney function, with an incidence rate of 10–20% in hospitalized patients and a mortality rate exceeding 50% in severely ill patients. Rhabdomyolysis-induced AKI is an important type of AKI. During rhabdomyolysis, a large amount of myoglobin is released into the bloodstream, filters through the glomeruli, deposits, and blocks the renal tubules, and simultaneously induces oxidative stress and vasoconstriction, leading to AKI in approximately 10–60% of rhabdomyolysis cases. Rhabdomyolysis-induced AKI is a clinical emergency requiring comprehensive intervention to halt the progression of kidney damage. Its core treatment methods include fluid resuscitation, urine alkalinization, diuretics to promote urine excretion, and correction of electrolyte imbalances. Current drug development for rhabdomyolysis-induced AKI mainly focuses on anti-inflammatory, anti-oxidative, and anti-apoptotic mechanisms.

[0003] Currently, there are few drugs developed for treating rhabdomyolysis-induced acute kidney injury (AKI). CN118910067A discloses a method for expressing and purifying recombinant human haptoglobin β subunits and its application in rhabdomyolysis-related acute kidney injury; CN113181196A discloses that saffron total glycosides tablets can prevent and treat acute kidney failure caused by rhabdomyolysis syndrome. In addition, CN119868326A discloses that MRS2578 can treat and alleviate cisplatin-induced AKI; CN119776516A discloses that RNF130 activator can improve renal function in mice with renal ischemia-reperfusion injury; CN119732962A discloses that trefoil glycosides inhibit acute kidney injury by upregulating Nrf2 / GPX4 in the glutathione pathway; and CN118512438A discloses that shikonin can improve the survival rate of mice with sepsis-induced AKI.

[0004] Although many drugs have been developed for acute kidney injury in recent years, the mortality rate of acute kidney injury has not improved significantly in the past 10 years. At the same time, there are few drugs developed for acute kidney injury that treat rhabdomyolysis. Therefore, there is an urgent need to develop drugs that can effectively treat AKI. Summary of the Invention

[0005] Studies have shown that the expression level of the endocrine FGF subfamily FGF23 is significantly increased in acute kidney injury (AKI), but the role of FGF23 in AKI remains unclear, and no related drugs have been developed. This invention discovers that intervention with FGF23 leads to excessive phosphate excretion, inducing hypophosphatemia. Therefore, this invention develops an FGF23 mutant through amino acid mutation, aiming to retain lower pFGFR activation activity to preserve AKI treatment activity while avoiding adverse reactions caused by overactivation. Based on this, the following technical solution is proposed.

[0006] First, this invention provides an FGF23 mutant (FGF23 partial agonist) with the following amino acid sequence: MLGARLRLWVCALCSVCSMSVLRAGGLIHLYTATAANSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLANGYDVYHSPQYHFLVSLGAAKAAFLPGAAAPPYSQFLSRRNEIPLIHFNTPIPRQHTQSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI.

[0007] Furthermore, the present invention provides a nucleic acid encoding the FGF23 mutant.

[0008] Furthermore, the present invention provides biological materials containing the FGF23 mutant or the nucleic acid.

[0009] Preferably, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or cell.

[0010] Furthermore, the present invention provides the application of the FGF23 mutant, the nucleic acid, or the biomaterial in the preparation of a pharmaceutical; the pharmaceutical is used to prevent or treat acute kidney injury or to prevent or treat hypophosphatemic rickets; preferably, the pharmaceutical is used to prevent or treat rhabdomyolysis-induced acute kidney injury.

[0011] Furthermore, the present invention provides a medicine containing the FGF23 mutant, or the nucleic acid, or the biological material.

[0012] Preferably, the medicine further includes pharmaceutically acceptable excipients.

[0013] Preferably, the excipients include, but are not limited to, fillers, excipients, lubricants, wetting agents, diluents, etc.

[0014] Preferably, the drug further includes a drug carrier; the drug carrier is selected from at least one of liposomes or lipid nanoparticles, polymer carriers, protein-based carriers, inorganic nanoparticles, biomimetic / cell membrane carriers, microbubbles, micromotors or nanomotors.

[0015] Preferably, the medicine also includes other drugs for the prevention or treatment of acute kidney injury or for the treatment of hypophosphatemic rickets.

[0016] Preferably, the drug is used for the prevention or treatment of acute kidney injury or for the prevention or treatment of hypophosphatemic rickets; preferably, the drug is used for the prevention or treatment of rhabdomyolysis-induced acute kidney injury.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides an FGF23 mutant, a novel partial agonist of FGF23 designed by mutating the amino acid sites of wild-type FGF23. This FGF23 mutant retains its activity in treating acute kidney injury (AKI) while avoiding adverse reactions caused by phosphate metabolism. Experimental verification shows that this FGF23 mutant exhibits excellent preventive and therapeutic effects against acute kidney injury both in vitro and in vivo, demonstrating broad application prospects. Attached Figure Description

[0018] Figure 1 It is FGF23 R176Q / R179Q Significantly improved AKI but resulted in hypophosphatemia; where A is serum creatinine test result; B is blood urea nitrogen test result; C is phosphate test result; D is KIM-1 and Ngal level test result; E is H&E staining result; *p<0.05, **p<0.01, ***p<0.001.

[0019] Figure 2 These are the results of in vitro and in vivo activity verification of the partial agonist FGF23; where A represents the activation level of FGFR1 by PAF23; and B represents the significant inhibition of FGF23 by PAF23. R176Q / R179Q Results of FGFR activation ability; C represents the effect of PAF23 on phosphate reabsorption; ***p<0.001.

[0020] Figure 3 The results show that the partial agonist of FGF23 significantly improved AKI without affecting phosphate metabolism; where A is the result of serum creatinine measurement; B is the result of blood urea nitrogen measurement; C is the result of phosphate measurement; D is the result of KIM-1 and Ngal level measurement; E is the result of H&E staining; **p<0.01, ****p<0.0001.

[0021] Figure 4This is the result of FGF23 partial agonists significantly improving renal fibrosis; where A represents the expression level of α-SMA in renal tissue; and B represents the results of Sirius red and Masson staining.

[0022] Figure 5 The results show that the partial agonist of FGF23 significantly inhibited renal cell apoptosis; where A represents the mRNA level of Bcl-2; B represents the mRNA level of Bax; C represents the protein levels of Bax, Bcl-2, and cleaved-caspase 3; D represents the TUNEL staining results; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0023] Figure 6 The results are from the prevention of hypophosphatemic rickets by partial agonists of FGF23; where A represents mouse body size; B represents mouse weight; C represents mouse tail length; D represents serum phosphate level; E represents mouse bone density; F represents mouse bone mineral density; G represents mouse bone volume fraction; H represents the ratio of bone surface area to bone volume; *p<0.05, ***p<0.001, ****p<0.0001.

[0024] Figure 7 This represents the results of FGF23 partial agonist therapeutic intervention in improving AKI; where A is the serum creatinine result; B is the blood urea nitrogen result; C is the phosphate result; D is the KIM-1, Ngal, and α-SMA level result; E is the H&E staining, Sirius red, and Masson trichrome staining result; *p<0.05, ***p<0.001, ****p<0.0001. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0026] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. Furthermore, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as gene sequencing, plasmid sequencing, and molecular weight determination.

[0027] Example 1 FGF23 R176Q / R179Q It can significantly improve acute kidney injury (rhabdomyolysis) but causes hypophosphatemia. FGF23 R176Q / R179Q The amino acid sequence of the protein is as follows: MLGARLRLWVCALCSVCSMSVLRAYPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDV YHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRQHTQSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI; Encoding FGF23 R176Q / R179Q The nucleotide sequence of the protein is as follows: .

[0028] Male C57BL / 6J mice were randomly divided into 4 groups (n=6 per group): Control group (NC): Healthy C57BL / 6J mice; Acute kidney injury (rhabdomyolysis) group (AKI): C57BL / 6J mice were given 8 mL / kg of 50% glycerol saline solution via intramuscular injection, and the AKI mouse model was obtained after 30 h. FGF23 R176Q / R179Q Treatment group (AKI+FGF23) R176Q / R179Q): 3 mg / kg FGF23 was injected intraperitoneally 2 hours before AKI model establishment. R176Q / R179Q The protein was administered prophylactically, and therapeutically at 6 h and 24 h after modeling. FGF23 R176Q / R179Q Treatment + FGF23 neutralizing antibody (KRN23) intervention group (AKI+FGF23) R176Q / R179Q +KRN23): FGF23 was injected intraperitoneally 2 hours before AKI model establishment. R176Q / R179Q The mixture of protein and KRN23 (mass ratio 1:8) was administered again at 6 h and 24 h after modeling.

[0029] Mice were sacrificed 30 hours later, and serum and kidney tissue were collected. The tissues were fixed in 4% formalin, embedded in paraffin, sectioned to a thickness of 4 μm, stained with hematoxylin and eosin (H&E), and examined under a microscope. FGF23 R176Q / R179Q It is a protein mutant with FGF23 stability modification. In this embodiment, FGF23 is administered via intraperitoneal injection. R176Q / R179Q The activity of this protein in AKI was evaluated.

[0030] The results showed that FGF23 R176Q / R179Q The intervention significantly alleviated the elevation of serum creatinine (sCr), blood urea nitrogen (BUN), and phosphate (Pi) levels in AKI mice. Figure 1 AC), but notably, phosphate levels were significantly lower than in the control group mice. In kidney tissue, KIM-1 and Ngal are classic biomarkers of kidney loss, and FGF23... R176Q / R179Q The administration of this drug also significantly alleviated the elevation of KIM-1 and Ngal levels. Figure 1 D). H&E staining showed significant damage to mouse kidney tissue, including tissue edema, cast formation, and inflammatory cell infiltration. FGF23 was administered. R176Q / R179Q The symptoms were relieved. Figure 1 E). This indicates FGF23's tolerance to hydrolases. R176Q / R179Q It showed good therapeutic activity against AKI.

[0031] To further demonstrate that this therapeutic activity originates from exogenously administered FGF23 R176Q / R179Q Protein, in this embodiment, FGF23 R176Q / R179Q Another group of AKI mice were administered a mixture of FGF23 neutralizing antibody (KRN23) at a ratio of 1:8. Results showed that FGF23 was significantly effective in almost all parameters. R176Q / R179Q The therapeutic activity of all of them was reversed by KRN23. Figure 1 ).

[0032] The above data indicates that FGF23R176Q / R179Q While it can significantly improve AKI, it causes excessive phosphate excretion, inducing the key adverse reaction of hypophosphatemia. Therefore, directly administering FGF23... R176Q / R179Q It is not suitable for the treatment of AKI.

[0033] Example 2: In vitro and in vivo activity verification of FGF23 partial agonist (FGF23 mutant PAF23) The amino acid sequence of the FGF23 partial agonist is as follows: The nucleotide sequence encoding a partial agonist of FGF23 is: MLGARLRLWVCALCSVCSMSVLRAGGLIHLYTATAANSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLANGYDVYHSPQYHFLVSLGAAKAAFLPGAAAPPYSQFLSRRNEIPLIHFNTPIPRQHTQSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI; .

[0034] In this embodiment, the activity of the p-FGFR signaling pathway was verified in the L6 cell line co-transfected with FGFR1c and αKlotho. The steps are as follows: Lentiviral packaging of the FGFR1c and αKlotho genes was completed in 293T cells. The supernatant containing viral particles was collected, filtered through a 0.45 μm filter, resuspended in PBS, and stored at -80°C. L6 cells in logarithmic growth phase were trypsinized and their density adjusted to 1 × 10⁻⁶ cells / cells. 5Cells / well were seeded in 6-well plates. After incubation at 37°C and 5% CO2 for 12-14 hours, cells were infected with the lentiviral supernatant to obtain an L6 cell line stably co-expressing FGFR1c and αKlotho. An equal amount of transfected cells (1×10⁶ cells / well) were then used to seed the L6 cells. 5 Cells (per well) were seeded in 12-well plates. Different concentration gradient protein stimulation groups were set up and added to the corresponding wells. The cells were incubated at 37°C for 5 minutes. Cells were collected immediately after stimulation and analyzed by Western blotting (WB).

[0035] Experimental results showed that, at the same concentration, PAF23 significantly reduced the activation level of FGFR1 compared to FGF23. R176Q / R179Q ( Figure 2 A). Furthermore, in PAF23 and FGF23 R176Q / R179Q Under co-stimulatory conditions, PAF23 can significantly inhibit FGF23. R176Q / R179Q The ability to activate FGFR. More importantly, even PAF23 and FGF23 R176Q / R179Q Increasing the proportion of p-FGFR will not cause the activation of p-FGFR to disappear completely. Figure 2 B).

[0036] To further investigate the effect of PAF23 on phosphate excretion, this example was tested in C57BL / 6J wild-type mice.

[0037] Male C57BL / 6J mice were randomly divided into 4 groups (n=6 per group): Control group (NC): Healthy C57BL / 6J mice; Saline control group (AKI): Each C57BL / 6J mouse was intraperitoneally injected with 200 μL of saline; FGF23 R176Q / R179Q Intervention group (FGF23) R176Q / R179Q ): Each healthy C57BL / 6J mouse was intraperitoneally injected with 3 mg / kg of FGF23. R176Q / R179Q protein; PAF23 intervention group (PAF23): Each healthy C57BL / 6J mouse was intraperitoneally injected with 3 mg / kg of PAF23 protein.

[0038] Single intraperitoneal injection of FGF23 R176Q / R179Q After 24 hours of PAF23 protein administration, the serum phosphate levels in mice were measured in this example. The results showed that, compared with FGF23, PAF23 phosphate levels decreased. R176Q / R179Q Unlike other phosphates, PAF23 does not inhibit phosphate reabsorption. Figure 2 C).

[0039] Example 3: FGF23 partial agonist (FGF23 mutant PAF23) significantly improved acute kidney injury. Male C57BL / 6J mice were randomly divided into 5 groups (n=6 per group), as follows: Control group (NC): Healthy C57BL / 6J mice; Acute kidney injury (rhabdomyolysis) group (AKI): C57BL / 6J mice were given 8 mL / kg of 50% glycerol saline solution via intramuscular injection, and the AKI mouse model was obtained after 30 h. FGF23 R176Q / R179Q Treatment group (AKI+FGF23) R176Q / R179Q ): 3 mg / kg FGF23 was injected intraperitoneally 2 hours before AKI model establishment. R176Q / R179Q The protein was administered prophylactically, and therapeutically at 6 h and 24 h after modeling. PAF23 treatment group (AKI+PAF23-3 mg / kg): 3 mg / kg PAF23 protein was administered prophylactically via intraperitoneal injection 2 hours before AKI model establishment, and therapeutically at 6 hours and 24 hours after model establishment; PAF23 treatment group (AKI+PAF23-6 mg / kg): 6 mg / kg PAF23 protein was administered prophylactically via intraperitoneal injection 2 hours before AKI model establishment, and therapeutically at 6 hours and 24 hours after model establishment.

[0040] like Figure 3 As shown in AC, PAF23 exhibits similarities to FGF23. R176Q / R179Q Similar therapeutic activity significantly alleviated the elevation of serum sCr, BUN, and phosphate levels in mice with rhabdomyolysis-induced AKI. Notably, PAF23's therapeutic effect on AKI was similar to that of FGF23. R176Q / R179Q The levels are comparable, but do not lead to excessive phosphate excretion, thus successfully avoiding the potential side effect of hypophosphatemia. In kidney tissue, Western blot analysis showed (…). Figure 3 D) PAF23 significantly reduced the expression levels of KIM-1 and Ngal in kidney tissue, with effects comparable to FGF23. R176Q / R179Q Quite similar. H&E staining shows ( Figure 3 E) PAF23 significantly reduced cast formation, decreased inflammatory cell infiltration, and improved renal tubular epithelial cell damage. All of these data indicate that PAF23 improves AKI without affecting phosphate metabolism.

[0041] Example 4: A partial agonist of FGF23 (FGF23 mutant PAF23) significantly inhibited renal fibrosis induced by rhabdomyolysis in acute kidney injury. Male C57BL / 6J mice were randomly divided into 5 groups (n=6 per group), as follows: Control group (NC): Healthy C57BL / 6J mice; Acute kidney injury (rhabdomyolysis) group (AKI): C57BL / 6J mice were given 8 mL / kg of 50% glycerol saline solution via intramuscular injection, and the AKI mouse model was obtained after 30 h. FGF23 R176Q / R179Q Treatment group (AKI+FGF23) R176Q / R179Q ): 3 mg / kg FGF23 was injected intraperitoneally 2 hours before AKI model establishment. R176Q / R179Q The protein was administered prophylactically, and therapeutically at 6 h and 24 h after modeling. PAF23 treatment group (AKI+PAF23-3 mg / kg): 3 mg / kg PAF23 protein was administered prophylactically via intraperitoneal injection 2 hours before AKI model establishment, and therapeutically at 6 hours and 24 hours after model establishment; PAF23 treatment group (AKI+PAF23-6 mg / kg): 6 mg / kg PAF23 protein was administered prophylactically via intraperitoneal injection 2 hours before AKI model establishment, and therapeutically at 6 hours and 24 hours after model establishment.

[0042] In a rhabdomyolysis-induced acute kidney injury model, significant mild collagen deposition was observed in the renal interstitium of mice, indicating that rhabdomyolysis induces early renal fibrosis. α-SMA is a classic biomarker of fibrosis. Following treatment with PAF23 protein, this study examined the expression level of α-SMA in renal tissue and found that FGF23... R176Q / R179Q Both PAF23 intervention significantly inhibited renal fibrosis levels, and Sirius red and Masson staining further confirmed this. Figure 4 ).

[0043] Example 5: A partial agonist of FGF23 (FGF23 mutant PAF23) significantly inhibited apoptosis of renal cells following rhabdomyolysis-induced AKI. Male C57BL / 6J mice were randomly divided into 4 groups (n=6 per group): Control group (NC): Healthy C57BL / 6J mice; Acute kidney injury (rhabdomyolysis) group (AKI): C57BL / 6J mice were given 8 mL / kg of 50% glycerol saline solution via intramuscular injection, and the AKI mouse model was obtained after 30 h. FGF23 R176Q / R179Q Treatment group (AKI+FGF23) R176Q / R179Q ): 3 mg / kg FGF23 was injected intraperitoneally 2 hours before AKI model establishment. R176Q / R179Q The protein was administered prophylactically, and therapeutically at 6 h and 24 h after modeling. PAF23 treatment group (AKI+PAF23): 3 mg / kg PAF23 protein was administered prophylactically via intraperitoneal injection 2 hours before AKI model establishment, and therapeutically at 6 hours and 24 hours after model establishment.

[0044] Apoptosis, as one of the core regulatory pathways of programmed cell death, plays a crucial role in the development and progression of acute kidney injury (AKI). Therefore, this study evaluated the effect of PAF23 protein on apoptosis after AKI. Bax and Bcl-2 are key biomarkers of apoptosis; therefore, this study measured the mRNA and protein levels of Bax and Bcl-2. The results showed that Bcl-2 expression significantly decreased and Bax expression significantly increased after AKI. R176Q / R179Q Intervention with PAF23 protein significantly reversed these processes. Furthermore, the level of caspase cleavage directly reflects the level of apoptosis; PAF23 protein significantly inhibited caspase cleavage, a finding also confirmed by TUNEL staining.

[0045] Example 6: Prevention of hypophosphatemic rickets by a partial FGF23 agonist (FGF23 mutant PAF23). Male C57BL / 6J mice were randomly divided into 3 groups (n=6 per group): Adeno-associated virus vector control group (AAV-mcherry group): Healthy C57BL / 6J mice were infected with AAV-mcherry empty vector virus (serotype 9) via a single injection into the tail vein for three weeks; FGF23 R176Q / R179Q Viral infection group (AAV-FGF23) R176Q / R179Q Group 1: AAV-FGF23 was administered via a single intravenous injection via the tail vein. R176Q / R179Q Healthy C57BL / 6J mice were infected with the virus for three weeks; PAF23 virus infection group (AAV-PAF23 group): Healthy C57BL / 6J mice were infected with AAV-PAF23 virus via a single injection into the tail vein for three weeks.

[0046] The AAV overexpression virus used employed pHBAAV-CMV-MCS-T2A-mcherry as a vector, and the AAV-FGF23R176Q / R179Q and AAV-PAF23 viruses encoded FGF23R176Q / R179Q and... The nucleotide sequence of PAF23 was inserted between the NheⅠ and BanHI sites of the AAV-mcherry empty vector virus, and the target fragment was recovered. After ligation and transformation of the target fragment with the vector, the virus was packaged using a three-plasmid adeno-associated virus system consisting of a vector plasmid carrying the target gene, a pAAV-RC vector plasmid, and a pHelper vector plasmid. The three plasmids were co-transfected into AAV-293 cells using Lipofiter™ transfection reagent. Cell pellet was collected 72 h after transfection. A 37°C water bath and liquid nitrogen were prepared, and centrifuge tubes containing cells were subjected to three freeze-thaw cycles in liquid nitrogen and a 37°C water bath. Cell debris was removed by centrifugation at 4°C, 2000 rpm, and 5 min, and the lysate containing AAV particles was collected to obtain crude virus extract. 0.1 μL of Benonase enzyme was added to each 1 mL of crude virus extract, and the mixture was incubated at 37°C for 1 h to remove cellular genome and residual plasmid DNA from the viral solution. The mixture was centrifuged at 600 rpm, 4°C, and 10 min, and the supernatant was collected. The viral supernatant was purified using the Biomiga AAV purification kit. 4 mL of the obtained AAV viral sample was added to an ultrafiltration tube and centrifuged at 1400 rpm for 30 min to obtain approximately 1 mL of AAV. The purified virus was collected and stored at -80°C.

[0047] To verify the long-term safety of the PAF23 protein, this example demonstrates the overexpression of FGF23 in mice using AAV-9 virus. R176Q / R179Q Genes and PAF23 genes were used to further induce high expression of these two proteins in mice.

[0048] Three weeks after infection, overexpression of FGF23 was found. R176Q / R179Q Mice expressing the PAF23 gene exhibited a markedly short body size, accompanied by weight loss, shorter tail length, and decreased serum phosphate levels; however, this was not observed in mice overexpressing the PAF23 gene. Figure 6 AD).

[0049] Furthermore, in this embodiment, micro-CT was used to assess bone quality in mice. Combining bone mineral density (BMD), bone volume fraction (BV / TV), and bone surface area to bone volume ratio (BS / BV), this embodiment found that FGF23 was overexpressed. R176Q / R179Q Mice expressing the PAF23 gene developed rickets symptoms, while the bone density of mice overexpressing the PAF23 gene was almost identical to that of the control group. Figure 6 EH).

[0050] Example 7: Improvement of acute kidney injury by a partial FGF23 agonist (FGF23 mutant PAF23) under therapeutic administration. Male C57BL / 6J mice were randomly divided into 4 groups (n=6 per group): Control group (NC): Healthy C57BL / 6J mice; Acute kidney injury (rhabdomyolysis) group (AKI): C57BL / 6J mice were given 8 mL / kg of 50% glycerol saline solution via intramuscular injection, and the AKI mouse model was obtained after 30 h. The low-dose PAF23 treatment group (AKI + PAF23 0.05 mg / kg): therapeutic intervention was performed by intraperitoneal injection of 0.05 mg / kg of PAF23 protein at 2 h and 24 h after the AKI model was established; High-dose PAF23 treatment group (AKI+PAF23 0.2 mg / kg): Therapeutic intervention was performed by intraperitoneal injection of 0.2 mg / kg PAF23 protein 2 h and 24 h after AKI model establishment.

[0051] AKI is an acute and sudden clinical emergency; therefore, therapeutic administration holds significant clinical application potential for drug development in this disease. Based on this, in this embodiment, AKI mice were administered high-dose (0.2 mg / mL) and low-dose (0.05 mg / mL) PAF23 via tail vein at 2 h and 24 h post-modeling, respectively, to evaluate whether therapeutic administration of PAF23 could alleviate AKI.

[0052] The study results showed that therapeutic administration of PAF23 also significantly reduced elevated serum sCr and BUN levels. Figure 7 (AB), indicating that PAF23 can alleviate kidney function impairment. Furthermore, PAF23 treatment restored renal filtration function and reduced phosphate levels without causing excessive phosphate excretion. Figure 7 C), this result is similar to that of prophylactic administration.

[0053] Next, this embodiment analyzed the kidney tissue. Western blotting was used to detect the expression levels of KIM-1, Ngal, and α-SMA in mouse kidney tissue after therapeutic administration of PAF23. The results showed that these biomarkers were significantly alleviated. Figure 7D). H&E staining results showed that during AKI, renal cells flattened, renal tubules dilated and necrosis occurred, casts formed, and renal interstitial edema developed; treatment with PAF23 significantly reduced these renal injuries. Sirius red and Masson trichrome staining also confirmed the efficacy of PAF23 in reducing tissue fibrosis. Figure 7 E). In summary, these data clearly demonstrate that therapeutic tail vein administration of PAF23 also has the ability to alleviate AKI.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An FGF23 mutant, characterized in that, MLGARLRLWVCALCSVCSMSVLRAGGLIHLYTATAANSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLANGYDVYHSPQYHFLVSLGAAKAAFLPGAAAPPYSQFLSRRNEIPLIHFNTPIPRQHTQSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI.

2. A nucleic acid encoding the FGF23 mutant.

3. A biological material containing the FGF23 mutant of claim 1 or the nucleic acid of claim 2.

4. The biomaterial of claim 3, wherein, The biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium, or a cell.

5. Use of the FGF23 mutant of claim 1, the nucleic acid of claim 2, or the biological material of claim 3 or 4 in the manufacture of a pharmaceutical product; the pharmaceutical product is for preventing or treating acute kidney injury or for preventing or treating hypophosphatemic rickets; preferably, the pharmaceutical product is for preventing or treating acute kidney injury induced by rhabdomyolysis.

6. A medicine, characterized in that, The pharmaceutical product contains the FGF23 mutant of claim 1, or the nucleic acid of claim 2, or the biological material of claim 3 or 4.

7. The pharmaceutical product according to claim 6, characterized in that The pharmaceutical product further contains a pharmaceutically acceptable excipient.

8. The pharmaceutical product according to claim 7, characterized in that The pharmaceutical product further contains a drug carrier; the drug carrier is selected from at least one of liposomes or lipid nanoparticles, polymeric carriers, protein-based carriers, inorganic nanoparticles, biomimetic / cell membrane carriers, microbubbles, micromotors, or nanomotors.

9. The pharmaceutical product according to claim 6, characterized in that The pharmaceutical product further contains other drugs for preventing or treating acute kidney injury or for preventing or treating hypophosphatemic rickets.

10. The pharmaceutical product according to any one of claims 6 to 9, characterized in that, The pharmaceutical product is for preventing or treating acute kidney injury or for preventing or treating hypophosphatemic rickets; preferably, the pharmaceutical product is for preventing or treating acute kidney injury induced by rhabdomyolysis. The pharmaceutical product is for preventing or treating acute kidney injury or for preventing or treating hypophosphatemic rickets; preferably, the pharmaceutical product is for preventing or treating acute kidney injury induced by rhabdomyolysis.

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