Use of neuron-derived neurotrophic factors in the preparation of drugs for the prevention and treatment of kidney stones

Abstract

This invention belongs to the field of biomedical technology, specifically disclosing the use of neuron-derived neurotrophic factors in the preparation of drugs for the prevention and treatment of kidney stones. Using a glyoxylate-induced mouse model of kidney calcium deposition, this invention demonstrates that neuron-derived neurotrophic factors can significantly reduce kidney calcium deposition, alleviate renal tubular structural damage, and improve renal function indicators. This achieves comprehensive intervention in the entire process of kidney stone disease, from crystal formation to tissue damage to functional abnormalities, exhibiting superior technical effects compared to single stone-dissolving or stone-expelling strategies, and possesses significant application value and clinical translational potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of neuron-derived neurotrophic factors in the preparation of drugs for the prevention and treatment of kidney stones. Background Technology

[0002] Urinary tract stones (including kidney stones and ureteral stones) are one of the most common diseases in urology, and their incidence is on the rise globally. Kidney stone patients often experience severe lower abdominal pain, hematuria, and urinary tract infections. In severe cases, they can cause kidney damage or even kidney failure, placing a significant burden on both patients' quality of life and the healthcare system.

[0003] Currently, the clinical treatment of kidney stones still mainly relies on surgical and minimally invasive physical removal methods, including extracorporeal shock wave lithotripsy (ESWL), ureteroscopic or nephroscopic lithotripsy (URS / RIRS), and percutaneous nephrolithotomy (PCNL). While these techniques have achieved high success rates in stone removal, they still generally suffer from limitations in indications, high rates of residual stones after fragmentation, and the need for repeat surgeries or complications in some patients.

[0004] For example, ESWL is effective for smaller, less dense stones, but its clearance rate is limited for larger, harder, or unusually located stones. While PCNL is considered the gold standard for treating large kidney stones, the procedure still carries risks of bleeding, infection, or damage to the renal parenchyma. Even with advancements in flexible endoscopic techniques and lasers, tiny stone fragments after lithotripsy may remain in the renal calyces or collecting system, posing a significant risk of recurrence.

[0005] Besides surgical and minimally invasive techniques, there is also research on oral medications for dissolving stones (such as alkalizing urine for uric acid stones) and adjuvant drugs. However, current systemic drug therapies mainly focus on improving metabolism or promoting the natural expulsion of stones, with limited research on preventing stone formation.

[0006] Therefore, the mainstream treatment currently focuses on physical lithotripsy and fragment removal techniques, while molecular drug treatment strategies to truly prevent stone formation are still in the exploratory stage.

[0007] Neuron-derived neurotrophic factor (NDNF) was initially identified as a neurotrophic factor that promotes neuronal migration (particularly gonadotropin-releasing hormone neurons), growth, and survival. Furthermore, it participates in angiogenesis, promoting endothelial cell survival and angiogenesis. Recent studies have also revealed a novel role for NDNF in the cerebral cortex. It is expressed in specific inhibitory interneurons that play a crucial role in the regulation of sleep microcircuits and memory consolidation.

[0008] However, existing research mainly focuses on the role of NDNF in inhibiting renal cell carcinoma, while its potential regulatory role in the formation of kidney stones has not been systematically revealed. Summary of the Invention

[0009] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by providing the use of neuronal-derived neurotrophic factors in the preparation of drugs for the prevention and treatment of kidney stones. Studies have found that neuronal-derived neurotrophic factors can significantly reduce glyoxylate-induced calcium salt deposition in the kidneys of mice, alleviate renal tubular structural damage, and improve renal function indicators.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect is to provide the use of neuron-derived neurotrophic factors in the preparation of drugs for the prevention and treatment of kidney stones.

[0011] The second aspect involves providing the use of genes encoding neurotrophic factors for the preparation of drugs for the prevention and treatment of kidney stones.

[0012] The third aspect involves providing the use of recombinant expression vectors containing genes encoding neurotrophic factors for the preparation of drugs for the prevention and treatment of kidney stone disease.

[0013] The fourth aspect concerns the use of recombinant bacteria containing genes encoding neurotrophic factors for the preparation of drugs for the prevention and treatment of kidney stones.

[0014] Furthermore, the kidney stone disease is kidney damage caused by calcium salt deposition.

[0015] Furthermore, the calcium salt deposition includes the deposition of calcium oxalate crystals in the renal tubules or renal interstitium.

[0016] Furthermore, the drug is used to reduce damage to the renal tubular structure.

[0017] Furthermore, the drug is used to improve renal function indicators, which are selected from one or more of serum creatinine levels and blood urea nitrogen levels.

[0018] Furthermore, the drug exerts its effect by inhibiting glyoxylate-induced nucleation or growth of renal calcium salt crystals.

[0019] Furthermore, the drug is configured as a dosage form for injection.

[0020] Compared with the prior art, the beneficial effects of the present invention are: (1) The use of the neuron-derived neurotrophic factor provided by this invention in the preparation of drugs for the prevention and / or treatment of kidney stones can produce significant and stable biological effects in vivo. By preparing the neuron-derived neurotrophic factor into a pharmaceutically acceptable formulation and applying it to a glyoxylate-induced mouse kidney stone model, the experimental results showed that compared with the control group, the calcium salt deposition in the kidneys of the treated mice was significantly reduced and the crystal distribution range was significantly reduced, suggesting that the neuron-derived neurotrophic factor can effectively inhibit the formation and development of stones.

[0021] (2) Histopathological analysis showed that after treatment with mouse neuron-derived neurotrophic factor recombinant protein (Ndnf), the structural damage of renal tubular epithelial cells in mice was significantly reduced, and pathological changes such as renal tubular dilation, epithelial shedding and interstitial inflammatory infiltration were significantly improved. This indicates that the protein can not only inhibit crystal deposition, but also protect the structural integrity of renal tissue and reduce secondary damage induced by crystals.

[0022] (3) The results of the detection of renal function-related biochemical indicators showed that after treatment with Ndnf recombinant protein, the serum creatinine, urea nitrogen and other indicators of mice decreased significantly compared with the model group, and the renal function level was effectively improved, further proving that the technical solution has a significant effect on functional recovery.

[0023] (4) The Ndnf recombinant protein provided by this invention can reduce calcium salt deposition, alleviate renal tubular structural damage and improve renal function indicators, thus achieving comprehensive intervention on the entire process of kidney stone disease from crystal formation to tissue damage to functional abnormality. It shows excellent technical effects in preventing and treating kidney stones and has significant application value and clinical translation potential. Attached Figure Description

[0024] Figure 1 The overexpression of human NDNF protein and mouse Ndnf gene in cells was verified; Figure 2 The effect of Ndnf recombinant protein on glyoxylate-induced serum creatinine levels in mice; Figure 3 The effect of Ndnf recombinant protein on glyoxylate-induced serum urinary nitrogen levels in mice; Figure 4 To compare the effects of different treatment groups on kidney tissue, the experiment was divided into three groups: Saline (saline control group), Gly (glyoxylate-induced model group), and Gly+Ndnf (glyoxylate + Ndnf treatment group). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] definition In this specification, the expressions “containing” and “comprising” include concepts such as “containing”, “comprising”, “substantially constituted by” and “consisting solely of”.

[0027] The term "treatment or prevention" refers to a method aimed at achieving a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relieving symptoms, reducing disease severity, stabilizing the condition (i.e., preventing deterioration), delaying or slowing disease progression, improving or alleviating the condition, and achieving detectable or undetectable partial or complete remission. Another meaning of "treatment" is extending a patient's survival compared to the expected survival rate without treatment. Therefore, "treatment" is an intervention aimed at altering the pathological process of a disease.

[0028] The terms "therapeutic or preventative effective amount," "effective amount," or "sufficient amount" refer to an amount of an active agent sufficient to induce a specific biological state, effect, and / or response. Specifically, in this invention, it refers to an amount sufficient to achieve the desired outcome, such as an effective treatment of sepsis, when administered to a subject, including mammals, such as humans. The effective amount of the agents described herein can vary depending on factors such as the subject's disease state, age, sex, and weight. As will be understood by those skilled in the art, dosages or treatment regimens can be adjusted to provide an optimal therapeutic response.

[0029] The "homology" of nucleotide sequences refers to the degree of similarity between two or more comparable nucleotide sequences. Therefore, the higher the similarity between two nucleotide sequences, the greater their identity or similarity. The level of nucleotide sequence identity can be determined, for example, using FASTA, a sequence analysis tool, with default parameters. Alternatively, it can be determined using the BLAST algorithm based on Karlin and Altschul. A program called BLASTX has been developed based on such a BLAST algorithm. The specific methods for these resolutions are well-known and can be found on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0030] The reagents provided by this invention include human NDNF recombinant protein and mouse Ndnf recombinant protein. The reagent used in the mouse treatment experiment was a mouse Ndnf protein solution, the amino acid sequence of which is shown in SEQ ID No. 1 and SEQ ID No. 2.

[0031] Neuron-derived neurotrophic factor (NDNF) was initially identified as a neurotrophic factor that promotes neuronal migration (especially gonadotropin-releasing hormone neurons), growth, and survival. Furthermore, it participates in angiogenesis, promoting endothelial cell survival and angiogenesis. Recent studies have also revealed a novel role for NDNF in the cerebral cortex. It is expressed in specific inhibitory interneurons that play a crucial role in the regulation of sleep microcircuits and memory consolidation. In the field of kidney-related research, existing studies mainly focus on the role of NDNF in inhibiting renal cell carcinoma. Compared with normal kidney tissue, NDNF expression is significantly downregulated in both renal cell carcinoma cell lines and tissues. In vitro cell experiments show that restoring NDNF expression in renal cell carcinoma cells significantly inhibits the proliferation, migration, and invasion of cancer cells. Meanwhile, the potential regulatory role of NDNF in kidney stone formation and the stone-kidney tissue interaction has not yet been systematically elucidated.

[0032] This invention is the first to discover that mouse-derived Ndnf protein can significantly reduce glyoxylate-induced calcium salt deposition in mouse kidneys, alleviate renal tubular structural damage, and improve renal function indicators, thus possessing potential application value as a drug for the prevention and treatment of kidney stones and related kidney injuries.

[0033] This invention, through a glyoxylate-induced mouse model of kidney stones, demonstrates that exogenous supplementation with Ndnf protein significantly reduces the crystal adhesion rate in mouse kidneys, decreases the crystal deposition area in kidney tissue, and lowers the levels of inflammatory factors, exhibiting excellent preventive and therapeutic effects. This effect is not limited to calcium oxalate stones but can also be extended to calcium phosphate stones, uric acid stones, cystine stones, or magnesium ammonium phosphate stones.

[0034] In some embodiments, the NDNF protein can be prepared in pharmaceutically acceptable formulations, including but not limited to injections, sustained-release formulations, or targeted delivery formulations. The dosage can be adjusted based on animal experimental results and clinical needs. Those skilled in the art can obtain the NDNF protein using conventional protein expression and purification techniques based on the disclosure in this specification, and implement the present invention accordingly.

[0035] Therefore, this invention not only provides a new use for NDNF protein, but also reveals its unique mechanism of action in regulating the microenvironment of stone formation, with clear technical effects and feasibility.

[0036] This invention further provides the use of the gene encoding the NDNF protein in the preparation of drugs for the prevention and / or treatment of kidney stones. In this invention, the encoding gene itself is not used directly as a drug, but rather as an intermediate in the preparation of the NDNF protein. After transcription and translation in a suitable expression system, the encoding gene can produce the NDNF protein, thereby providing a source of NDNF protein for subsequent applications. The encoding gene includes: a nucleotide sequence encoding the NDNF protein, and a variant sequence that has high homology (e.g., ≥90%, preferably ≥95%, more preferably ≥99%) and retains the same or substantially the same biological function. These functional variants can still achieve the technical effect of inhibiting stone formation without changing the core structure of the NDNF protein.

[0037] In some implementations, the encoding gene can be introduced into host cells (such as Escherichia coli, yeast, or mammalian cells), and after culture and expression, the NDNF protein can be obtained through conventional protein purification techniques.

[0038] This invention also provides the use of a recombinant expression vector containing the NDNF coding gene in the preparation of drugs for the prevention and / or treatment of kidney stones. The recombinant expression vector places the NDNF coding gene under the control of appropriate regulatory elements, enabling its efficient expression in host cells. The vector typically includes elements such as a promoter, coding sequence, and transcription termination signal. Those skilled in the art can select different expression systems (such as prokaryotic or eukaryotic systems) for construction as needed.

[0039] In some embodiments, after the recombinant expression vector is introduced into host cells, the NDNF protein is expressed via fermentation or cell culture, and then purified to obtain the NDNF protein product. Engineered expression systems are used to improve the efficiency and yield of NDNF protein preparation.

[0040] This invention also provides the use of recombinant bacteria containing the NDNF encoding gene in the preparation of drugs for the prevention and treatment of kidney stones. The recombinant bacteria, as a production system, can be used to continuously express and provide NDNF protein. Specifically, by introducing the NDNF encoding gene into a suitable host bacterium (such as *Escherichia coli*), a recombinant strain capable of stably expressing the NDNF protein is constructed. Under appropriate culture conditions, this strain can continuously produce NDNF protein, thereby serving as a protein source for subsequent preparation of drugs for the prevention and treatment of kidney stones.

[0041] In some implementations, bacterial cells or fermentation products containing NDNF protein can be obtained through fermentation culture, and the NDNF protein can be further extracted and purified. Alternatively, recombinant bacteria can be used to express NDNF protein in the in vivo environment to achieve a continuous supply of protein.

[0042] The following specific embodiments illustrate the use of the neuron-derived neurotrophic factor provided by the present invention in the preparation of drugs for the prevention and treatment of kidney stones.

[0043] The NDNF / Ndnf protein used in this invention is prepared through eukaryotic cell expression. Specifically, the nucleic acid sequence encoding NDNF / Ndnf is constructed into a pcDNA3.1 expression vector with a C-terminal 6xHis tag. The recombinant vector is then integrated into HEK293 cells at an appropriate density via transient transfection for overexpression. After the cells have been cultured for an appropriate time, they are collected, washed with PBS, and lysed thoroughly using RIPA lysis buffer containing a protease inhibitor. NDNF / Ndnf has already been synthesized in large quantities in the cells (e.g., ...). Figure 1 As shown in the figure, the protein sample was then initially purified by nickel-NTA affinity chromatography, followed by desalting and concentration via ultrafiltration centrifugation. The concentrated protein sample was mixed with a lyophilization protectant and filtered through a 0.22 μm filter membrane for sterilization. It was then placed in sterile lyophilization vials for lyophilization to prepare a lyophilized powder, which was stored at -20 °C for long-term storage. When needed, the powder is diluted with physiological saline to obtain the NDNF / Ndnf-6xHis protein solution formulation.

[0044] The purified NDNF / Ndnf protein was dialyzed to remove impurities and then freeze-dried to prepare a lyophilized powder, thereby improving protein stability and shelf life. For use, the lyophilized powder is dissolved in physiological saline to prepare an NDNF / Ndnf drug formulation suitable for in vivo administration.

[0045] Human NDNF-6xHis amino acid sequence: QKLPTRDEELFQMQIRDKAFFHDSSVIPDGAEISSYLFRDTPKRYFFVVEEDNTPLSVTVTPCDAPLEWKLSLQELPEDRSGEGSGDLEPLEQQKQQIINEEGTELFSYKGNDVEYFISSSSPSGLYQLDLLSTEKDTHFKVYATTTPESDQPYPELPYDPRVDVTSLGRTTVTLAWKPSPTASLLKQPIQYCVVINKEHNFKSLCAVEAKLSADDAFMMAPKPGLDFSPFDFAHFGFPSDNSGKERSFQAKPSPKLGRHVYSRPKVDIQKICIGNKNIFTVSDLKPDTQYYFDVFVVNINSNMSTAYVGTFARTKEEAKQKTVELKDGKITDVFVKRKGAKFLRFAPVSSHQKVTFFIHSCLDAVQIQVRRDGKLLLSQNVEGIQQFQLRGKPKAKYLVRLKGNKKGASMLKILATTRPTKQSFPSLPEDTRIKAFDKLRTCSSATVAWLGTQERNKFCIYKKEVDDNYNEDQKKREQNQCLGPDIRKKSEKVLCKYFHSQNLQKAVTTETIKGLQPGKSYLLDVYVIGHGGHSVKYQSKVVKTRKFCHHHHHH。

[0046] Mouse-derived Ndnf-6xHis amino acid sequence: .

[0047] Example 1 Six- to eight-week-old C57BL / 6 mice, regardless of sex and weighing 18-22 g, were selected and acclimatized for the experiment. A glyoxylate-induced mouse model of kidney stones was used as the disease model.

[0048] During the experiment, the mice were given intraperitoneal injections daily, as detailed below: Experimental group 1: Mice were injected with physiological saline into both the left and right abdominal cavities for 7 consecutive days as a control.

[0049] Experimental Group 2: Mice were injected intraperitoneally with glyoxylic acid in the left side at a dose of 80 mg / kg / day for 7 consecutive days to induce the formation of calcium oxalate stones in the kidneys. Experimental Group 3: Mice were injected intraperitoneally with glyoxylic acid at a dose of 80 mg / kg / day for 7 consecutive days to induce the formation of calcium oxalate stones in the kidneys; at the same time, mice were injected intraperitoneally with Ndnf recombinant protein preparation at a dose of 0.8 mg / kg / day for 7 consecutive days to intervene in glyoxylic acid-induced kidney stone formation and kidney damage.

[0050] Example 2 Seven days after continuous administration, the experimental mice were euthanized and dissected, and bilateral kidney tissue was collected. The left kidney tissue was fixed, embedded in paraffin, and prepared into tissue sections.

[0051] Von Kossa staining was used to detect calcium salt deposition in kidney tissue to assess the degree of kidney stone formation; hematoxylin-eosin (HE) staining and PAS staining were used to observe and evaluate renal tubular structure, inflammatory response and tissue damage.

[0052] Example 3 Mouse blood samples were collected, and the renal function of mice was assessed by detecting serum creatinine and blood urea nitrogen (BUN) levels, thus verifying the protective effect of Ndnf against glyoxylate-induced kidney injury from a functional perspective.

[0053] The results are as follows Figure 2 As shown, glyoxylic acid has a significant kidney-damaging effect, leading to a significant increase in serum creatinine and impairing kidney function; Ndnf has a significant protective effect, effectively inhibiting the increase in creatinine caused by glyoxylic acid and restoring kidney function indicators to near-normal levels.

[0054] like Figure 3 As shown, the blood urea nitrogen level in the glyoxylic acid group was significantly increased, indicating that glyoxylic acid successfully induced kidney damage. Compared with the Gly group, the blood urea nitrogen level in the Gly + Ndnf group was significantly decreased, indicating that Ndnf has a significant protective effect and can effectively inhibit the increase in blood urea nitrogen caused by glyoxylic acid, restoring renal function indicators to near-normal levels.

[0055] like Figure 4 As shown, the differences in renal pathological changes between the saline group (Saline), the glyoxylate (Gly)-induced model group, and the Ndnf treatment group (Gly+Ndnf) were compared. Von Kossa staining showed that glyoxylate significantly induced calcium deposition in kidney stones, while the addition of Ndnf significantly reduced these black stone particles. HE and PAS staining showed severe renal tubular damage, luminal dilation, and brush border detachment in the model group, while Ndnf treatment significantly improved renal tissue morphology and restored the integrity of the basement membrane. Masson staining showed a large amount of blue collagen fiber deposition in the model group, indicating severe fibrosis, while the degree of fibrosis was significantly reduced in the Ndnf group. In summary, the experimental results strongly demonstrate that Ndnf can effectively inhibit glyoxylate-induced kidney stone formation and alleviate the renal tissue damage and interstitial fibrosis caused by it.

[0056] For any points not covered above, existing technologies shall apply.

[0057] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of neuron-derived neurotrophic factors in the preparation of drugs for the prevention and treatment of kidney stones.

2. The use of genes encoding neurotrophic factors for the prevention and treatment of kidney stones.

3. The use of recombinant expression vectors containing genes encoding neurotrophic factors for the prevention and treatment of kidney stones.

4. Use of recombinant bacteria containing genes encoding neurotrophic factors for the prevention and treatment of kidney stones.

5. The use according to claim 1, characterized in that, The kidney stones mentioned are kidney damage caused by calcium salt deposition.

6. The use according to claim 5, characterized in that, The calcium salt deposition includes the deposition of calcium oxalate crystals in the renal tubules or renal interstitium.

7. The use according to claim 1, characterized in that, The drug is used to reduce damage to the renal tubular structure.

8. The use according to claim 1, characterized in that, The drug is used to improve renal function indicators, which are selected from one or more of serum creatinine levels and blood urea nitrogen levels.

9. The use according to claim 1, characterized in that, The drug works by inhibiting glyoxylate-induced nucleation or growth of calcium salt crystals in the kidneys.

10. The use according to claim 1, characterized in that, The drug is formulated as an injectable dosage form.

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