Peroxiredoxin 5 (Prdx5) as a pharmaceutical composition for improvement or treatment of kidney disease-associated hypertention

Abstract

The present invention relates to a pharmaceutical composition for lowering blood pressure, and more specifically, to the identification of a mechanism by which blood pressure is lowered by acting as an expression inhibitor of NaCl co-transporter (NCC), which is known to cause hypertension when Peroxiredoxin-5 (Prdx5) is not deficient and is normally activated in the kidney, and to a pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease that includes a substance acting on said mechanism as an active ingredient.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for lowering blood pressure, and more specifically, to the identification of a mechanism by which blood pressure is lowered by acting as an expression inhibitor of NaCl co-transporter (NCC), which is known to cause hypertension when Peroxiredoxin-5 (Prdx5) is not deficient and is normally activated in the kidney, and to a pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease that includes a substance acting on said mechanism as an active ingredient. Background Technology

[0002] Hypertension is known as the greatest risk factor for cardiovascular disease, a major cause of death worldwide. Since hypertension is influenced by various physiological systems, it cannot be attributed to a single etiology. Therefore, a multifaceted approach to treatment is also required.

[0003] Hypertension and renal disease share similar pathophysiologies and are bidirectionally related. In a large-scale chronic kidney disease (CKD) cohort study, increased extracellular fluid (ECF) volume was identified as an independent determinant of uncontrolled hypertension and resistant hypertension in CKD patients. Hypertension in CKD patients indicates impairment of the renal pressure-sodium excretion mechanism due to sodium retention, accompanied by ECF volume expansion.

[0004] In a 30-year study of Mendelian-type hypertension, changes in sodium processing in the kidneys, particularly in the thiazide-sensitive Na-Cl co-transporter (NCC) unique to the distal tubule (DCT), and altered function of the NCC were found to have a significant impact on blood pressure regulation, as described by the overactivation and inactivation of the NCC in Gordon's and Gitelman syndromes, respectively.

[0005] Significant progress has revealed several factors affecting the expression and activity of NCC. In particular, NCC activity is controlled by phosphorylation / dephosphorylation, while NCC expression is promoted by glycosylation and negatively regulated by ubiquitination. Studies have also revealed that parvalbumin is an unexpected NCC regulator.

[0006] In recent years, particularly with-no-lysine [K] (WNK) and its downstream target kinases, Ste20-related proline alanine rich kinase (SPAK) and oxidative stress responsive 1 (OSR1), have led to the discovery of new inhibitory molecules. Prior art literature

[0007] “Peroxiredoxin-5 Knockdown Accelerates Pressure Overload-Induced Cardiac Hypertrophy in Mice” (https: / / www.hindawi.com (2022), Chengyun Hu and 10 others) The problem to be solved

[0008] Accordingly, the objective of the present invention is to confirm that in the event of a deficiency of Peroxiredoxin 5 (Prdx5) in the kidney, the progression of renal fibrosis, a common characteristic of kidney disease, particularly chronic kidney disease (CKD), is accelerated by the existing function of Prdx5, which is known as an antioxidant protein, and in addition, the progression of hypertension is promoted; and consequently, to identify the mechanism by which hypertension is suppressed by lowering blood pressure through the action of an inhibitor of the expression and activity of NaCl co-transporter (NCC), which has been found to cause hypertension when Prdx5 is not deficient and is normally activated, thereby providing a composition for the prevention, improvement, or treatment of hypertension associated with kidney disease comprising a substance acting on this mechanism as a negative regulator of NCC.

[0009] Another objective of the present invention is to provide a composition for the prevention, improvement, or treatment of hypertension that can be usefully employed to inhibit the progression of hypertension as well as kidney damage caused by chronic kidney disease by inhibiting the expression and activity of NCC.

[0010] The objectives of the present invention are not limited to those mentioned above, and other unmentioned objectives will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0011] To achieve the objectives of the present invention described above, the present invention first provides a pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease comprising, as active ingredients, one or more of the following: mRNA of a gene encoding Peroxiredoxin 5 (Prdx5), a substance that promotes the expression of Prdx5, a substance that inhibits the decrease in the expression of Prdx5, and Prdx5.

[0012] In a preferred embodiment, the active ingredient reduces the phosphorylation expression of the WNK4-SPAK / OSR1-NCC axis.

[0013] In a preferred embodiment, the active ingredient inhibits the increase in blood pressure caused by Angiotensin II (Ang II) by inhibiting the activation of the NaCl co-transporter (NCC).

[0014] In a preferred embodiment, the formulation of the pharmaceutical composition is an injection.

[0015] In addition, the present invention provides a method for treating hypertension comprising the step of administering the above-described pharmaceutical composition to mammals other than humans.

[0016] In a preferred embodiment, the formulation of the pharmaceutical composition is an injection. Effects of the invention

[0017] According to the present invention described above, when Prdx5, known as an antioxidant protein in the kidney, is deficient, the existing function of Prdx5, namely its antioxidant function, is not exerted, thereby accelerating the progression of renal fibrosis, which is a general characteristic of kidney disease, particularly chronic kidney disease. In addition, by elucidating the mechanism by which it progresses to hypertension, a new use of Prdx5 is utilized, and hypertension associated with kidney disease can be prevented, improved, or treated by using a substance that increases the expression of Prdx5 acting on the above mechanism.

[0018] In addition, the present invention can be usefully employed to effectively suppress hypertension caused by kidney disease at a relatively early stage, thereby inhibiting the progression of hypertension caused by kidney damage.

[0019] In addition, the present invention presents a therapeutic target that effectively inhibits the progression to hypertension in the mechanism of progression to hypertension caused by kidney damage, and can be usefully utilized as a pharmaceutical composition that helps improve and treat hypertension caused by a decline in kidney function resulting from the administration of various drugs.

[0020] Therefore, it is expected that a new therapeutic strategy for the prevention, improvement, or treatment of hypertension associated with kidney disease can be established by using the present invention to target a gene expressing Prdx5, which acts as a negative regulator of NCC and is known to inhibit the expression and activity of NCC associated with kidney disease, and by using one or more of a substance or Prdx5 protein that induces the overexpression of Prdx5 or inhibits the decrease in expression.

[0021] These technical effects of the present invention are not limited to the scope mentioned above, and naturally include effects of the invention that a person skilled in the art can recognize from the description of specific details for implementing the invention that follows, even if not explicitly mentioned. Brief explanation of the drawing

[0022] Figure 1 is a schematic diagram regarding the role of Prdx5 in the distal tubules and signaling sites in the development of hypertension induced by Angiotensin II (Ang II). Figures 2a to 2c illustrate a comparison of the increase in blood pressure caused by Ang II in Prdx5 WT mice and KO mice. Figures 3a to 3h show the correlation between Ang II-induced kidney damage and the decrease in Prdx5 expression. Figures 4a to 4d illustrate a comparison of the results of oxidative stress generation in mouse kidneys of Ang II-infused Prdx5 KO vs WT. Figures 5a to 5c illustrate a comparison of the degree of renal fibrosis progression in Ang II-infused Prdx5 KO vs WT mice. Figures 6a and 6b show increased NCC phosphorylation in the kidneys of Ang II-infused Prdx5 KO mice and increased activity of the upstream signaling pathway With No Lysine[K](WNK4)-Ste20-related proline alanine rich kinase / oxidative stress responsive kinase (SPAK / OSR1). Figures 7a to 7e illustrate the activity of the WNK4-SPAK / OSR1-NCC axis following AngII treatment in 209 / mDCT (Distal convoluted tubule) cells overexpressing Prdx5. Figures 8a to 8d illustrate the activity of the WNK4-SPAK / OSR1-NCC axis following AngII treatment in 209 / mDCT cells with low expression of Prdx5. Specific details for implementing the invention

[0023] The terms used in this invention have been selected based on currently widely used general terms while considering their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been selected at the applicant's discretion, and in such cases, their meanings will be described in detail in the relevant description of the invention.

[0024] In cases where terms such as 'includes,' 'has,' and 'is accomplished' mentioned in the present invention are used, other parts may be added unless 'only' is used. When a component is expressed in the singular, it includes cases where it is in the plural unless specifically stated otherwise.

[0025] In interpreting the components, they are interpreted to include a margin of error even in the absence of a separate explicit statement.

[0026] The characteristic parts of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and may technically enable various interlocking and operation. Each embodiment may be implemented independently of one another or may be implemented together in an associated relationship.

[0027] Hereinafter, the technical configuration of the present invention will be described in detail with reference to the attached drawings and preferred embodiments.

[0028] However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Throughout the specification, the same reference numerals used to describe the present invention indicate the same components.

[0029] The technical feature of the present invention is to utilize a new use of Prdx5 obtained by elucidating the mechanism by which, when Prdx5, known as an antioxidant protein in the kidney, is deficient, its existing function—namely, antioxidant function—is not exerted, thereby accelerating the progression of renal fibrosis, a general characteristic of kidney disease, and leading to hypertension. Specifically, the invention provides a pharmaceutical composition capable of preventing, improving, or treating hypertension associated with kidney disease by elucidating the mechanism by which hypotension occurs when Peroxiredoxin-5 (Prdx5) is not deficient in the kidney and is normally activated, by acting as an expression inhibitor of NaCl co-transporter (NCC), which has been found to cause hypertension, and by utilizing a substance acting on the said mechanism.

[0030] In other words, the association between hypertension and renal disease is complex, multifaceted, and involves different mechanisms. These pathogenic mechanisms include sodium dysregulation, increased sympathetic nervous system activity, and alterations in the renin-angiotensin-aldosterone system. Furthermore, the medical treatment of hypertension associated with renal disease is complex, making the development of therapeutic agents difficult. The inventors discovered that Prdx5 acts as a mediator linking the complex mechanisms of renal disease to hypertension, and that Prdx5 regulates the function of the NCC, which is responsible for sodium reabsorption in the distal convex tubules. In renal fibrosis induced by Ang II, the downregulation of Prdx5—specifically the decrease in Prdx5 caused by Ang II in DCT—not only increased oxidative stress but also increased the activity of the WNK4-SPAK / OSR1 pathway. Consequently, the activity of the NCC, which plays a crucial role in sodium retention, increased. In this context, it was found that the decrease in Prdx5 caused by Ang II is one of the major causes of hypertension. From this, it was revealed that a deficiency in Prdx5 was associated with the expression and activity of NCC, which is known to induce hypertension, and that the efficacy of Prdx5 relies on a mechanism of action dependent on the WNK-SPAK / OSR1 pathway, the primary activator of NCC.

[0031] Accordingly, the pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease according to the present invention comprises, as active ingredients, one or more of the mRNA of the gene encoding peroxiredoxin-5 [Peroxiredoxin 5 (Prdx5)], a substance that promotes the expression of Prdx5, a substance that inhibits the decrease in the expression of Prdx5, and Prdx5.

[0032] Here, the active ingredient reduces the phosphorylation expression of the WNK4-SPAK / OSR1-NCC axis as shown in Fig. 1, and in particular, by inhibiting the activation of the NaCl co-transporter (NCC), it can inhibit the increase in blood pressure caused by Angiotensin II (Ang II).

[0033] Peroxiredoxin-5 (PRDX5) is a protein encoded by the PRDX5 gene located on chromosome 11 in humans, and it was known to possess antioxidant and cytoprotective functions during oxidative stress. Specifically, overexpression of human PRDX5 has been reported to inhibit peroxide accumulation induced by TNF-alpha, PDGF, and p53 in NIH3T3 and HeLa cells, and to reduce apoptosis caused by exogenous peroxides in various organelles of CHO, HT-22, and human tendon cells. Meanwhile, reduced expression of PRDX5 induces cellular susceptibility to oxidative damage and apoptosis is induced by etoposide, doxorubicin, MPP+, and peroxides, and PRDX5 in Drosophila melanogaster has been shown to promote longevity in addition to its antioxidant activity.

[0034] However, through the present invention, it has been revealed that peroxiredoxin-5 (PRDX5) acts as an inhibitor of NCC expression, which has been identified as causing hypertension, thereby lowering blood pressure and enabling the prevention, improvement, or treatment of hypertension.

[0035] As a result, the pharmaceutical composition of the present invention can be administered orally or parenterally and used to prevent, improve, or treat hypertension in mammalian patients with chronic kidney disease, including humans, as long as the active ingredient can reach the kidney that is the target.

[0036] As used in the present invention, the term "prevention" refers to any act of suppressing the occurrence of hypertension caused by kidney damage in a patient with chronic kidney disease or delaying its onset by administering a pharmaceutical composition according to the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of hypertension caused by kidney damage in a patient with chronic kidney disease by administering a pharmaceutical composition according to the present invention.

[0037] The pharmaceutical composition of the present invention may include one or more of the following as active ingredients: mRNA of the gene encoding Prdx5, a substance that promotes the expression of Prdx5, a substance that inhibits the decrease in the expression of Prdx5, and Prdx5; additionally, depending on the formulation, method of use, and purpose of use, it may further include pharmacologically acceptable carriers or excipients.

[0038] Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may additionally include other conventional additives such as antioxidants and buffers as needed. Additionally, diluents, dispersants, surfactants, binders, lubricants, etc., may be added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, the composition may be preferably formulated according to each component using the methods disclosed in Remington's literature. The pharmaceutical composition of the present invention may be formulated into injectables, inhalants, topical skin preparations, etc., as long as the active ingredient can adequately reach the kidney, although there are no particular restrictions on the formulation.

[0039] The pharmaceutical composition of the present invention may be administered orally or parenterally depending on the intended method; however, at the current level of technology, parenteral administration—such as topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or transdermal administration—would effectively deliver the active ingredient to the kidney. Therefore, the formulation of the pharmaceutical composition of the present invention may be an injectable; in this case, if the active ingredient is processed into a form suitable for delivery, such as by encapsulating it with nanoparticles or encapsulating it in liposomes, a greater amount of the active ingredient may reach the kidney, thereby enhancing the therapeutic effect.

[0040] The dosage of the pharmaceutical composition of the present invention is a pharmaceutically effective amount, which varies depending on the patient's condition and body weight, the severity of the disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by a person skilled in the art. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease with a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level may be determined based on factors including the patient's disease type and severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. Other pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

[0041] Specifically, the dosage of the pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease according to the present invention may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, a composition of the present invention comprising one or more of the following as active ingredients may be administered to an adult at a dose of about 0.1 ng to about 20 mg / kg, particularly 1 ng to about 2 mg / kg, per adult: mRNA of a gene encoding Peroxiredoxin 5 (Prdx5), a substance that promotes the expression of Prdx5, a substance that inhibits the decrease in expression of Prdx5. In some cases, the pharmaceutical composition of the present invention is preferably administered at a dose of about 0.0001 to 200 mg / kg per adult, particularly 0.001 to 20 mg / kg, and more specifically 0.01 to 2 mg / kg. The frequency of administration of the pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into several doses. However, since the dose may be increased or decreased depending on the route of administration, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0042] In addition, the method for treating hypertension according to the present invention may treat hypertension by including the step of administering the above-described pharmaceutical composition to treat hypertension in mammals other than humans. Here, as one embodiment, the step of administration may be performed by directly injecting the pharmaceutical composition into the kidney when the formulation is an injectable.

[0043] Example 1

[0044] To observe the correlation between the expression of Prdx5 and hypertension, a Prdx5 KO mouse model and an Ang II-induced hypertension model were constructed as follows, and the increase in blood pressure induced by Ang II in Prdx5 WT mice and KO mice was compared, and the results are shown in Figures 2a to 2c.

[0045] CRISPR / Cas9-mediated Prdx5 KO mice (C57BL / 6N-Prdx5em1Gmcr, endonuclase-mediated mutant, deletion) were produced by GEMCRO Inc. (Seoul, Korea) using Toolgen's technology on a C57BL / 6N background by deleting exons 3 through 6. 8-10 week old male Prdx5 WT and Prdx5 KO mice (n = 6, each group) were used to develop a hypertension model via subcutaneous injection of Ang II (Sigma-Aldrich, St. Louis, MO).

[0046] After inserting an osmotic minipump (Alzet MODEL 2004; DURECT, Cupertino, CA) with 1000 ng / kg / min or saline for 4 weeks, blood pressure was measured using Volume Pressure Recording (VPR). The tail-cuff method of the CODA non-invasive sphygmometer (Kent Scientific, Torrington, USA) was performed weekly for 4 weeks according to the manufacturer's instructions. In summary, mice were induced to enter the holder freely, movement was restricted, and acclimatization cycles were set to 5 times in a thermostat preheated for at least 5 minutes, with cycles per set set to 20 cycles prior to measurement. Only data marked as True for both normalized cycles and pass were used, and measurements with a standard deviation of 30 or greater were not used. After 4 weeks, blood was collected to measure the expression of renal injury markers. Tissues of the kidney, heart, and aorta were fixed in 10% formalin for immunohistochemistry (IHC) or frozen in an optimal cutting temperature (OCT) compound (Leica Biosystems, Richmond, IL, USA) for tissue slide preparation. Additionally, tissues were rapidly cooled in liquid nitrogen and stored at -70°C for protein expression, mRNA expression, and other experiments.

[0047] As shown in Figures 2a to 2c, when blood pressure was compared by injecting Ang II, a substance that induces hypertension, and Saline as a control for 4 weeks, it was confirmed that the increase in blood pressure caused by Ang II in Prdx5 KO mice, which have a defect in exon 3-exon 6 of Prdx5, was higher than in WT mice.

[0048] Example 2

[0049] Changes in blood, urine, and tissue markers of kidney injury in Ang II-infused WT vs Prdx5 KO mice were observed, and the results are shown in Figures 3a to 3h.

[0050] The levels of sCr, BUN, and NGAL, which are blood markers of renal injury, were determined, and the levels of uPCR and uACR, which are urinary markers, were measured. Histological damage was confirmed through PAS staining, and changes in Prdx5 expression in histologically caused by Ang II were investigated by comparing protein expression via IHC staining and western blotting with mRNA expression levels via real-time PCR.

[0051] As shown in Figures 3a to 3h, it was confirmed that there was more kidney damage in Ang II-infused Prdx5 KO mice by confirming that blood indicators of kidney damage, such as sCr, BUN, and NGAL, and urine indicators, such as uPCR and uACR, were higher than in W5T mice, and that there was a decrease in the expression of Prdx5 caused by Ang II, and that there was more tissue damage in the kidney tissue of Ang II-infused Prdx5 KO mice than in WT mice.

[0052] Example 3

[0053] The degree of oxidative stress in the kidneys of Ang II-infused WT vs Prdx5 KO mice was determined as follows, and the results are shown in Figures 4a to 4d.

[0054] To compare the increase in renal oxidative stress caused by Ang II, frozen renal tissue was sliced ​​into 8-μm pieces and prepared on slides, and the amount of superoxide anion, a type of ROS, was measured in the renal tissue. The sample slides were rinsed with pure water for 30 seconds and immediately stained with 5 μM DHE (dihydroethdium) for 20 minutes. Afterward, the slides were washed three times with pure water, immediately mounted, and DHE fluorescence (excitation 535 nm / emission 610 nm) was captured within 30 minutes using a fluorescence microscope (Nikon Eclipse Ni). Randomly selected fields of the stained images were examined (×400). To detect lipid peroxidation products in kidney tissue, specimens were reacted with an anti-HHE antibody (1 / 50 dilution, COSMO BIO., LTD, Tokyo, Japan), and to detect 3-NT (nitrotyrosine), known as a marker of peroxynitrite-mediated oxidative damage, were reacted with an anti-3-NT antibody (1 / 100 dilution). Tissue sections were then reacted with an HRP-conjugated secondary antibody and stained with DAB. Nuclei were counterstained with H&E.

[0055] As shown in Figures 4a to 4d, when observing the change in nitrotyrosine in the total amount of protein, proteins of approximately 28 kDa and 15 kDa were detected by the 3-nitrotyrosine antibody, and it was observed that the 15 kDa size band increased in the kidneys of Ang II-infused Prdx5 KO mice compared to WT.

[0056] As a result of observing the decrease in Prdx5 and the amount of oxidative stress generated by Ang II, it was confirmed that there was more oxidative stress generated in the kidneys of Ang II-infused Prdx5 KO mice compared to WT.

[0058] Example 4

[0059] The degree of accelerated renal fibrosis in Ang II-infused WT vs Prdx5 KO mice was compared by performing IHC, Western blot, and real-time PCR as follows, and the results are shown in Figures 5a to 5c.

[0060] To compare the degree of renal fibrosis in each group, IHC was performed on α-SMA and Fibronectin, known as fibrosis markers, and the protein expression levels of the fibrosis markers (α-SMA, Fibronectin, vimentin) in the total protein of the kidney were confirmed using Western blot.

[0061] As shown in Figures 5a to 5c, real-time PCR was used to confirm not only protein expression but also expression levels at the mRNA level, and it was confirmed that the expression of fibrosis markers in the kidneys of Ang II-infused Prdx5 KO mice was significantly increased.

[0062] In order to determine whether the increased blood pressure in Ang II-infused Prdx5 KO mice was associated with increased fibrosis, also known as kidney damage or CKD characteristics, the expression levels of fibrosis markers in the kidney tissues of each group were examined. As a result, it was evident that the expression of fibrosis markers was increased in the kidneys of Ang II-infused Prdx5 KO mice compared to WT mice.

[0063] Example 5

[0064] Changes in the activity of the WNK4-SPAK / OSR1-NCC axis signaling pathway associated with sodium excretion in the kidneys of Ang II-infused WT vs Prdx5 KO mice were observed by IF and Western blot, and the results are shown in Figures 6a and 6b.

[0065] As shown in Figures 6a and 6b, changes in the activity of NCC, which plays a role in sodium excretion during the mechanism of blood pressure elevation by Ang II, were confirmed through immunofluorescence staining (IF). It was found that Thr53 phosphorylation of NCC in the apical membrane of the kidney tissue of Ang II-infused Prdx5 KO mice was higher than in WT mice. In addition, increased activity due to NCC phosphorylation was observed in the total protein of the kidney tissue, and increased expression of pS PAK (Ser373) / p-OSR1 (Ser325), which is a phosphorylation important for the activity of the upstream signaling agent SPAK / OSR1.

[0066] In order to identify the cause of increased blood pressure due to Ang II-induced kidney damage in Prdx5 KO mice, the phosphorylation level of Thr53 in NCC, which is associated with impaired sodium excretion, was compared, and the activation level of WNK4-SPAK / OSR1, an upstream mechanism of NCC, was confirmed. As a result, it was evident that the activity of the WNK4-SPAK / OSR1-NCC axis was increased in the kidneys of Ang II-infused Prdx5 KO mice.

[0067] Example 6

[0068] Changes in the activity of the WNK4-SPAK / OSR1-NCC axis signaling pathway and changes in the amount of oxidative stress produced by Prdx5 overexpression in Ang II-treated 209 / mDCT cells were observed as follows using DHE fluorescence and Western blot, and the results are shown in Figures 7a to 7e.

[0069] To reconfirm the inhibitory effect of Prdx5 on the signaling activity of the WNK4-SPAK / OSR1-NCC axis in vitro, 209 / mDCT cells, which are distal convex tubule cells, were transiently overexpressed using a HA-tagged mouse Prdx5 expression vector and then treated with Ang II to confirm the phosphorylation-induced activity of the WNK4-SPAK / OSR1-NCC axis by Western blot.

[0070] As shown in Figures 7a to 7e, when Prdx5 was overexpressed, the phosphorylation of the WNK4-SPAK / OSR1-NCC axis was reduced compared to the mock, and it was confirmed that the DHE fluorescence, which is a probe for superoxide anion, was also reduced.

[0071] Example 7

[0072] Changes in the activity of the WNK4-SPAK / OSR1-NCC axis signaling pathway and changes in the amount of oxidative stress produced by Prdx5 low expression in Ang II-treated 209 / mDCT cells were observed by DHE fluorescence and Western blot, and the results are shown in Figures 8a to 8d.

[0073] Contrary to the results derived in Figures 7a to 7e, we wanted to reconfirm whether the activity of the WNK4-SPAK / OSR1-NCC axis signaling pathway increases when Prdx5 is underexpressed using siRNA.

[0074] As shown in Figures 8a to 8d, after injecting 25 nM of siCon and siPrdx5 into 209 / mDCT cells and treating them with Ang II, the activity of the WNK4-SPAK / OSR1-NCC axis by phosphorylation was confirmed. The results showed that a decrease in the expression of Prdx5 not only increased the activity of the WNK4-SPAK / OSR1-NCC axis but also increased the fluorescence of DHE, a probe for superoxide anion.

[0075] From the experimental results described above, it was found that among the causes of blood pressure elevation due to Ang II, a decrease in Prdx5 is associated with impaired renal function in sodium excretion in the distal tubules of the kidney, and that overexpression of Prdx5 can exhibit an effect of suppressing blood pressure elevation by inhibiting NCC activity. It was also revealed that the inhibitory effect of Prdx5 on NCC activity and its blood pressure-lowering effect are associated with incomplete lipid metabolism and oxidative stress, and that the mechanism is due to the inhibition of increased phosphorylation expression of the WNK4-SPAK / OSR1-NCC axis as a signaling signal, as shown in Figure 1. This clarified that Peroxiredoxin 5 (Prdx5) can act as an effective ingredient for preventing, improving, and / or treating hypertension associated with kidney disease.

[0076] Therefore, according to the present invention, since it is possible to lower blood pressure by sodium excretion through the induction of Prdx5 expression by utilizing the efficacy of Prdx5 as an inhibitor of NCC, it is highly possible to develop a drug for the prevention, improvement, or treatment of hypertension associated with kidney disease, as well as to apply it as an adjuvant for the treatment of general hypertension.

[0077] Although the present invention has been illustrated and described with preferred embodiments as described above, it is not limited to the embodiments described above, and various changes and modifications may be made by those skilled in the art within the scope of the invention without departing from the spirit of the invention.

Claims

Claim 1 A pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with renal disease, characterized by comprising one or more of the mRNA of the gene encoding Peroxiredoxin 5 (Prdx5) and Prdx5 as active ingredients, wherein said active ingredients reduce the phosphorylated expression of the WNK4-SPAK / OSR1-NCC axis in the distal tubule of the kidney. Claim 2 delete Claim 3 A pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease, wherein the active ingredient inhibits the increase in blood pressure caused by Angiotensin II (Ang II) by inhibiting the activation of the NaCl co-transporter (NCC) through the reduction of phosphorylation expression. Claim 4 A pharmaceutical composition for the prevention, improvement, or treatment of hypertension associated with kidney disease, characterized in that, in claim 1 or 3, the formulation of the pharmaceutical composition is an injectable. Claim 5 A method for treating hypertension comprising the step of administering the pharmaceutical composition of claim 1 or 3 to a mammal other than a human. Claim 6 A method for treating hypertension according to claim 5, characterized in that the formulation of the pharmaceutical composition is an injectable.

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