Screening method for hypoxanthine transporter inhibitors as a treatment or preventive agent for chronic kidney disease

Abstract

To provide a screening method for a human Na+ dependent hypoxanthine transporter inhibitor to be a candidate substance for an agent for treating or preventing chronic kidney disease, particularly xanthinuria.SOLUTION: The screening method includes the step for bringing a cell expressing human SLC23A3 (solute carrier family 23 member 3) gene product into contact with a test substance in the presence of hypoxanthine and sodium ions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening hypoxanthine transporter inhibitors as therapeutic or prophylactic agents for chronic kidney disease (CKD), particularly xanthinuria. [Background technology]

[0002] The human SLC23A3 (solute carrier family 23 member 3) gene, expressed in the straight part of the proximal tubule of the kidney, is SLC23 Na + It is a member of the dependent ascorbic acid transporter family and is registered as NM_001144889 (mRNA, 2322 bp). In humans, it is found in SLC23 Na + The SLC23A1-SLC23A4 family is known as a dependent ascorbic acid transporter family, and SLC23A1 and SLC23A2 are Na + While it was known that SLC23A4 is a pseudogene that transports L-ascorbic acid in a dependent manner, the function of SLC23A3 remained unknown until now (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Burzle, M et al: The sodium-dependent ascorbic acid transporter family SLC23. Mol Aspects Med 34(2-3): 436-454, 2013 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The function of the human SLC23A3 gene was previously unknown, but the inventors have now used a Xenopus oocyte expression system to express Na+ It was revealed that it is a dependent hypoxanthine transporter. Escherichia coli having a plasmid containing the human SLC23A3 gene is inhibited from growing in the presence of Na + while it can grow in the absence of Na + Therefore, it is considered that the SLC23A3 gene product is also a transporter that expresses hypoxanthine toxicity in the human kidney. Accordingly, an inhibitor of SLC23A3 gene function is considered to be a candidate substance for a renal protective agent.

[0005] An object of the present invention is to provide a method for screening a human Na + -dependent hypoxanthine transporter inhibitor, which is a candidate substance for a therapeutic or preventive agent for chronic kidney disease, particularly xanthinuria.

Means for Solving the Problems

[0006] The above problems can be solved by the following present invention: [1] (1) A step of contacting a cell expressing a human SLC23A3 (solute carrier family 23 member 3) gene product with a test substance in the presence of hypoxanthine and sodium ions (contact step) A method for screening an inhibitor of human SLC23A3 gene function, comprising the above steps. [2] When the cell is a microorganism, after the contact step, (2A) A step of culturing the microorganism in a state where the test substance is contacted in the presence of hypoxanthine and sodium ions and observing its growth (culture observation step), (3A) A step of selecting a test substance in which microbial growth is observed as a candidate substance (selection step) The screening method of [1], further comprising the above steps. [3] When the cell is an animal cell, after the contact step, (2B) A step of measuring the amount of hypoxanthine uptake in the animal cell in a state where the test substance is contacted in the presence of hypoxanthine and sodium ions (measurement step), (3B) Step of selecting a test substance that reduces the uptake amount of hypoxanthine as a candidate substance (selection step) The screening method of [1], further comprising [4] Step of confirming the inhibitory effect on human SLC23A3 gene function, the therapeutic or preventive effect on chronic kidney disease, or the therapeutic or preventive effect on xanthinuria using another evaluation system for the selected candidate substance (confirmation step) The screening method of [2] or [3], further comprising [5] The screening method of any one of [1] to [4], wherein the human SLC23A3 gene function inhibitor is a therapeutic or preventive drug for chronic kidney disease [6] The screening method of [5], wherein the chronic kidney disease is xanthinuria

Advantages of the Invention

[0007] According to the screening method of the present invention, it is possible to provide a screening method for a human Na+-dependent hypoxanthine transporter inhibitor that is a candidate substance for a therapeutic or preventive drug for chronic kidney disease, particularly xanthinuria. In particular, according to the screening method using a microorganism (typically Escherichia coli), which is a preferred embodiment of the screening method of the present invention, candidate substances can be selected using the presence or degree of growth of the microorganism as an index without measuring the uptake amount of hypoxanthine. Therefore, a large number of test substances can be screened quickly and at low cost. +

Brief Description of the Drawings

[0008] [Figure 1] It is a graph showing the uptake amount of hypoxanthine in Xenopus laevis oocytes microinjected with human SLC23A3 RNA in the absence or presence of Na+.

Embodiments for Carrying Out the Invention

[0009] <00叭15>The screening method of the present invention is ​(1) A step of bringing cells expressing the human SLC23A3 gene product into contact with the test substance in the presence of hypoxanthine and sodium ions (contact step) Includes.

[0010] If the cells in the contact step are microorganisms (for example, E. coli), the screening method of the present invention is: (1) A step of bringing a microorganism expressing the human SLC23A3 gene product into contact with the test substance in the presence of hypoxanthine and sodium ions (contact step), (2A) Following the contact step, the microorganism is cultured while maintaining the contact state and its growth is observed (culture observation step), (3A) A step in selecting test substances in which microbial growth is observed as candidate substances (selection step) Includes. The aforementioned screening method may be, if desired, (4) A step to confirm the effect of the selected candidate substance on inhibiting human SLC23A3 gene function, on treating or preventing chronic kidney disease, or on treating or preventing xanthinuria using a different evaluation system (confirmation step) It can further include:

[0011] If the cells in the contact step are animal cells (for example, Xenopus oocytes), the screening method of the present invention is: (1) A step of bringing animal cells expressing the human SLC23A3 gene product into contact with the test substance in the presence of hypoxanthine and sodium ions (contact step), (2B) Following the contact step, a step of measuring the amount of hypoxanthine uptake in the animal cells while maintaining the contact state (measurement step), (3B) A step to select a test substance that reduces the uptake of hypoxanthine as a candidate substance (selection step) Includes. The aforementioned screening method may be, if desired, (4) For the selected candidate substance, using another evaluation system, a step of confirming the inhibitory effect on human SLC23A3 gene function, the therapeutic or preventive effect on chronic kidney disease, or the therapeutic or preventive effect on xanthinuria (confirmation step) may further be included.

[0012] Hereinafter, the screening method of the present invention will be described in detail. After explaining the contact step (1) common to microorganisms and animal cells, the culture observation step (2A) and the selection step (3A) in one embodiment of the present invention using microorganisms as cells, and the measurement step (2B) and the selection step (3B) in another embodiment of the present invention using animal cells as cells will be described in this order, and finally the confirmation step (4) common to microorganisms and animal cells will be explained.

[0013] 《Contact step (1)》 In the contact step (1) of the screening method of the present invention, cells expressing the human SLC23A3 gene product (hereinafter sometimes referred to as screening cells) and a test substance are brought into contact with each other in the presence of hypoxanthine and sodium ions.

[0014] The human SLC23A3 (solute carrier family 23 member 3) gene is a member of the SLC23 Na + -dependent ascorbic acid transporter family and is registered as NM_001144889 (mRNA, 2322 bp). In humans, as the SLC23 Na + -dependent ascorbic acid transporter family, SLC23A1 to SLC23A4 are known. It was known that SLC23A1 and SLC23A2 transport L-ascorbic acid in a Na + -dependent manner, and SLC23A4 is a pseudogene, but the function of SLC23A3 has been unknown until now. As shown in Example 2 described later, the present inventor has now clarified that the human SLC23A3 gene product is a Na + -dependent hypoxanthine transporter using the Xenopus oocyte expression system.

[0015] For example, when the human SLC23A3 gene is expressed in animal cells, Na + In the presence of Na, hypoxanthine transport occurs, taking extracellular hypoxanthine into the cell, but intracellular hypoxanthine accumulation is toxic, therefore Na + In its presence, cell proliferation is inhibited. Similarly, when the human SLC23A3 gene is expressed in microorganisms, Na + In the presence of Na, hypoxanthine transport occurs, taking extracellular hypoxanthine into the cell. However, hypoxanthine accumulation in microorganisms is toxic, so + In its presence, the growth of microorganisms is inhibited.

[0016] The cells that can be used in the screening method of the present invention are not particularly limited, as long as they can be introduced in a state in which the human SLC23A3 gene can be expressed inside the cell and the human SLC23A3 gene product can exert its function. Examples include microorganisms such as Escherichia coli, animal cells such as oocytes (preferably mammalian cells), and plant cells.

[0017] When using microorganisms as the aforementioned cells, microorganisms expressing the human SLC23A3 gene product can be obtained by using known gene transfer methods, such as inserting the human SLC23A3 gene into a suitable expression vector and transforming the microorganism using that expression vector, or by introducing the human SLC23A3 gene into the microorganism by electroporation or the like and incorporating the gene into its genome.

[0018] When using animal or plant cells as the aforementioned cells, animal or plant cells expressing the human SLC23A3 gene product can be obtained by known gene transfer methods, such as injecting the human SLC23A3 gene into the cells by microinjection and expressing it within the cells; inserting the human SLC23A3 gene into a suitable expression vector and transforming the cells using that expression vector; or introducing the human SLC23A3 gene into the cells by electroporation and incorporating the gene into the genome.

[0019] The test substances that can be used in the screening method of the present invention are not particularly limited, but include, for example, various compounds registered in the Chemical File, a group of compounds obtained by combinatorial chemistry technology (Terrett NK et al., Tetrahedron, 51, 8135-73, 1995), a group of random peptides created by applying phage display methods (Felici, F. et al., J.Mol.Biol., 222, 301-310, 1991), etc.; proteins, nucleic acid molecules, peptides, antibodies; culture supernatants or cell extracts of microorganisms or plant or animal cells, natural components derived from plants or marine organisms, animal tissue extracts, bodily fluids in living organisms; and furthermore, compounds that have been chemically or biologically modified from compounds selected by the screening method of the present invention.

[0020] Contact between screening cells and the test substance in the presence of hypoxanthine and sodium ions can be carried out, for example, by initiating the culture of microorganisms (typically E. coli) using a medium containing the test substance, hypoxanthine, and sodium ions, if the cells are microorganisms. Alternatively, the contact can be carried out by preparing a microplate with the test substance pre-dispensed into each well, and then adding microorganisms suspended in a medium containing hypoxanthine and sodium ions to each well and initiating the culture.

[0021] If the cells are animal cells (typically Xenopus oocytes), the contact involves, for example, injecting a small amount of human SLC23A3 RNA synthesized by in vitro transcription into the animal cells, culturing them for a predetermined time (more than 36 hours for Xenopus oocytes) to express the human SLC23A3 gene, and then using the test substance. 3 This can be carried out by replacing or adding a culture medium containing H-hypoxanthine and sodium ions.

[0022] In the contact step of the screening method of the present invention, the amount of cells used (e.g., cell count / culture medium volume), hypoxanthine concentration, sodium ion concentration, etc., can be appropriately determined by referring to the examples described later. The concentration of the test substance cannot be specified in general, as it differs in, for example, its type, degree of purification, and strength of activity, but a person skilled in the art can appropriately determine it by conducting preliminary experiments at several different concentrations.

[0023] In the contact step of the screening method of the present invention, in addition to the test group to which the test substance is added, a negative control in which the test substance is not added may be optionally included, or Na may be used instead of the test substance. + A positive control can be prepared by adding a known inhibitor of the dependent hypoxanthine transporter (i.e., the human SLC23A3 gene product) (e.g., dipyridamole).

[0024] 《Culture observation process (2A), selection process (3A)》 In the screening method of the present invention, in a culture observation step (2A) in one embodiment of the present invention using microorganisms as cells, following the contact step (1), the microorganisms are cultured in contact with the test substance in the presence of hypoxanthine and sodium ions, and their growth is observed. Subsequently, in the selection step (3A) in this embodiment, test substances in which microbial growth is observed are selected as candidate substances.

[0025] In the culture observation step described above, the method for observing the growth of microorganisms is not particularly limited as long as it is a method that can determine whether or not growth is occurring and / or the degree of growth. Examples include observing turbidity by visual inspection, quantifying turbidity by absorbance, and determining the extent of growth by centrifuging the culture solution and determining the amount of microorganisms that precipitate.

[0026] As mentioned earlier, when the human SLC23A3 gene is expressed in microorganisms, Na + In the presence of Na, hypoxanthine transport occurs, taking extracellular hypoxanthine into the cell. However, hypoxanthine accumulation in microorganisms is toxic, so + In its presence, microbial growth is inhibited (negative control without the test substance). On the other hand, the human SLC23A3 gene product Na + In the positive control group, when the human SLC23A3 gene was expressed in microorganisms, hypoxanthine transport was inhibited, and therefore, hypoxanthine accumulation did not occur within the microorganisms. + Microorganisms can still grow in the presence of other substances.

[0027] In the above selection step, among the test groups to which the test substance was added, Na + Test substances in which microbial growth was observed in the presence of the substance were selected as candidate substances. The selected candidate substance is the human SLC23A3 gene product, Na + It is a candidate substance for inhibiting the dependent hypoxanthine transporter. + Since the dependent hypoxanthine transporter is thought to be a transporter that exhibits hypoxanthine toxicity, Na + Dependent hypoxanthine transporter inhibitors are considered potential candidates for renal protective agents, i.e., for the treatment or prevention of chronic kidney disease, particularly xanthinuria.

[0028] By applying multiple compounds chemically or biologically modified from the obtained candidate substances as lead compounds to the screening method of the present invention again as test substances, more effective candidate substances can be obtained.

[0029] Measurement process (2B) and selection process (3B) In the screening method of the present invention, in a measurement step (2A) in one embodiment of the present invention using animal cells as cells, following the contact step (1), the amount of hypoxanthine uptake in the animal cells is measured while the test substance is in contact with the animal cells in the presence of hypoxanthine and sodium ions. Subsequently, in the selection step (3A) in this embodiment, a test substance that reduces the amount of hypoxanthine uptake is selected as a candidate substance.

[0030] In the aforementioned measurement step, the method for measuring the amount of hypoxanthine uptake is not particularly limited as long as it is a known hypoxanthine measurement method, but for example, it can be carried out using hypoxanthine labeled with a labeling compound such as a radioactive isotope.

[0031] When the human SLC23A3 gene is expressed in animal cells, Na + In the presence of Na, hypoxanthine transport occurs, which involves taking extracellular hypoxanthine into the cell. + In the presence of the test substance, the uptake of hypoxanthine increases (negative control without the test substance). On the other hand, the human SLC23A3 gene product Na + In positive controls with a hypoxanthine transporter-dependent inhibitor, hypoxanthine transport is inhibited even when the human SLC23A3 gene is expressed in animal cells, therefore Na + In its presence, the uptake of hypoxanthine decreases.

[0032] In the selection step, among the test groups to which the test substance was added, Na was selected in comparison to the hypoxanthine uptake amount of the negative control. +Test substances that reduce hypoxanthine uptake in the presence of [substance name] are selected as candidate substances. The selected candidate substance is the human SLC23A3 gene product, Na + It is a candidate substance for inhibitors of the dependent hypoxanthine transporter. As mentioned above, Na + Since the dependent hypoxanthine transporter is thought to be a transporter that exhibits hypoxanthine toxicity, Na + Dependent hypoxanthine transporter inhibitors are considered potential candidates for renal protective agents, i.e., for the treatment or prevention of chronic kidney disease, particularly xanthinuria.

[0033] By applying multiple compounds chemically or biologically modified from the obtained candidate substances as lead compounds to the screening method of the present invention again as test substances, more effective candidate substances can be obtained.

[0034] 《Confirmation process (4)》 In the confirmation step (4) of the screening method of the present invention, the selected candidate substance is used to confirm its effect on inhibiting human SLC23A3 gene function, its therapeutic or preventive effect on chronic kidney disease, or its therapeutic or preventive effect on xanthinuria using a different evaluation system.

[0035] For example, a candidate substance selected by one aspect of the screening method of the present invention using microorganisms as cells is applied to another aspect of the screening method of the present invention using animal cells as cells, Na + By confirming that the uptake of hypoxanthine decreases in the presence of Na, the inhibitory effect on human SLC23A3 gene function can be reconfirmed. Conversely, a candidate substance selected by one aspect of the screening method of the present invention using animal cells can be applied to another aspect of the screening method of the present invention using microorganisms as cells, and Na + By confirming that microorganisms proliferate in the presence of this substance, the inhibitory effect on human SLC23A3 gene function can be reconfirmed.

[0036] Furthermore, the therapeutic or preventive effects of the candidate substance on chronic kidney disease, particularly xanthinuria, can be confirmed using known evaluation systems for such treatments or preventive agents. In this process, the effects of the candidate substance can be confirmed, if desired, in comparison with known therapeutic or preventive agents. [Examples]

[0037] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0038] Example 1: Cloning of the human SLC23A3 gene Human SLC23A3 cDNA was cloned by nested RT-PCR using total RNA derived from human kidney. Table 1 shows the primer sets used in the first and second nested PCRs. Cloning was performed according to standard procedures, except that E. coli was cultured in LB medium without NaCl during the cloning of the PCR product. The typical composition of LB medium is 1% (w / v) tryptone, 0.5% (w / v) yeast extract, and 1% (w / v) sodium chloride.

[0039] [Table 1]

[0040] The obtained cDNA sequence (SEQ ID NO: 5) matched the sequence of the human SLC23A3 (solute carrier family 23 member 3) gene, which is registered as NM_001144889 (mRNA, 2322 bp).

[0041] Example 2: Expression of the human SLC23A3 gene in Xenopus oocytes and Na + Confirmation of Dependent Hypoxanthine Transporter Activity》 1. In vitro transfer from SLC23A3 clone vector Since the SLC23A3-pCR4TOPO plasmid has a T7 RNA polymerase binding sequence at its 5' end, the SLC23A3-pCR4TOPO plasmid was cleaved at the SpeI restriction enzyme site located at its 3' end to form a linear chain. SLC23A3 RNA was synthesized in vitro using the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific), the SLC23A3-pCR4TOPO plasmid was degraded with Turbo DNase (Thermo Fisher Scientific), the RNA was purified using the MegaClear kit (Thermo Fisher Scientific), and a Poly A tail was added to the RNA using the Poly A tailing kit (Thermo Fisher Scientific). The concentration was adjusted to 1 μg / μL using distilled water and stored at -80°C.

[0042] 2. Preparation of Xenopus oocytes Under anesthesia, the abdomen of female African clawed frogs was incised, and a portion of the ovarian mass was removed. The wound was then closed with 4-0 surgical absorbable sutures. After the ovarian capsule was crushed with forceps, oocytes were isolated by shaking in 10 mL of 2 mg / mL collagenase / OR2 solution at room temperature for 2 hours. When the oocytes were transferred to ND96 solution containing gentamicin, the follicular cells surrounding the oocytes detached, making them suitable for microinjection. Oocytes that had matured to stage 6 size were selected under a microscope.

[0043] 3. Microinjection of RNA into Xenopus oocytes A glass capillary was pulled with a puller, the needle-shaped tip was broken off with tweezers, and dry-heat sterilized at 180°C for 2 hours to be used as a glass needle. A small amount of silicone oil was filled into the glass needle and attached to a Manual Oocyte Microinjection Pipette (Drummond Scientific), and the Microinjection Pipette was attached to a manipulator. RNA solution or distilled water placed on Parafilm was drawn into the glass needle. ND96 solution was placed on a petri dish lined with gauze, and the Xenopus oocytes prepared in step 2 were placed on it. The glass needle was inserted into the equator of the Xenopus oocytes, and 50 nL of RNA or distilled water was injected. The Xenopus oocytes were transferred to ND96 solution in which NaCl was replaced with choline Cl or NMDG (N-methyl-D-glucamine) (Na-free ND96 solution) and cultured at 18°C ​​for 48 hours or more.

[0044] 4. 3 H-hypoxanthine uptake experiment Ten Xenopus oocytes, cultured for over 48 hours in a sodium-free ND96 solution, were placed in each well of a 24-well plate. 500 μL of 10 nmol / L solution was used per well. 3 After adding H-hypoxanthine / ND96 solution and allowing uptake for 2 minutes, the cells were washed with approximately 1 mL of ice-cooled ND96 solution five times. One Xenopus oocyte was transferred to one liquid scintillation vial, and eight cells were transferred from one well. 100 μL of 5% sodium dodecyl sulfate solution was added to a liquid scintillation vial and allowed to dissolve overnight. 1 mL of liquid scintillation cocktail was added, and the dpm was measured using a scintillation counter.

[0045] 5.Results The results are shown in Figure 1. In oocytes that were not injected with SLC23A3 RNA (control), Na + The presence or absence of [the substance] did not change the amount of hypoxanthine uptake. On the other hand, in oocytes injected with the same RNA, Na +With or without Na, greater hypoxanthine uptake was observed compared to the control, and Na + Non-existence and Na + In comparison with the presence of Na, + A significant increase in uptake was observed in the presence of the substance. From these results, it appears that SLC23A3 expressed in the human kidney is Na + It was confirmed to be a dependent hypoxanthine transporter.

[0046] Example 3: Expression of the human SLC23A3 gene in Escherichia coli and Na + Confirmation of dependent E. coli growth》 During the cloning of the PCR product of SLC23A3, E. coli containing the SLC23A3-pCR4TOPO plasmid was colonized on NaCl-free LB agar medium (containing kanamycin). One colony was then picked and added to NaCl-free LB solution (containing kanamycin) to prepare a bacterial suspension. A certain amount of this bacterial suspension was added to NaCl-added LB solution (containing kanamycin) and NaCl-free LB solution (containing kanamycin), and the cells were incubated overnight at 37°C and 225 rpm. In this example and in the following Example 4, the "NaCl-added LB solution" is a standard LB medium (1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride). Furthermore, since standard LB medium contains hypoxanthine derived from yeast extract, it is not necessary to add hypoxanthine separately.

[0047] In a NaCl-free LB solution, E. coli containing the SLC23A3-pCR4TOPO plasmid was able to grow, and turbidity of the culture medium was observed. In contrast, in a NaCl-added LB solution, growth was inhibited, the turbidity of the culture medium was reduced, and less E. coli precipitate was obtained by centrifugation of the culture medium. This result is the human SLC23A3 gene product Na + Dependent hypoxanthine transporter, Na +Because it exhibits toxicity due to hypoxanthine accumulation in E. coli in the presence of this gene product, inhibiting the growth of E. coli expressing this gene product, and using this culture system for screening, Na + This study demonstrates that a test substance that restores the growth of E. coli in the culture medium containing the substance could be a candidate for a human SLC23A3 gene function inhibitor.

[0048] Example 4: Na using Escherichia coli expressing the human SLC23A3 gene + Screening for inhibitors of the activity of the dependent hypoxanthine transporter. E. coli containing the SLC23A3-pCR4TOPO plasmid is allowed to colonize on NaCl-free LB agar medium (containing kanamycin). One colony is then picked and added to NaCl-free LB solution (containing kanamycin) to prepare a bacterial suspension. 2 μL of DMSO is added to each well of an Assay-Ready Plate obtained from the University of Tokyo's Pharmaceuticals Research Institute. 100 μL of NaCl-added LB solution (containing kanamycin) is then added, and the drug is dissolved by rotation and shaking. 100 μL of the above bacterial suspension is then added to each well, sealed with a sterile gas-permeable plate seal (SureSeal Breathable, Sterile; BM instrument), and incubated overnight at 37°C. The absorbance at 660 nm is measured to identify wells where bacterial growth has occurred. [Industrial applicability]

[0049] The screening method of the present invention can be used in the development of therapeutic or preventive drugs for chronic kidney disease, particularly xanthinuria.

Claims

1. (1) Express the human SLC23A3 (solute carrier family 23 member 3) gene product. A step of bringing a microorganism into contact with a test substance in the presence of hypoxanthine and sodium ions (contact step), (2A) A step of culturing the microorganisms in contact with the test substance in the presence of hypoxanthine and sodium ions and observing their growth (cultivation observation step), and (3A) A step in which test substances in which microbial growth is observed are selected as candidate substances (selection step) A screening method for human SLC23A3 gene function inhibitors, including [specific example].

2. (4) A step to confirm the inhibitory effect on human SLC23A3 gene function of the selected candidate substance using a different evaluation system (confirmation step) The screening method according to claim 1, further comprising:

3. The screening method according to claim 1 or 2, wherein the human SLC23A3 gene function inhibitor is a drug for the treatment or prevention of chronic kidney disease.

4. The screening method according to claim 3, wherein the chronic kidney disease is xanthinuria.

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