JP7553105B2 - Methods for predicting the onset of focal segmental glomerulosclerosis - Google Patents

Description

Application of Article 30, paragraph 2 of the Patent Act Posted on the following website on November 9, 2020 https://www2. aeplan. co. jp/mbsj2020/ https://conference-apps-online. net/web/mbsj2020/abstract. html? sid=186 https://conference-apps-online. net/web/mbsj2020/abstract. html? sid=186&ano=2214 https://conference-apps-online. NET/WEB/MBSJ2020/poster.html Presented at the 43rd Annual Meeting of the Molecular Biology Society of Japan on December 3, 2020

The present invention relates to a method for estimating the onset time of focal segmental glomerulosclerosis. More specifically, the present invention relates to a method for estimating the onset time of focal segmental glomerulosclerosis caused by a TRPC6 mutation, based on functional analysis of TRPC6.

Focal segmental glomerulosclerosis (FSGS) is an intractable kidney disease that develops into steroid-resistant nephrotic syndrome and progresses to renal failure. Clinically, FSGS causes hypoproteinemia due to increased protein in urine and decreased protein in blood, and symptoms such as swelling (edema) and ascites. In advanced cases, fluid accumulates in the lungs, abdomen, heart, and scrotum. Increased blood cholesterol can lead to renal failure, thrombosis, infections, etc. In FSGS, focal segmental sclerosing lesions are observed in the glomeruli under light microscopy, and widespread loss of foot processes of glomerular epithelial cells is observed under electron microscopy. In Japan, FSGS is classified as primary nephrotic syndrome (designated intractable disease 222). Renal survival rates (non-dialysis rate) for FSGS are reported to be 85.3% at 10 years, 60.1% at 15 years, and 33.5% at 20 years, and the long-term prognosis is poor. There are various causes of this disease, but the TRPC6 (Transient Receptor Potential Canonical 6) channel has been identified as one of the causative genes of familial FSGS (Non-Patent Documents 1 and 2). Since then, various TRPC6 mutations have been identified in FSGS patients of various races and genders, but the age of onset of FSGS is widely distributed from infants to adults (Non-Patent Documents 3 to 7), and no correlation has been found between the position of the mutation or the type of amino acid substituted and the age of onset. TRPC6 mutations account for approximately 6% of familial FSGS and approximately 2% of non-familial FSGS (Non-Patent Document 8). TRPC6 is a nonselective cation channel that is expressed in the cell membrane of glomerular podocytes and is thought to regulate the filter function in the renal glomerulus by being involved in the influx of cations (mainly sodium and calcium ions), but the relationship between TRPC6 mutations and the age at onset of FSGS is unclear (Non-Patent Documents 1 and 2). Recent studies have reported that TRPC6 mutants involved in the onset of FSGS have abnormalities in the inactivation mechanism inherent to TRPC6 (Non-Patent Document 9).

Winn et al., Science, 308, 1801-1804, 2005 Reiser et al., Nat Genetics, 37, 739-744, 2005 Heeringa et al., PLos One, 4, e7771, 2009 Buscher et al., Clinical Nephrology, 78, 47-53, 2012 Mir et al., Nephrol Dial Transplant, 27, 205-209, 2012 Hofstra et al., Nephrol Dial Transplant, 28, 1830-1838, 2013 Hirono Suzuki, Takuji Yamada, Japanese Journal of Pediatric Nephrology, 32, 37-42, 2019 Santin et al., Nephrol Dial Transplant, 24, 3089-3096, 2009 Polat et al., J Am Soc Nephrol, 30, 1587-1603, 2019

The present invention aims to clarify the relationship between the function of TRPC6 mutants and the time of onset, and to provide a method for predicting the time of onset of focal segmental glomerulosclerosis caused by TRPC6 mutations.

The inventors expressed various TRPC6 mutants in HEK293 cells and analyzed the degree of inactivation of TRPC6 current and the amount of electricity per unit cell membrane area for 60 seconds after activation (also called "total current density") using the patch clamp method. As a result, TRPC6 (R175W) with a mutation identified from a patient with a young age at onset of FSGS was inactivated more slowly and had a higher total current density for 60 seconds after activation than TRPC6 (R175Q) with a mutation identified from a patient with an older age at onset of FSGS. In addition, a similar tendency was obtained when comparing different mutants (R895C and R895L) with different onset ages. In other words, TRPC6 with a mutation that leads to an early onset of FSGS (juvenile type) was hardly inactivated once activated, resulting in an increase in the total current density, while a mutation that leads to a late onset of FSGS (adult type) tended to be slightly more difficult to inactivate than the wild type. Further analysis revealed that mutations that result in a significant increase in the amount of electricity passing through the TRPC6 channel upon activation (particularly the total current density (amount of electricity per unit cell membrane)) were associated with a lower age at onset of FSGS, even if the delay in inactivation was only slight, whereas mutations that result in a slight increase in the amount of electricity passing through the TRPC6 channel upon activation (particularly the total current density (amount of electricity per unit cell membrane)) were associated with a higher age at onset of FSGS, even if the delay in inactivation was severe. Based on these findings, the inventors conducted further investigations and completed the present invention.

That is, the present invention relates to the following.
[1] A method for predicting the onset time of focal segmental glomerulosclerosis caused by TRPC6 mutations, comprising the following steps:
(1) To evaluate in vitro the degree of increase in the electrical charge passing through the TRPC6 channel upon activation in TRPC6 mutants in subjects with TRPC6 mutations, and (2) to correlate the degree of increase in the electrical charge passing through the TRPC6 channel with the onset of focal segmental glomerulosclerosis.
[2] The method of [1] in which the electrical quantity is the electrical quantity per unit cell membrane.
[3] The method of [1] or [2], wherein the increase in the amount of electricity passing through the TRPC6 channel is due to delayed inactivation of the TRPC6 channel.
[4] The method of [1] or [2], wherein the increase in the amount of electricity passing through the TRPC6 channel is due to an increase in the peak current of the TRPC6 channel.
[5] Any of the methods described in [1] to [4], wherein the TRPC6 mutation is a missense mutation in the intracellular domain of TRPC6.
[6] The method according to [5], wherein the intracellular domain is an ankyrin repeat domain or a coiled-coil domain.
[7] The method of any one of claims 1 to 6, wherein the TRPC6 mutation is an amino acid substitution at glycine at position 109, arginine at position 175, histidine at position 218, or arginine at position 895.

This invention makes it possible to predict the age of onset and progression of FSGS caused by TRPC6 mutations, and is expected to provide useful insight into the treatment of FSGS patients.

Schematic diagram summarizing examples of TRPC6 mutations reported to be associated with FSGS. 1 is a graph showing the time course of TRPC6 current when HEK293 cells expressing wild-type TRPC6 or TRPC6 mutants (R175Q, R175W) were stimulated with carbachol. HEK293 cells also co-express muscarinic receptors (M ₁ R). The asterisk indicates noise due to a ramp wave for investigating the current-voltage characteristics. This is a graph comparing the time course of inactivation of TRPC6 channels between wild-type TRPC6 and TRPC6 mutants (R175Q and R175W). The black circles represent wild-type TRPC6, the triangles represent R175Q, and the squares represent R175W. 1 is a graph showing the time course of TRPC6 current when HEK293 cells expressing wild-type TRPC6 or a TRPC6 mutant (H218L) were stimulated with carbachol, with the ordinate representing current (pA) and the abscissa representing time (ms). This graph compares the electrical charge (total current density) (pC/pF) per unit cell membrane for 60 seconds after activation when HEK293 cells expressing wild-type TRPC6 or TRPC6 mutants (G109S, R175Q, R175W, H218L, R895C, R895L) were stimulated with carbachol. "Young" indicates mutations that cause FSGS at a young age, and "Adult" indicates mutations that cause FSGS in adults.

The present invention provides a method for predicting the onset time of focal segmental glomerulosclerosis caused by a TRPC6 mutation (hereinafter referred to as the method of the present invention), which comprises the following steps:
(1) To evaluate in vitro the degree of increase in the electrical charge passing through the TRPC6 channel upon activation in TRPC6 mutants in subjects with TRPC6 mutations, and (2) to correlate the degree of increase in the electrical charge passing through the TRPC6 channel with the onset of focal segmental glomerulosclerosis.

"Focal segmental glomerulosclerosis (FSGS)" is an intractable kidney disease that develops into steroid-resistant nephrotic syndrome and progresses to renal failure. Clinically, FSGS causes hypoproteinemia due to an increase in protein in the urine and a decrease in protein in the blood, and symptoms such as swelling (edema) and ascites are presented. In advanced cases, fluid accumulates in the lungs, abdomen, heart, and scrotum. Increased blood cholesterol leads to renal failure, thrombosis, infections, etc. In FSGS, focal segmental sclerosing lesions are observed in the glomeruli under light microscopy, and widespread loss of foot processes of glomerular epithelial cells is observed under electron microscopy. In this specification, "onset" of FSGS means the presentation of the above-mentioned pathological lesions (i.e., focal segmental sclerosing lesions in the glomeruli).

TRPC6 (Transient Receptor Potential Canonical 6) is one of the nonselective cation channels that allows cations such as Na ⁺ (sodium ion) and Ca ²⁺ (calcium ion) to permeate from outside the cell to inside the cell. TRPC6 is activated by agonist stimulation of G protein-coupled receptors (GPCRs) such as α1-adrenergic receptor, muscarinic acetylcholine receptor, and AT1R, and allows cations such as Na ⁺ and Ca ²⁺ to permeate into the cell. The TRPC6 used in the present invention is usually TRPC6 from humans and non-human mammals, and preferably human TRPC6. A representative amino acid sequence of wild-type human TRPC6 is shown in SEQ ID NO: 1 (GenBank accession no. NP_004612.2). In this specification, when a region or amino acid position in human TRPC6 is indicated, the amino acid sequence represented by SEQ ID NO: 1 is used as a reference.

In this specification, the intracellular region of TRPC6 refers to the intracellular region located at the N-terminus and C-terminus of TRPC6, and corresponds to amino acids 1 to 438 and 728 to 931 of the amino acid sequence represented by SEQ ID NO:1. The ankyrin repeat region refers to an ankyrin-like repeat sequence of about 33 amino acid residues, and corresponds to amino acids 97 to 250 of the amino acid sequence represented by SEQ ID NO:1. The ankyrin repeat region of TRPC6 contains four ankyrin-like sequences (Ank1, Ank2, Ank3, Ank4), of which Ank1 corresponds to amino acids 97 to 126 of the amino acid sequence represented by SEQ ID NO:1, Ank2 corresponds to amino acids 127 to 160 of the amino acid sequence represented by SEQ ID NO:1, Ank3 corresponds to amino acids 161 to 217 of the amino acid sequence represented by SEQ ID NO:1, and Ank4 corresponds to amino acids 218 to 250 of the amino acid sequence represented by SEQ ID NO:1. The intracellular region on the C-terminal side of TRPC6 contains a calmodulin-binding domain (CaM-binding domain: CBD) and a coiled-coil domain (CC). The calmodulin-binding domain corresponds to amino acids 853 to 874 of the amino acid sequence shown in SEQ ID NO:1, and the coiled-coil domain corresponds to amino acids 879 to 920 of the amino acid sequence shown in SEQ ID NO:1.

As used herein, "TRPC6 mutation" refers to a mutation that results in an amino acid sequence that differs from the amino acid sequence of wild-type TRPC6. The mutation may be a missense mutation or a nonsense mutation, but is preferably a missense mutation. The number of amino acids that result in a mutation is not particularly limited, but is usually 5 or less (i.e., 5, 4, 3, 2, or 1), and is preferably 1. That is, the TRPC6 mutation is preferably a missense mutation that replaces one amino acid in TRPC6 with another amino acid.

The region in TRPC6 in which the mutation occurs is not particularly limited, but is preferably an intracellular region, more preferably an ankyrin repeat region, a calmodulin-binding region, or a coiled-coil region, and even more preferably an ankyrin repeat region (e.g., Ank1, Ank2, Ank3, Ank4) or a coiled-coil region.

The specific positions of the amino acids substituted by the mutations are not particularly limited. For example, in the N-terminal intracellular region, proline 15, arginine 68, serine 89, glycine 109, asparagine 110, proline 112, cysteine 121, asparagine 125, aspartic acid 130, methionine 132, asparagine 143, histidine 145, glycine 162, arginine 163, arginine 164, arginine 165, arginine 166, arginine 167, arginine 168, arginine 169, arginine 170, arginine 171, arginine 172, arginine 173, arginine 174, arginine 175, arginine 176, arginine 177, arginine 178, arginine 179 ... Examples of the amino acids that can be substituted include lysine, tyrosine at position 173, arginine at position 175, histidine at position 218, serine at position 270, arginine at position 360, leucine at position 395, and alanine at position 404. Examples of the amino acids that can be substituted include glycine at position 757, leucine at position 780, lysine at position 874, glutamic acid at position 875, glutamine at position 889, arginine at position 895, and glutamic acid at position 897. The amino acids substituted by mutation are preferably glycine at position 109, arginine at position 175, histidine at position 218, or arginine at position 895. The type of amino acid substituted is not particularly limited.

In one embodiment, the TRPC6 mutation is a known mutation that has already been reported to be associated with FSGS (see Figure 1). In one embodiment, the TRPC6 mutation is a novel mutation that has not previously been reported to be associated with FSGS.

"Activation" of an ion channel refers to the process of opening the ion channel. When the TRPC6 channel is activated, cations such as Na ⁺ and Ca2 ⁺ flow into the cell from outside the cell via TRPC6.

The "amount of electricity passing through the TRPC6 channel" refers to the time-integrated value of the current generated by the influx of cations into the cell via TRPC6 after activation of the TRPC6 channel. In one embodiment, the amount of electricity passing through the TRPC6 channel is the amount of electricity passing through the TRPC6 channel during a certain period of time (e.g., 5 seconds or more and 120 seconds or less, preferably 20 seconds or more and 80 seconds or less (e.g., 60 seconds)) after activation of the TRPC6 channel.

In the present invention, the "electrical quantity passing through the TRPC6 channel" is preferably the electric quantity passing through the TRPC6 channel per unit cell membrane. The "electrical quantity per unit cell membrane" is the value obtained by dividing the electric quantity passing through the TRPC6 channel by the amount of cell membrane. The amount of cell membrane can be the cell membrane capacity, the cell membrane area, the amount of cell membrane components, etc., but is preferably the cell membrane capacity. The cell membrane capacity can be calculated from the capacitance current immediately after the cell membrane is broken in the patch clamp method described later. In addition, in this specification, the electric quantity per unit cell membrane may be referred to as the "total current density". In addition, since the electric capacity per 1 μm ² of the cell membrane is approximately equivalent to 0.01 pF, the cell membrane area can be calculated from the cell membrane capacity.

"Inactivation" of an ion channel refers to the process of closing an ion channel that has been opened by activation. Wild-type TRPC6 has an autoinhibitory (brake) mechanism called Ca ²⁺ -dependent inactivation (CDI), which inactivates the channel in an intracellular Ca ²⁺ -dependent manner. When Ca ²⁺ flows into the cell from outside the cell via TRPC6 due to activation of TRPC6 and the intracellular Ca ²⁺ concentration increases, TRPC6 is immediately inactivated by CDI, the channel is closed, and the intracellular Ca ²⁺ concentration rapidly decreases. It has been reported that CDI of TRPC6 occurs when calmodulin bound to Ca ²⁺ interacts with the coiled-coil domain of TRPC6 in a bridging manner (Non-Patent Document 9).

The "delayed inactivation" of an ion channel means that the process of closing an ion channel opened by activation is impaired due to a mutation in the ion channel, etc., and the ion channel opened by activation is not closed (i.e., remains open) or takes longer to close than a normal ion channel (e.g., a wild-type ion channel). When CDI is impaired due to a mutation in TRPC6, even after Ca ²⁺ flows from the outside of the cell into the cell due to activation of TRPC6 and the intracellular Ca ²⁺ concentration increases, the TRPC6 channel is not closed or takes longer to close than the wild-type TRPC6, and as a result, it takes longer for the intracellular Ca ²⁺ concentration to decrease than the wild-type TRPC6. The delayed inactivation of the TRPC6 channel causes an increase in the amount of electricity (preferably the amount of electricity per unit cell membrane) passing through the TRPC6 channel upon TRPC6 activation.

In the method of the present invention, first, the degree of increase in the amount of electricity passing through the TRPC6 channel upon activation of the TRPC6 mutant possessed by a subject having a TRPC6 mutation is evaluated in vitro. The fact that a subject has a TRPC6 mutation can be determined by isolating genomic DNA or mRNA containing the TRPC6 coding region from the subject, analyzing the base sequence of the TRPC6 coding region, translating it into a deduced amino acid sequence, and comparing it with the amino acid sequence of wild-type TRPC6 by a well-known molecular biology technique. In one embodiment, the subject in the method of the present invention is a human who has a TRPC6 mutation and has not yet developed FSGS (e.g., a newborn baby in which a mutation was found in the TRPC6 gene immediately after birth). The degree of increase in the amount of electricity passing through the TRPC6 channel upon activation of the TRPC6 mutant can be evaluated by analyzing the cation channel function of the TRPC6 mutant using an electrophysiological technique (e.g., patch clamp method). For example, a transfectant in which a TRPC6 mutant is forcibly expressed in an appropriate mammalian cell and a control transfectant in which a wild-type TRPC6 is forcibly expressed are prepared, and a stimulus for activating TRPC6 is applied to these cells. The time-dependent change in the current generated between the inside and outside of the cell due to the activation and subsequent inactivation of TRPC6 is measured by the patch clamp method, and the amount of electricity passing through the TRPC6 channel is calculated by time integration, and compared between the TRPC6 mutant and the wild-type TRPC6. When calculating the amount of electricity per unit cell membrane, the cell membrane capacitance is determined from the capacitive current immediately after the cell membrane is broken, and the amount of electricity is divided by the cell membrane capacitance. Mammalian cells forcibly expressing TRPC6 mutants and wild-type TRPC6 are cells in which the activation and subsequent inactivation of TRPC6 can be evaluated. As described above, TRPC6 is activated by agonist stimulation of GPCRs such as α1-adrenergic receptors, muscarinic acetylcholine receptors, and AT1R. In addition, the CDI of TRPC6 occurs when calmodulin bound to Ca ²⁺ interacts with TRPC6. Therefore, it is preferable to forcibly express TRPC6 mutants and wild-type TRPC6 in mammalian cells (preferably human cells, more preferably human kidney cells or cell lines derived from human kidney cells) expressing GPCRs such as α1-adrenergic receptors, muscarinic acetylcholine receptors, AT1R, and calmodulin, and measure the time-dependent change in the current generated between the inside and outside of the cell when stimulated with an agonist of the GPCR. For example, TRPC6 mutants and wild-type TRPC6 are forcibly expressed in cell lines (e.g., HEK293 cells) derived from human kidney cells expressing muscarinic acetylcholine receptors (e.g., M1R) and calmodulin, and measure the time-dependent change in the current generated between the inside and outside of the cell when stimulated with an agonist (e.g., carbachol) for the muscarinic acetylcholine receptor, and calculate the amount of electricity passing through the TRPC6 channel by time integration.

When wild-type TRPC6 is expressed, Ca ²⁺ flows into the cell from outside the cell through TRPC6 activated by stimulation such as GPCR agonist, and the intracellular Ca ²⁺ concentration rises, and TRPC6 is immediately inactivated by CDI, the channel is closed, and the intracellular Ca ²⁺ concentration drops rapidly. Therefore, the amount of electricity passing through the TRPC6 channel after stimulation such as GPCR agonist (preferably the amount of electricity per unit cell membrane) is relatively small compared to the TRPC6 mutant. In the TRPC6 mutant, the factor that the amount of electricity passing through the TRPC6 channel at the time of activation is increased more than that of the wild-type TRPC6 is not particularly limited. In one embodiment, the factor is due to the delayed inactivation of the TRPC6 channel. When a TRPC6 mutant that causes delayed inactivation is expressed, after the intracellular cation concentration is increased by stimulation such as a GPCR agonist, the TRPC6 channel does not close or takes a longer time to close, and as a result, the intracellular cation concentration remains high for a longer time compared to wild-type TRPC6, and the current generated between the inside and outside of the cell remains high, and as a result, the amount of electricity passing through the TRPC6 channel (preferably, the amount of electricity per unit cell membrane) becomes relatively larger compared to wild-type TRPC6. In another embodiment, the reason why the amount of electricity passing through the TRPC6 channel during activation in the TRPC6 mutant is greater than that of the wild-type TRPC6 is due to an increase in peak current. When a TRPC6 mutant that causes a higher peak current compared to wild-type TRPC6 is expressed, even if the speed of inactivation of the TRPC6 channel is equivalent to that of the wild-type TRPC6, the amount of electricity passing through the TRPC6 channel (preferably, the amount of electricity per unit cell membrane) becomes relatively larger compared to wild-type TRPC6.

Next, the degree of increase in the amount of electricity passing through the TRPC6 channel upon activation of the TRPC6 mutant is correlated with the onset of FSGS. As shown in the Examples below, in the case of a TRPC6 mutant that causes a significant increase in the amount of electricity (preferably the amount of electricity per unit cell membrane) passing through the TRPC6 channel after activation by stimulation such as a GPCR agonist compared to wild-type TRPC6 (e.g., a TRPC6 mutant with a large degree of delay in inactivation, a TRPC6 mutant with a peak current significantly higher than the wild type), the onset of FSGS is relatively early and the possibility of juvenile FSGS is high, whereas in the case of a TRPC6 mutant that causes a slight increase in the amount of electricity (preferably the amount of electricity per unit cell membrane) passing through the TRPC6 channel after activation by stimulation with a GPCR agonist compared to wild-type TRPC6 (e.g., a TRPC6 mutant with a small degree of delay in inactivation, a TRPC6 mutant with a peak current slightly higher than the wild type), the onset of FSGS is relatively late and the possibility of adult FSGS is high. That is, the onset of FSGS can be estimated based on the negative correlation between the degree of increase in the amount of electricity (preferably the amount of electricity per unit cell membrane) passing through the TRPC6 channel upon activation and the period until the onset of FSGS. Furthermore, if the amount of electricity (preferably the amount of electricity per unit cell membrane) passing through the TRPC6 channel upon activation of a TRPC6 mutant is equivalent to that of wild-type TRPC6, it can be estimated that the likelihood of developing FSGS is low.

In general, the main treatment for FSGS is corticosteroid therapy, and in cases of resistance to steroids, immunosuppressants (such as cyclosporine) are used. However, in cases of juvenile FSGS, renal failure often occurs early, and in such cases, renal replacement therapy (dialysis, kidney transplantation) is used. Therefore, if the degree of increase in the amount of electricity passing through the TRPC6 channel when the TRPC6 mutant is activated in a subject is large, the onset of FSGS is relatively early, and it is determined that there is a high possibility of developing FSGS at a young age, the treatment plan can be adjusted to start preparation for renal replacement therapy early. On the other hand, if the degree of increase in the amount of electricity passing through the TRPC6 channel when the TRPC6 mutant is activated in a subject is small, the onset of FSGS is relatively late, and it is determined that there is a high possibility of developing FSGS after reaching adulthood, since the time to onset of FSGS and the subsequent course are relatively slow, preventive measures such as improving lifestyle habits and checking the condition through frequent medical examinations may be taken.

All references cited herein, including publications, patent documents, and the like, are hereby incorporated by reference to the same extent as if each was individually and specifically incorporated by reference and specifically set forth in its entirety.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited thereto.

1. Methods (TRPC6 mutants)
The following TRPC6 mutants identified in FSGS patients were expressed in HEK293 cells, and the TRPC6 channel current after receptor stimulation was measured using the whole-cell voltage-clamp method, and ^{Ca2+ -dependent} inactivation (CDI) was analyzed.

(Molecular Biology)
All TRPC6 channels were constructed from human TRPC6 (GenBank accession no. NM_004621) in the pIRESn vector, which was modified from pIRES2 (Invitrogen) by deleting the EGFP coding region from the IRES. Mutations in TRPC6 were generated by overlap extension PCR using mutagenic primers.

(Cell Culture)
HEK293 cells were obtained from ATCC and maintained in DMEM (Gibco) supplemented with 10% FBS and penicillin/streptomycin (Invitrogen) at 37°C and 5% _CO2 . Transfection of TRPC6 expression vectors for electrophysiological analysis was performed with SuperFect (Qiagen) according to the manufacturer's recommendations. Transfected HEK293 cells were used within 48 hours.

(Electrophysiological analysis)
HEK293 cells expressing TRPC6 mutants were subjected to whole-cell voltage clamp. Current signals were recorded by a low-noise patch clamp amplifier (AxoPatch 200B; Axon Instruments). Signals were filtered at 2 kHz and digitized at 1 kHz. The internal solution contained 120 mM CsOH, 120 mM aspartate, 20 mM CsCl, 2 mM MgCl2 _, 1 mM EGTA, 0.3 mM CaCl2 _, 2 mM ATP-Na2 _, 0.1 mM GTP, 10 mM HEPES, and 10 mM glucose, with a pH of 7.2 (adjusted with Tris base) and approximately 290 mOSm. Fire-polished patch pipettes had a resistance of 5-8 MΩ when backfilled with the internal solution. The external solution contained 140 mM NaCl, 5 mM KCl, 1.8 mM _CaCl2 , 1.2 mM _MgCl2 , 10 mM HEPES, and 10 mM glucose, with a pH of 7.4 and 300 mOsm (adjusted with glucose). To examine TRPC6 channel currents, NaCl in the external solution was replaced with the same concentration of N-methyl-D-glucose (NMDG). Currents were generated by applying 100 μM carbamylcholine chloride (carbachol, CCh; Sigma). Currents were recorded at a holding potential of -50 mV. During the experiment, HEK293 cells were continuously perfused with gravity-fed external solution at a flow rate of 0.5 ml/min. Current density (= current value/cell membrane capacitance) was calculated as the amount of ionic current per unit of cell membrane. Cell membrane capacitance was calculated from the capacitive current immediately after cell membrane rupture.

2. Results Wild-type TRPC6 channels were activated by carbachol stimulation, producing a current signal due to Ca ²⁺ influx, and then immediately inactivated (Ca ²⁺ -dependent inactivation: CDI) (Fig. 2). All of the analyzed TRPC6 mutants showed a higher amount of electricity passing through the TRPC6 channel, especially the amount of electricity per unit cell membrane (total current density), for 60 seconds after carbachol stimulation compared to wild-type TRPC6 (Fig. 5). TRPC6 (R175W), which has a mutation identified from a patient with a young FSGS onset, was inactivated more slowly than TRPC6 (R175Q), which has a mutation identified from a patient with an old FSGS onset (Figs. 2 and 3), and the amount of electricity passing through the TRPC6 channel, especially the amount of electricity per unit cell membrane, for 60 seconds after activation was higher (Figs. 2 and 5). A similar tendency was confirmed when comparing R895L, which has a young onset age, with R895C, which has an old onset age (Fig. 5). The H218L mutation, which is associated with a younger age at onset of FSGS, showed only a slight delay in inactivation (Fig. 4), but the peak current was increased and the amount of electricity passing through the TRPC6 channel upon activation, especially the amount of electricity per unit cell membrane (total current density), was significantly increased (Fig. 5).The R895C mutation, which is associated with a older age at onset of FSGS, showed a severe delay in inactivation, but only a slight increase in the amount of electricity passing through the TRPC6 channel upon activation, especially the amount of electricity per unit cell membrane (total current density) (Fig. 5). These results suggest that subjects with TRPC6 mutations that result in a significant increase in the amount of electricity passing through the TRPC6 channel after TRPC6 activation, particularly the amount of electricity per unit cell membrane (total current density), compared to wild-type TRPC6, may have a shorter period until they develop postnatal FSGS and may be of the young-onset type, whereas subjects with TRPC6 mutations that result in a slight increase in the amount of electricity passing through the TRPC6 channel after TRPC6 activation, particularly the amount of electricity per unit cell membrane (total current density), may be of the adult-onset type and may be of the longest period until they develop postnatal FSGS.

This invention makes it possible to predict the age of onset and progression of FSGS caused by TRPC6 mutations, and is expected to provide useful insight into the treatment of FSGS patients.

Claims (7)

A method for evaluating the function of a TRPC6 mutant to predict the onset time of focal segmental glomerulosclerosis caused by a TRPC6 mutation, comprising the steps of:
(1) Evaluating in vitro the degree of increase in the time-integrated value of the current generated by the influx of cations into the cell via TRPC6 per unit cell membrane (hereinafter referred to as "total current density") for TRPC6 mutants in subjects with TRPC6 mutations over a certain period of time after activation of the TRPC6 channel, and (2) comparing the degree of increase in total current density obtained in the evaluation in step (1) with the criterion that the greater the degree of increase in total current density, the earlier the onset of focal segmental glomerulosclerosis. The method of claim 1 , wherein the increase in total current density is due to delayed inactivation of TRPC6 channels. The method of claim 1 , wherein the increase in total current density is due to an increase in the peak current of a TRPC6 channel. The method of claim 1, wherein the comparison in step (2) is performed when the speed of inactivation of the TRPC6 channel of the TRPC6 mutant is equivalent to that of wild-type TRPC6. The method according to any one of claims 1 to 4, wherein the TRPC6 mutation is a missense mutation in the intracellular domain of TRPC6. The method according to claim 5, wherein the intracellular domain is an ankyrin repeat domain or a coiled-coil domain. The method according to any one of claims 1 to 6, wherein the TRPC6 mutation is an amino acid substitution at glycine 109, arginine 175, histidine 218, or arginine 895.