Medicine for treating renal fibrosis and application thereof

Abstract

The invention discloses a medicine for treating renal fibrosis and application thereof, and the medicine for treating renal fibrosis comprises an inhibitor capable of inhibiting epithelial-mesenchymal transition of renal tubular epithelial cells HK-2 stimulated by TGF-beta1; the invention proves that the Kisspeptin analogue TAK-448 obviously improves the epithelial-mesenchymal transition of renal tubular epithelial cells HK-2 stimulated by TGF-beta1, and improves the renal fibrosis of UUO and FA mice, so that the Kisspeptin analogue TAK-448 has wide clinical application prospects, has huge potential in preparation of medicines for treating and / or improving renal fibrosis diseases, and can be used for preparing medicines for treating and / or improving renal fibrosis diseases. The industrial utilization value is high.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a medicine for treating renal fibrosis and an application thereof. Background Art

[0002] Chronic kidney disease is a disease characterized by kidney damage or persistent decline in kidney filtration function caused by a variety of causes, affecting 8%-16% of the world's population. Renal fibrosis is considered to be the final common pathway and main pathological basis for the progression of almost all types of chronic kidney disease to end-stage renal disease, and its occurrence and development stem from multiple pathological processes. Since renal fibrosis is a complex process involving multiple factors and pathways, there is a lack of specific and effective treatments in clinical practice. Therefore, it is of great clinical significance to continue to explore and develop effective drugs for the treatment of renal fibrosis. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention provides a drug for treating renal fibrosis and its application.

[0004] A drug for treating renal fibrosis, comprising an inhibitor capable of inhibiting epithelial-mesenchymal transition of renal tubular epithelial cells HK-2 stimulated by TGF-β1.

[0005] Preferably, the inhibitor comprises the kisspeptins analog TAK-448.

[0006] Preferably, the sequence of TAK-448 is WYPDTFGLQ.

[0007] Preferably, the chemical structural formula of TAK-448 is

[0008]

[0009] The application of the above inhibitor in the preparation of a drug for treating TGF-β1-induced epithelial-mesenchymal transition of renal tubular epithelial cells.

[0010] The above inhibitor is used in the preparation of medicine for treating kidney pathological damage and renal interstitial collagen fiber deposition caused by ureteral obstruction.

[0011] The above inhibitor is used in the preparation of medicine for treating renal pathological damage and renal interstitial collagen fiber deposition caused by folic acid injection.

[0012] The use of the above inhibitor in the preparation of drugs for treating renal fibrosis caused by ureteral obstruction.

[0013] The use of the above inhibitor in the preparation of drugs for treating renal fibrosis caused by folic acid injection.

[0014] The present invention demonstrates that kisspeptin analogue TAK-448 significantly improves epithelial-mesenchymal transition of renal tubular epithelial cells HK-2 stimulated by TGF-β1, and significantly improves renal fibrosis in UUO and FA mice, so that the present invention has broad clinical application prospects, and has great potential in the preparation of drugs for treating and / or improving renal fibrosis diseases, and has high industrial utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 A is the immunoblotting results of KISS1 and epithelial-mesenchymal transition marker proteins (Fibronectin, α-SMA) in HK-2 cells stimulated with different concentrations of TGF-β1 (0, 2.5, 5, 10 and 20 ng / μL) for 48 h; B is the immunoblotting results of KISS1 and epithelial-mesenchymal transition marker proteins (Fibronectin, Collagen-Ι) in kidney tissue of UUO mice; C is the statistical analysis of the KISS1 protein expression results in Figure B, n=4, data are expressed as Mean±SDs, analyzed by Student's t-tests, ***: P<0.001. TGF-β1, transforming growth factor β1; UUO, unilateral ureteral obstruction; α-SMA, smooth muscle actin.

[0017] Figure 2 Immunoblotting was used to detect the protein expression of epithelial-mesenchymal transition-related indicators (Fibronectin, E-cadherin, α-SMA) in HK-2 cells treated with different concentrations of KPA (0, 5, 10 and 20 nM) for 48 h. TGF-β1, transforming growth factor β-1; α-SMA, α-smooth muscle actin; KPA, kisspeptin analog (KP analog, KPA) TAK-448.

[0018] Figure 3A in the middle is the picture of the left kidney tissue taken after euthanasia on the 7th day after UUO modeling; B is the picture of blood taken from the orbital vein of mice to measure the blood Scr level; C is the picture of blood taken from the orbital vein of mice to measure the blood BUN level, n=3, data are expressed as Mean±SDs, and one-way ANOVA analysis was used for inter-group comparison, *: P<0.05, **: P<0.01, ***: P<0.001, ns: not significant. UUO, unilateral ureteral obstruction; Scr, creatinine; BUN, urea nitrogen; KPA, kisspeptin analog (KP analog, KPA) TAK-448.

[0019] Figure 4 A is a representative image of Masson staining, scale bar = 100μm or 50μm; B is a quantitative analysis of the collagen area fraction in A, n = 4, data are expressed as Mean±SDs, and one-way ANOVA analysis was used for inter-group comparison, ****: P<0.0001, ns: not significant; C is a representative image of HE staining showing the pathological changes in the kidney tissue of mice in each group, scale bar = 100μm or 50μm; D is a quantitative analysis of the tubular injury score based on HE staining in C, n = 4, data are expressed as Mean±SDs, and one-way ANOVA analysis was used for inter-group comparison, **: P<0.01, ****: P<0.0001, ns: not significant. UUO, unilateral ureteral obstruction; Masson, Masson staining; HE, hematoxylin-eosin; KPA, kisspeptin analog (KP analog, KPA) TAK-448.

[0020] Figure 5Figure A is a representative image of immunohistochemical staining of Collagen-Ⅰ, Fibronectin and α-SMA in the tubulointerstitium of mice in each group, scale bar = 100 μm; Figure BD is a quantitative analysis of the protein levels of Collagen-Ⅰ, Fibronectin and α-SMA in the tubulointerstitium based on Figure A, n = 3, data are expressed as Mean ± SDs, and one-way ANOVA was used for comparison between groups, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant; Figure E is a graph of the levels of fibrotic proteins (Fibronectin, Vimentin and α-SMA) in the renal tissues of mice in each group detected by immunoblotting experiments; Figure FH is a statistical analysis of the results of Figure E, n = 4, data are expressed as Mean ± SDs, and one-way ANOVA was used for comparison between groups. ANOVA analysis, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant; IK is the expression level of Fn1, Vim, and Acta2 mRNA in the kidney tissue of each group of mice detected by RT-qPCR, and the statistics are the results of 4 technical repetitions. The data are expressed as Mean ± SDs. One-way ANOVA analysis was used for inter-group comparison, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. UUO, unilateral ureteral obstruction; α-SMA, α-smooth muscle actin; RT-qPCR, reverse transcription real-time quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KPA, kisspeptin analog (KPanalog, KPA) TAK-448.

[0021] Figure 6 A is the weight growth curve of mice obtained by weighing mice regularly after FA modeling, n=3, data are expressed as Mean±SDs, and one-way ANOVA analysis was used for inter-group comparison, *: P<0.05, ns: not significant; B is the picture of kidneys of mice taken after euthanasia 28 days after modeling; C is the picture of blood Scr measured by orbital venous blood of mice, D is the picture of blood BUN measured by orbital venous blood of mice, n=3, data are expressed as Mean±SDs, and one-way ANOVA analysis was used for inter-group comparison, *: P<0.05, **: P<0.01, ***: P<0.001, ns: not significant. FA, folic acid; BUN, blood urea nitrogen; Scr, serum creatinine; KPA, kisspeptin analog (KP analog, KPA) TAK-448.

[0022] Figure 7 A is a representative image of Masson staining, scale bar = 100 μm or 50 μm; B is a quantitative analysis of the collagen area fraction in Figure A, n = 3, data are expressed as Mean ± SDs, and one-way ANOVA was used for comparison between groups, *: P < 0.05, **: P < 0.01, ****: P < 0.0001, ns: not significant; C is a representative image of HE staining showing renal tissue pathological damage in each group, scale bar = 100 μm or 50 μm; D is a statistical analysis of the renal tubular pathological damage score based on Figure C, n = 3, data are expressed as Mean ± SDs, and one-way ANOVA was used for comparison between groups, *: P < 0.05, **: P < 0.01, ****: P < 0.0001, ns: not significant. FA, folic acid; Masson, Masson staining; HE, hematoxylin-eosin; KPA, kisspeptin analog (KP analog, KPA) TAK-448.

[0023] Figure 8Figure A is a representative image of immunohistochemical staining of Fibronectin, α-SMA and Vimentin in the tubulointerstitium of mice in each group, scale bar = 100 μm; Figure BD is a quantitative analysis of the protein levels of Fibronectin, α-SMA and Vimentin in the tubulointerstitium based on Figure A, n = 3, data are expressed as Mean ± SDs, and one-way ANOVA was used for comparison among the groups, *: P < 0.05, ***: P < 0.001, ****: P < 0.0001, ns: not significant; E is the level of fibrosis proteins (Fibronectin, E-cadherin, Vimentin, α-SMA) in the renal tissue of each group of mice detected by immunoblotting experiment; FI is the statistical analysis of the results of Figure E, n=3, data are expressed as Mean±SDs, and one-way ANOVA analysis was used for inter-group comparison, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, ns: not significant; JM is the expression level of Fn1, Col1a1, Vim and Acta2 mRNA in the kidney of FA mice detected by RT-qPCR, n=3, data are expressed as Mean±SD, and one-way ANOVA analysis was used for inter-group comparison, ****: P<0.0001, ns: not significant. FA, folic acid; α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KPA, kisspeptin analog (KP analog, KPA) TAK-448. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The modification process of TAK-448 is as follows: ① amidation reaction occurs on L-tryptophan for modification, ② acetylation modification occurs on the amino group of tyrosine, ③ hydroxylation modification of the 4R configuration of proline, ④ 1-azaglycosyl modification occurs on glycine, which usually refers to replacing a carbon atom with a nitrogen atom in the glycosyl structure and acylation reaction occurs, ⑤ imino (methylamino) methyl modification occurs on the N5 position of ornithine, and the remaining amino acids are not specially modified.

[0026] The sequence of TAK-448 is WYPDTFGLQ.

[0027] Example 1 KISS1 is involved in renal fibrosis

[0028] This example verifies that KISS1 is highly expressed in renal tubular epithelial cells with epithelial-mesenchymal transition and fibrotic renal tissue, among which KISS1 expression is upregulated in human proximal tubular epithelial cells (HK-2) stimulated by TGF-β1 and in the UUO renal fibrosis mouse model.

[0029] In this example, HK-2 cells were stimulated with different concentrations of TGF-β1 (0, 2.5, 5, 10 and 20 ng / μL) to establish an epithelial-mesenchymal transition cell model. The protein expression of epithelial-mesenchymal transition-related indicators (Fibronectin, Collagen-Ⅰ) and KISS1 before and after stimulation was detected by immunoblotting. The experimental results are shown in Figure 2. Figure 1 As shown in A. The results showed that Fibronectin and Collagen-Ⅰ were upregulated in HK-2 cells stimulated by TGF-β1, indicating that the cell model of epithelial-mesenchymal transition was successfully constructed. At the same time, with the increase of TGF-β1 concentration, the protein expression of KISS1 also gradually increased, indicating that KISS1 protein may be involved in the epithelial-mesenchymal transition process of HK-2 cells.

[0030] In this example, a mouse model of renal fibrosis was further constructed by unilateral ureteral obstruction surgery (UUO: Unilateral Uretera Obstruction, renal interstitial fibrosis model). On the 7th day after the surgery, the protein expressions of fibrosis-related markers (Fibronectin, α-SMA) and KISS1 in the experimental group (UUO) and the control group (Sham) were detected by immunoblotting. The experimental results are shown in Figure 2. Figure 1 BC. The results showed that Fibronectin and α-SMA were upregulated in the UUO animal model, indicating that the fibrosis animal model was successfully constructed. At the same time, the protein expression of KISS1 was also upregulated in UUO mice, suggesting that KISS1 protein may be involved in the renal fibrosis of UUO mice.

[0031] These experimental results confirmed the increased expression of KISS1 protein in fibrotic renal tissue both in vitro and in vivo, suggesting that KISS1 is involved in renal fibrosis.

[0032] Example 2 Kisspeptin analog (KP analog, KPA) TAK-448 significantly improves epithelial-mesenchymal transition of renal tubular epithelial cells stimulated by TGF-β1

[0033] Example 1 has proved that KISS1 is involved in renal fibrosis at both the cellular and animal levels. This example further explores the mitigating effect of KPA on epithelial-mesenchymal transition at the cellular level. HK-2 cells stimulated with TGF-β1 were treated with different concentrations of KPA (0, 5, 10 and 20 nM) for 48 h. The protein expression of epithelial-mesenchymal transition-related indicators (Fibronectin, E-cadherin, α-SMA) in HK-2 cells after KPA treatment was detected by immunoblotting. The results showed that ( Figure 2 ), after TGF-β1 stimulation, the expression of Fibronectin, E-cadherin, and α-SMA proteins in HK-2 cells increased significantly, indicating that TGF-β1 stimulated HK-2 cells to successfully construct a fibrotic cell model. After administration of different concentrations of KPA, the expression of epithelial-mesenchymal transition-related indicator proteins decreased significantly and showed a concentration-dependent manner, indicating that KPA can significantly inhibit TGF-β1-induced epithelial-mesenchymal transition of renal tubular epithelial cells HK-2.

[0034] Example 3 Kisspeptin analog (KP analog, KPA) TAK-448 improves renal morphology and renal function in UUO mice

[0035] This example further explores the effect of KPA on renal fibrosis. Example 3 verifies the effect of KPA on renal morphology and renal function in a fibrotic UUO mouse model.

[0036] In the UUO mouse model experiment of this embodiment, the mice were randomly divided into 7 groups, with 4 mice in each group, namely sham operation group (sham), Sham + KPA group (Sham-KPA, KPA dosage was 0.6 mg / (kg·day)), model group (UUO), UUO + KPA low-dose group (UUO-KPA, KPA dosage was 0.15 mg / (kg·day)), UUO + KPA medium-dose group (UUO-KPA, KPA dosage was 0.3 mg / (kg·day)), UUO + KPA high-dose group (UUO-KPA, KPA dosage was 0.6 mg / (kg·day)) and positive control group, UUO + enalapril (UUO-Enalapril, Enalapril dosage was 10 mg / (kg·day)). KPA was dissolved in saline and administered by subcutaneous injection, and enalapril was dissolved in sodium carboxymethylcellulose (CMC-Na) and administered by gavage. Administration began 24 hours after UUO surgery and was once a day until 7 days after surgery. Mouse kidney tissue was collected 7 days later. After administration, the mice were anesthetized, and blood was collected from the eye sockets for the detection of blood creatinine (Scr) and urea nitrogen (BUN). At the same time, the left (blocked) kidney was removed and photographed.

[0037] Mouse kidney tissue morphology ( Figure 3 A) showed that compared with the Sham group, the UUO group had ureteral dilatation and severe renal tissue damage, and urine squeezed the renal tissue until only a thin layer of cortex remained, indicating that the model was successful; compared with the UUO group, the degree of ureteral dilatation and renal tissue squeezing in the UUO+different concentrations of KPA group were less than those in the corresponding UUO group, indicating that KPA can reduce renal tissue damage after UUO surgery and play a protective role in the kidney. In addition, Scr( Figure 3 B) and BUN( Figure 3 C) The test results showed that compared with the Sham group, the Scr level in the UUO group was significantly increased, indicating that UUO modeling damaged the function of the kidney. After administration of KPA 0.6 mg / (kg·day), the Scr and BUN levels were significantly reduced, indicating that KPA has a protective effect on renal function.

[0038] Example 4 Kisspeptin analog (KP analog, KPA) TAK-448 significantly improves renal interstitial collagen fiber deposition and renal pathological damage in UUO mice

[0039] In this example, in the UUO mouse model constructed above, the effects of KPA on renal interstitial collagen fiber deposition and renal pathological damage were further detected by Masson staining and HE staining. The results of Masson staining showed that there was a large amount of collagen fiber deposition in the renal interstitium of UUO mice, and the collagen fiber deposition was significantly improved after administration of different concentrations of KPA, indicating that KPA can effectively alleviate collagen deposition in the renal tissue of UUO mice ( Figure 4 AB), and has a certain concentration dependence. The degree of tubular interstitial damage in HE staining was judged by inflammation score, tubular dilatation score, and tubular cast score. The scoring criteria are: 0 points, normal kidney (no damage); 1 point, mild damage (≤10%); 2 points, moderate damage (11%-25%); 3 points, severe damage (26%-45%); 4 points, highly severe damage (46%-75%); 5 points, large area damage (>75%). The results showed that KPA effectively reduced tubular epithelial cell atrophy, brush border loss, tubular dilatation and tubular collapse in the UUO mouse model in a dose-dependent manner ( Figure 4 CD). The above results suggest that KPA can significantly improve renal pathological damage in UUO mice.

[0040] Example 5 Kisspeptin analog (KP analog, KPA) TAK-448 improves renal fibrosis in UUO mice

[0041] This example further detected the improvement effect of KPA on renal fibrosis in UUO mice. In the UUO mouse model constructed above, this example first detected the effect of KPA on the expression of fibrosis marker proteins (Collagen-Ⅰ, Fibronectin and α-SMA) by immunohistochemistry, and then detected the effect of different concentrations of KPA on the expression of fibrosis marker proteins (Fibronectin, Vimentin and α-SMA) by immunoblotting experiments, and detected the changes in fibrosis markers (Fn1, Vim, Acta2) mRNA by RT-qPCR experiments. The results showed that the expression of fibrosis marker proteins in the kidney tissue of UUO mice ( Figure 5 AH) and mRNA levels ( Figure 5 After administration of different concentrations of KPA, these indicators were significantly reduced, indicating that KPA can effectively alleviate renal fibrosis in UUO mice.

[0042] Example 6 Kisspeptin analog (KP analog, KPA) TAK-448 significantly improves renal morphology and renal function in FA mice

[0043] In the FA mouse model experiment of this embodiment, the mice were randomly divided into 4 groups, 3 mice in each group, Saline group, Saline+KPA group (Saline-KPA, KPA dosage was 0.6mg / (kg·day)), model group (FA FA: 250mg / kg), FA+KPA group (FA-KPA FA: 250mg / kg, KPA dosage was 0.6mg / (kg·day)). FA was dissolved in 300mM sodium bicarbonate and administered by intraperitoneal injection. KPA was dissolved in physiological saline and administered by subcutaneous injection. KPA administration started 18 days after FA model establishment and was administered once a day for a total of 10 days. After the administration, the mice were anesthetized, and blood was collected from the eye sockets for the detection of blood creatinine (Scr) and urea nitrogen (BUN). At the same time, the left and right kidneys were taken, weighed, and photographed.

[0044] Mouse weight growth curve results ( Figure 6 A) shows that after FA modeling, the weight of mice decreased significantly, and the weight loss was improved after administration of KPA. Figure 6 B) showed that compared with the Saline group, the kidneys in the FA group were atrophied and scarred on the kidney surface, indicating that the model was successful; compared with the FA group, the kidney size, weight and kidney surface smoothness of the FA+KPA group were better than those of the FA group, and were close to the morphology of the kidneys in the Saline group, indicating that KPA can reduce kidney tissue damage after FA modeling and play a protective role in the kidneys. In addition, Scr( Figure 6 C) and BUN( Figure 6 D) The test results showed that compared with the Saline group, the Scr and BUN levels in the FA group were significantly increased, indicating that FA modeling damaged the function of the kidneys, while after KPA administration, Scr and BUN decreased significantly, indicating that KPA has a protective effect on renal function. The above experimental results confirm that KPA can significantly improve the morphological changes and renal function of FA mice.

[0045] Example 7 Kisspeptin analog (KP analog, KPA) TAK-448 improves renal interstitial collagen fiber deposition and renal pathological damage in FA mice

[0046] In this example, in the FA mouse model constructed above, the effects of KPA on renal interstitial collagen fiber deposition and renal pathological damage were further detected by Masson staining and HE staining. Figure 7 AB) and HE staining results (Figure CD) showed that KPA significantly improved collagen deposition and tubulointerstitial damage caused by high-dose FA. The above results are consistent with the results of the UUO mouse model, indicating that KPA can significantly improve the deposition of renal interstitial collagen fibers and renal pathological damage caused by FA injury.

[0047] Example 8 Kisspeptin analog (KP analog, KPA) TAK-448 significantly improves renal fibrosis in FA mice

[0048] This example further detected the improvement effect of KPA on renal fibrosis in FA mice. In the FA mouse model constructed above, this example detected the effect of KPA on the expression of fibrosis marker proteins (Fibronectin, α-SMA and Vimentin) by immunohistochemistry, detected the effect of KPA on the expression of fibrosis marker proteins (Fibronectin, E-cadherin, Vimentin, α-SMA) by immunoblotting, and detected the changes in fibrosis markers (Fn1, Collal, Vim, Acta2) mRNA by RT-qPCR. The results showed that the expression of fibrosis marker proteins in the kidney tissue of FA mice ( Figure 8 AI) and mRNA( Figure 8 JM) levels were significantly increased, while these indicators were significantly downregulated after KPA treatment, indicating that KPA significantly reduced the expression of fibrosis markers and alleviated renal fibrosis in FA mice.

[0049] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0050] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A drug for treating renal fibrosis, characterized in that: The invention includes an inhibitor capable of inhibiting epithelial-mesenchymal transition of HK-2 renal tubular epithelial cells stimulated by TGF-β1.

2. The drug for treating renal fibrosis according to claim 1, characterized in that: The inhibitors include the kisspeptins analog TAK-448.

3. The drug for treating renal fibrosis according to claim 2, characterized in that: The sequence of TAK-448 is WYPDTFGLQ.

4. The drug for treating renal fibrosis according to claim 2, characterized in that: The chemical structural formula of TAK-448 is 5. Use of the inhibitor according to any one of claims 1 to 2 in the preparation of a drug for treating TGF-β1-induced epithelial-mesenchymal transition of renal tubular epithelial cells.

6. Use of the inhibitor according to any one of claims 1 to 2 in the preparation of a drug for treating renal pathological damage and renal interstitial collagen fiber deposition caused by ureteral obstruction.

7. Use of the inhibitor according to any one of claims 1 to 2 in the preparation of a drug for treating renal pathological damage and renal interstitial collagen fiber deposition caused by folic acid injection.

8. Use of the inhibitor according to any one of claims 1 to 2 in the preparation of a drug for treating renal fibrosis caused by ureteral obstruction.

9. Use of the inhibitor according to any one of claims 1 to 2 in the preparation of a drug for treating renal fibrosis caused by folic acid injection.

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