Frozen Block Tissue Staining for Eye Structure of Zebrafish Embryo.

Histology is a technique used to study the morphology of cell structures by cutting samples into thin sections. Histological cross-section and staining are the techniques needed to visualize the morphology of cell tissues. A suitable tissue staining experiment was created to observe changes in the retinal layer in zebrafish embryos. Zebrafish have a human-like visual system, retina, and eye structures. Due to the small size of zebrafish and undeveloped bones in the embryonic stage, the resistance through cross-section is inevitably small. Here, we present optimized protocol changes in eye tissue of zebrafish using frozen blocks.

Clinical Usefulness of BACT Count and BACT-Info Flag of UF-5000 for Screening for Urinary Tract Infection and Prediction of Gram-Negative Bacteria.

BACKGROUND: A rapid and reliable screening test for urinary tract infection (UTI) is needed to reduce the turn-around time and to rule out negative results of urine culture. The aim of this study was to evaluate the performance of BACT count and BACT-Info flag of the UF-5000 for screening for UTI. METHODS: A total of 1,063 urine specimens from April to September 2019 were included in this study. We evaluated the diagnostic performance of white blood cell (WBC) count, BACT count, BACT-Info flag, and UTI flag in UF-5000 by comparing with the urine culture results. RESULTS: Of the urine specimens, 16.7% were culture-positive (≥ 105 CFU/mL) with 15 being yeast positive. A BACT count of > 685.3/µL showed the best diagnostic performance with 93.8% sensitivity and 90.2% specificity. We confirmed that the combination of BACT count (685.3/µL) and BACT-Info flag would be appropriate to use in a clinical laboratory (sensitivity 91.5%, specificity 90.5%). Based on this combination, the sensitivity and specificity of the Gram-negative flag were 95.5% and 94.8%. CONCLUSIONS: We recommend the use of a combination of BACT count (685.3/µL) and BACT-Info for UTI diagnosis. This combination is more appropriate for Gram-negative bacteria, and it would be useful for selecting empirical treatment.

HL3501, a Novel Selective A3 Adenosine Receptor Antagonist, Lowers Intraocular Pressure (IOP) in Animal Glaucoma Models.

PURPOSE: The A3 adenosine receptor (A3AR) is a known therapeutic target for glaucoma treatment. In this study, we developed HL3501 and examined its selectivity profile and in vitro and in vivo effects. METHODS: For the rabbit model, intraocular pressure (IOP) was increased by laser photocoagulation of the trabecular meshwork (TM). The rabbits were then topically treated with HL3501, latanoprost, timolol, or vehicle for 3 weeks. For the mouse model, HL3501, latanoprost, or vehicle was administered following induced IOP elevation by dexamethasone (Dex). The IOP of all rabbits and mice was measured. Electroretinography was performed on both eyes of dark-adapted anesthetized mice on days 0 and 21. The mice's eyes were enucleated at the end of the treatment for immunofluorescence staining. RESULTS: HL3501 was highly specific to the A3AR and inhibitory of A3AR function. In the rabbit glaucoma model, HL3501 and latanoprost significantly decreased the IOP. In the Dex-treated mouse model, HL3501 and latanoprost significantly decreased the IOP and increased the b-wave amplitude as compared with the vehicle treatment. HL3501 and latanoprost also inhibited fibronectin and α-smooth muscle actin expression induced by Dex treatment. CONCLUSIONS: HL3501 had effects similar to those of latanoprost in reducing ocular hypertension in animal models. HL3501 could be used as a novel approach to treat glaucoma. TRANSLATIONAL RELEVANCE: HL3501 is a novel preclinical compound targeting the A3 adenosine receptor, which may also be a new treatment option to fill the unmet needs of many glaucoma patients.

Cevimeline-induced anti-inflammatory effect through upregulations of mucins in the ocular surface of a dry eye mouse model.

This study aimed to investigate the effects of various concentrations of cevimelines (CVMs) and compare them with commercial drugs in a murine model of dry eye. The experimental mouse model used male and female NOD.B10.H2b mice over 12 weeks of age. Desiccation stress was performed at 30-40% ambient humidity, and subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide was performed four times a day for 10 days. The efficacy of various concentrations of CVMs (seven experimental groups) was first evaluated, and then 2% CVM was compared with commercial drugs, such as cyclosporine A (CsA), diquafosol (DQS), and rebamipide (REB) (seven experimental groups). The clinical changes, including tear production, corneal irregularity, and fluorescein staining, were measured after the instillation of various concentrations of CVMs and commercial drugs for 0, 3, 5, 7, and 10 days. Histological changes, such as corneal detachment, conjunctival goblet cell and mucin density staining, were assessed by staining the cornea or conjunctiva with hematoxylin-eosin, periodic acid-Schiff, and alcian blue. The expression of inflammatory markers and mucin factors was detected by immunohistochemistry and immunofluorescence in the lacrimal gland, cornea, and conjunctiva. Tear production was significantly increased in the 2% CVM group and was similar to that in the DQS and REB groups (P < 0.05). The corneal smoothness and fluorescein staining score were significantly improved in the 2% CVM group and were similar to those in the REB group (P < 0.05). Corneal epithelial cells were significantly decreased in the 2% CVM group, with similar observations made in the DQS and REB groups (P < 0.05). The conjunctival goblet cells and mucin density recovered in the 2% CVM group were similar to those in the CsA and REB groups (P < 0.05). The 2% CVM group showed suppressed expression of inflammatory factors in the lacrimal gland and was comparable to that seen in the CsA and REB groups. The expression of mucin factors was upregulated in the cornea and conjunctiva of the 2% CVM group and was similar to that of the CsA and REB groups. In conclusion, administration of CVM resulted in recovery or clinical and histological improvement of the murine dry eye model, and all the observed parameters were comparable to those with commercial drugs.

Development of a zebrafish screening model for diabetic retinopathy induced by hyperglycemia: Reproducibility verification in animal model.

This study aimed at creating a zebrafish screening model for diabetic retinopathy, and evaluated the effects of aflibercept, which is being used to treated diabetic retinopathy. A morphological change occurred at 160 mM of glucose. The survival and hatching rate decreased in a dose-dependent manner. In the 130 mM glucose group, the retinal vessel diameter was more than double that in the normal group. The zebrafish embryo morphology changed in 200 µg/mL and 400 µg/mL at aflibercept. The survival and hatching rate decrease at 400 µg/mL. Aflibercept 100 µg/mL was a nontoxic and effective dose for the zebrafish diabetic retinopathy model. The expression of diabetic retinopathy inflammatory markers was increased in hyperglycemia. But the inflammation was improved by aflibercept in the zebrafish eye. In a zebrafish diabetic retinopathy model, the diameters of retinal vessels were reduced after treatment with aflibercept, and molecular biological and histopathological efficacy was confirmed. This model can serve for screening of new drug candidates for treatment of in diabetic retinopathy.

Drug Delivery Strategies for Enhancing the Therapeutic Efficacy of Toxin-Derived Anti-Diabetic Peptides.

Toxin peptides derived from the skin secretions of amphibians possess unique hypoglycemic activities. Many of these peptides share cationic and amphipathic structural similarities and appear to possess cell-penetrating abilities. The mechanism of their insulinotropic action is yet not elucidated, but they have shown great potential in regulating the blood glucose levels in animal models. Therefore, they have emerged as potential drug candidates as therapeutics for type 2 diabetes. Despite their anti-diabetic activity, there remain pharmaceutical challenges to be addressed for their clinical applications. Here, we present an overview of recent studies related to the toxin-derived anti-diabetic peptides derived from the skin secretions of amphibians. In the latter part, we introduce the bottleneck challenges for their delivery in vivo and general drug delivery strategies that may be applicable to extend their blood circulation time. We focus our research on the strategies that have been successfully applied to improve the plasma half-life of exendin-4, a clinically available toxin-derived anti-diabetic peptide drug.

Preclinical Evaluation of UDCA-Containing Oral Formulation in Mice for the Treatment of Wet Age-Related Macular Degeneration.

As a posterior ocular disease, wet age-related macular degeneration (WAMD) has been known to be related to vision loss, accompanying ocular complications. The intravitreous injection of VEGF antibodies has been reported to be an effective treatment to relieve symptoms of WAMD. However, the limitations of this treatment are high costs and invasiveness. For this reason, oral delivery route can be considered as a cost-effective way and the safest method to deliver drug molecules to the eyes. Accordingly, ursodeoxycholic acid (UDCA) was included in the oral formulation as the potential substance for the cure of WAMD in the animal model. Various pharmacological activities, such as antioxidant or anti-inflammatory effects, have been reported for UDCA and recent reports support the effects of UDCA in ocular treatment. However, due to poor water solubility and low pKa (around 5.0), it has been challenging to formulate aqueous solution of UDCA in the neutral pH range. In the present study, we confirmed the aqueous solubility of the oral UDCA formulation and performed a preclinical study, including pharmacokinetic profiling and WAMD model efficacy study in mice after oral administration of the drug solution. The results demonstrated that the formulation improved bioavailability of UDCA and efficiently delivered UDCA to the eye tissues after oral absorption. UDCA formulation was found to have inhibitory effects of choroidal neovascularization with a functional recovery in mice retinas. Taken together, our results suggest that the oral UDCA formulation could be used as a potent supplement for the cure of WAMD and related retinal diseases.

Sulglycotide ameliorates inflammation in lipopolysaccharide-stimulated mouse macrophage cells by blocking the NF-κB signaling pathway.

Objective: Several studies demonstrated that sulglycotide has anti-inflammatory and anti-cancer effects. However, the effect of sulglycotide is limited to gastric mucosal tissues and cells and underlying molecular mechanisms are not clear. This study estimated the effect of sulglycotide on lipopolysaccharide (LPS)-induced inflammatory responses in the macrophage cell line, RAW 264.7 and elucidated the molecular mechanisms. Materials and methods: The inhibitory effect of sulglysotide on LPS-induced oxidative stress and inflammatory reactions were determined by Immunofluorescence staining, ELISA, Western blotting and RT-PCR. Results: Our results show that sulglycotide has the ability to inhibit inflammatory mediators and cytokine production as well as reactive oxygen species (ROS) generation. This effect can be the result from regulating the activation of nuclear factor-kappa B (NF-κB) through blocking mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Conclusions: These results indicate that sulglycotide could be an anti-inflammatory and anti-oxidative compound that may be a useful candidate for treatment of inflammatory diseases.

Potential Albumin-Based Antioxidant Nanoformulations for Ocular Protection against Oxidative Stress.

Amongst various drug administration methods, ophthalmic drug delivery has been a useful way for the treatment of eye-related diseases. However, therapeutic efficacy of ocular therapy for anterior or posterior eye segments through topical administration is considerably challenged by the number of anatomical and physiological barriers in the eyes affecting ocular bioavailability. In this respect, advanced biocompatible nanoformulations make it possible to improve drug delivery to the target sites and enhance ocular bioavailability of ophthalmic medicines. Various ocular diseases have been reported to be related to oxidative stresses in tissues, and polyphenolic compounds have been known for their antioxidant activities in various tissues, including the eyes. Despite drug efficacy, poor water solubility and intrinsic color of the compounds limit the drug's inclusion into the development of ocular medicine. In the present study, we investigated the antioxidant protectant efficacy of rosmarinic or ursolic acid in the retinal epithelial cells, as compared to those of curcumin, by forming nanospheres with bovine serum albumin. Our results demonstrate that antioxidant-containing nanoformulations provide a significantly higher drug solubility and decreased ROS (reactive oxygen species) production in the retinal epithelial cells. Finally, we also found that albumin-based nanoformulations could improve bioavailability and increase antioxidant activity of rosmarinic or ursolic acid in the retina to be applied as efficient ocular protectant.

Catechin solubilization by spontaneous hydrogen bonding with poly(ethylene glycol) for dry eye therapeutics.

Catechin exhibits various pharmacological effects, yet its poor aqueous solubility limits its clinical use. Here, we investigate a facile catechin solubilization method via spontaneous hydrogen bonding between catechin and poly(ethylene glycol) (PEG). The method is extremely simple in that mixing PEG with catechin followed by lyophilization completely converts insoluble catechin to soluble PEG/catechin nanoscale complexes. This solubilized catechin formulation is useful for preparing eyedrop medicine, and we demonstrate that the solubilized catechin exhibits therapeutic effect upon dry eye diseases.

Evaluation of epithelial transport and oxidative stress protection of nanoengineered curcumin derivative-cyclodextrin formulation for ocular delivery.

Ocular drug delivery has been a well-known route for the drug administration for the treatment of ocular diseases. However, numerous anatomical and physiological barriers prevailing in the eye itself create considerable challenges for achieving the necessitated therapeutic efficacy along with ocular bioavailability. However, recent advances in nanoengineered strategies hold definite promises in terms of devising improved ophthalmic medicines for the effective drug delivery to target the sites with enhanced ocular bioavailability. Curcumin, a hydrophobic polyphenol yellow colored compound, and its metabolic reduced product, tetrahydrocurcumin (THC), have been known for their beneficial pharmacological functions, such as anti-inflammatory or anti-oxidant activities at various tissue sites. However, the low aqueous solubility of these compounds results in their poor bioavailability, thereby limiting their widespread application. Therefore, in the present study, we investigated the changes in drug solubility by forming inclusion complexes with different derivatives of hydroxypropyl (HP)-cyclodextrins (CD). To this end, the spray drying technique was used for nanoengineering curcumin or THC-loaded formulations to improve the stability of formulations during the storage. The formulations were characterized in terms of physicochemical properties and cellular permeability. The results demonstrated that the encapsulation of curcumin (or THC) into the HP-CDs significantly increased the drug solubility and enhanced the corneal and retinal epithelial permeability. Curcumin or THC complexes in HP-CDs with improved bioavailability also induced anti-oxidant activity (SOD1, CAT1, and HMOX1) in higher levels in the ocular epithelial cells and showed oxidative protection effects in rabbit cornea tissues that will boost up their application in ocular medicine.

A New Ophthalmic Pharmaceutical Formulation, Topical Sulglycotide, Enhances the Ocular Mucin Secretion in Desiccation Stress-Mediated Dry Eye Disease.

Purpose: The aim of this study was the investigation of the effect of sulglycotide (SOS), a polysulfated glycopeptide derived from porcine duodenal mucin, for the treatment of dry eye disease. Methods: NOD.B10.H2b mice were exposed to an air draft for 10 days, and, simultaneously, scopolamine hydrobromide was injected subcutaneously. The mice were randomly divided into nine groups as follows: four kinds of SOS formulations and three kinds of commercial medicine. After 10 days of treatment, we estimated the effect of treatment on tear production, epithelium stabilization, mucin secretion, and inflammation. Results: The desiccation stress significantly decreased tear production and corneal epithelium stabilization, as well as markedly decreased the numbers of goblet cells and mucin-stained cells in conjunctiva. However, the topical 4% SOS eye drops markedly increased tear production and corneal stabilization, which recovered to baseline levels. In addition, topical 4% SOS significantly induced an increase in the numbers of goblet cells and the expression of membrane-associated mucins including MUC1, MUC4, and MUC16, as well as the gel-forming mucin, MUC5AC. Furthermore, SOS formulations provided anti-inflammatory improvement in a dose-dependent manner. Conclusions: In summary, we suggest that a new ophthalmic pharmaceutical formulation, topical sulglycotide, enhances the ocular mucin secretion in dry eye disease and can be used as a new ophthalmic pharmaceutical material to treat dry eye disease.

RGN-259 (thymosin ß4) improves clinically important dry eye efficacies in comparison with prescription drugs in a dry eye model.

This study evaluated the clinical activity of RGN-259 (thymosin ß4) in comparison with cyclosporine A (CsA), diquafosol (DQS), and lifitegrast (LFA) in a murine model of dry eye. The model was NOD.B10-H2b mice in a 30-40% humidified environment together with daily scopolamine hydrobromide injections for 10 days. After desiccation stress, all drugs were evaluated after 10 treatment days. RGN-259 increased tear production similar to that in the DQS- and LFA-treated mice while CsA was inactive. RGN-259 improved corneal smoothness and decreased fluorescein staining similar to that of LFA group while CsA and DQS were inactive. Corneal epithelial detachment was reduced by RGN-259, and DQS and LFA showed similar activity but the CsA was inactive. RGN-259 increased conjunctival goblet cells and mucin production comparable to that seen with CsA, while DQS and LFA were inactive. RGN-259 reduced the over-expression of inflammatory factors comparable to that with CsA and LFA, while DQS was inactive. RGN-259 increased mucin production comparable to that observed with CsA, while DQS and LFA were inactive. In conclusion, RGN-259 promoted recovery of mucins and goblet cells, improved corneal integrity, and reduced inflammation in a dry eye mouse model and was equal to or more effective than prescription treatments.

Microplasma Jet Arrays as a Therapeutic Choice for Fungal Keratitis.

The clinical impact of microplasma jets on rabbit eyes infected by Candida albicans has been investigated. Arrays of such jets produce low-temperature plasma micro-columns suitable for ophthalmic therapeutics and fungal infections, in particular, and the technology is capable of being scaled to surface areas of at least 10 cm2. Keratitis was induced in the right central corneas of rabbits, whereas the left eyes served as a normal group. The rabbits were divided into the plasma non-treated group (control) and plasma treatment group. Histologic analyses of both groups showed marked reductions in the thickness, angiogenesis, and opacity of all rabbit corneas following plasma treatment. Indeed, for treatment times beyond 14 days, infected eyes exhibited no significant differences from the normal group. Healing of rabbit eyes infected by Candida albicans apparently proceeds by disrupting corneal epithelial proliferation, and by reducing fibrotic changes in the stroma. This study demonstrates that low-temperature plasma jets are remarkably effective in healing Candida albicans-infected corneas, thereby providing a promising medical treatment option for keratitis.

Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B.

Retinal vascular endothelial growth factor (VEGF) increased by neovascularization is well known as a pathogenic factor in ocular neovascular diseases. However, it is still unclear how retinal neurons are damaged by VEGF. The aims of this study are to demonstrate the inflammatory protein expression regulated by VEGF using mouse photoreceptor-derived cells and the protective effect of quercetin against VEGF-induced inflammatory response. Expression and phosphorylation of protein and expression of mRNA were detected by immunoblot and reverse transcriptase polymerase chain reaction. VEGF-induced degradation of limiting membrane and translocation of nuclear factor kappa B (NF-κB) were analyzed by immunocytochemistry. VEGF treatment activated angiogenic signaling pathway in photoreceptor cells. In addition, adhesion molecules and matrix metalloproteinases were increased in VEGF-treated photoreceptor cells. All these events were reversed by quercetin. Zona occludins-1 and ß-catenin decreased by VEGF were recovered by quercetin. NF-κB signaling pathway regulated by VEGF through phosphorylations of mitogen-activated protein kinases (MAPK) and protein kinase B (Akt) was suppressed by quercetin. These results suggest that quercetin suppressed VEGF-induced excessive inflammatory response in retinal photoreceptor cells by inactivation of NF-κB signals through inhibition of MAPKs and Akt. These data may provide a basic information for development of pharmaceuticals or nutraceuticals for treatment of retinal diseases caused by excessive VEGF.

Anti-inflammatory effect of hydroxyproline-GQDGLAGPK in desiccation stress-induced experimental dry eye mouse.

The purpose of this study has been the investigation of the effect of novel peptide hydroxyproline-GQDGLAGPK (Hyp-GQDGLAGPK) in desiccation stress-induced dry eye mouse model and compared medicines for dry eye disease including cyclosporine, diquafosol and sodium hyaluronate. Seventy eight NOD.B10.H2 b mice were injected with scopolamine and exposed to an air draft for 10 days, and then the mice were treated with normal saline (n = 13), 1% Hyp-GQDGLAGPK (n = 13), 0.05% cyclosporine (n = 13), 3% diquafosol (n = 13), and 0.1% hyaluronate (n = 13) for 10 days. Thirteen mice were used for histopathologic analysis at DS 10d. The desiccation stress significantly decreased tear production, but the topical treatment of Hyp-GQDGLAGPK recovered to the baseline levels, which was similar to cyclosporine and diquafosol. In addition, Hyp-GQDGLAGPK improved facilitating epithelium stabilization including the corneal irregularity score, fluorescein score and detachment of the corneal epithelium. These improvements in stabilization of the corneal epithelium was superior to that in the cyclosporine and sodium hyaluronate groups. Furthermore, desiccation stress markedly induced expression of autoimmune inflammation-related factors in the lacrimal glands, but it was significantly suppressed by Hyp-GQDGLAGPK treatment. Overall, we found that novel peptide Hyp-GQDGLAGPK has multi-functional effects such as stabilizing the tear film and inhibiting inflammation.

Therapeutic Efficacy of Nanocomplex of Poly(Ethylene Glycol) and Catechin for Dry Eye Disease in a Mouse Model.

Purpose: We investigated the possibility of the nanocomplex of poly(ethylene glycol) (PEG) and catechin as a new biomedical material to treat dry eye disease. Methods: NOD.B10.H2b mice were exposed to an air draft and injected with scopolamine for 10 days. Ten days later, the mice were treated with normal saline (n = 11), 1% catechin (n = 11), 1% PEG (n = 11), and 1% catechin/PEG nanocomplex solution mixture containing catechin and PEG at weight ratios of 1:1 (CP1, n = 11), 1:5 (CP5, n = 11), and 1:10 (CP10, n = 11). All treatments were administered five times a day for 10 days. We estimated the effect of PEG/catechin nanocomplexes on inflammation, tear production, epithelium stabilization, and goblet cell density. Results: Desiccation stress significantly decreased tear production and increased the corneal irregularity score. Furthermore, desiccation stress markedly increased the detached epithelium and decreased the numbers of conjunctival goblet cells. In addition, the expression of proinflammatory-related factors was markedly induced by desiccation stress in the lacrimal glands. However, the PEG/catechin nanocomplex effectively induced an increase in tear production, stabilization of the corneal epithelium, and an increase in conjunctival goblet cells and anti-inflammatory improvements in a PEG dose-dependent manner. Conclusions: In this study, we found that PEG may increase bioavailability of catechin. Therefore, the PEG/catechin nanocomplex can be used as a new biomedical material to treat dry eye disease through stabilization of the tear film and inhibition of inflammation.

Effects of silk fibroin in murine dry eye.

The study aimed to investigate the effects of silk fibroin in a mouse model of dry eye. The experimental dry eye mouse model was developed using more than twelve-weeks-old NOD.B10.H2b mice exposing them to 30-40% ambient humidity and injecting them with scopolamine hydrobromide for 10 days. Tear production and corneal irregularity score were measured by the instillation of phosphate buffered saline or silk fibroin. Corneal detachment and conjunctival goblet cell density were observed by hematoxylin and eosin or periodic acid Schiff staining in the cornea or conjunctiva. The expression of inflammatory markers was detected by immunohistochemistry in the lacrimal gland. The silk group tear production was increased, and corneal smoothness was improved. The corneal epithelial cells and conjunctival goblet cells were recovered in the silk groups. The expression of inflammatory factors was inhibited in the lacrimal gland of the silk group. These results show that silk fibroin improved the cornea, conjunctiva, and lacrimal gland in the mouse model of dry eye. These findings suggest that silk fibroin has anti-inflammatory effects in the experimental models of dry eye.

Association of genetic polymorphisms of angiopoietin-like 4 with severity of posttransplant proteinuria in kidney allograft recipients.

BACKGROUND: Proteinuria is a hallmark of glomerular injury, and persistent proteinuria is associated with graft failure in kidney transplant patients. Recently, it is known that the level of circulating angiopoietin-like 4 (ANGPTL4) is elevated in the patients with human nephrotic syndrome, in which ANGPTL4 is responsible for relieving proteinuria. PURPOSE: The purpose of this study is to determine effects of clinical factors and genetic polymorphism of ANGPTL4 on proteinuria after kidney transplantation. METHODS: A total of 282 patients out of 400 renal transplant patients between 2008 and 2012 at St. Vincent Medical Center, CA were studied in a retrospective study design. The level of proteinuria was measured by random urine protein to creatinine ratio, and divided into two groups (Group 1: UPC<500mg/day, Group 2: ≥500mg/day). Single nucleotide polymorphisms of ANGPTL4 genes (rs1044250, rs2278236, rs116843064, rs11672433, rs4076317) were determined by real time PCR with sequence specific primers. RESULTS: Among various clinical factors, only delayed graft function, mTOR inhibitor use and fish oil use were significantly associated with posttransplant proteinuria. Statistical differences were found in genetic polymorphisms of ANGPTL4 (rs1044250, rs2278236) in regards to proteinuria among tested patients. rs1044250 (C/T, T228M, missense mutation) alleles showed multiple significant differences (Group 1 vs. Group 2: C vs. T: OR=1.893, CI=1.322-2.710, p<0.001). Similar trends were found in rs2278236 (A/G) alleles with statistical significances (Group 1 vs. Group 2: A vs. G: OR=0.620, CI=0.443-0.867, p=0.005). With multiple logistic regression, rs1044250 was still a significant risk factor of moderate/severe proteinuria (p=0.021). CONCLUSIONS: This study suggests that the presence of C allele of rs1044250 and G allele of rs2278236 in ANGPTL4 gene is associated with higher risk of moderate/severe proteinuria in renal transplant patients.