Characterizing Glomerular Barrier Dysfunction with Patient-Derived Serum in Glomerulus-on-a-Chip Models: Unveiling New Insights into Glomerulonephritis.

Glomerulonephritis (GN) is characterized by podocyte injury or glomerular filtration dysfunction, which results in proteinuria and eventual loss of kidney function. Progress in studying the mechanism of GN, and developing an effective therapy, has been limited by the absence of suitable in vitro models that can closely recapitulate human physiological responses. We developed a microfluidic glomerulus-on-a-chip device that can recapitulate the physiological environment to construct a functional filtration barrier, with which we investigated biological changes in podocytes and dynamic alterations in the permeability of the glomerular filtration barrier (GFB) on a chip. We also evaluated the potential of GN-mimicking devices as a model for predicting responses to human GN. Glomerular endothelial cells and podocytes successfully formed intact monolayers on opposite sides of the membrane in our chip device. Permselectivity analysis confirmed that the chip was constituted by a functional GFB that could accurately perform differential clearance of albumin and dextran. Reduction in cell viability resulting from damage was observed in all serum-induced GN models. The expression of podocyte-specific marker WT1 was also decreased. Albumin permeability was increased in most models of serum-induced IgA nephropathy (IgAN) and membranous nephropathy (MN). However, sera from patients with minimal change disease (MCD) or lupus nephritis (LN) did not induce a loss of permeability. This glomerulus-on-a-chip system may provide a platform of glomerular cell culture for in vitro GFB in formation of a functional three-dimensional glomerular structure. Establishing a disease model of GN on a chip could accelerate our understanding of pathophysiological mechanisms of glomerulopathy.

Efficacy of Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles in Ameliorating Cisplatin Nephrotoxicity, as Modeled Using Three-Dimensional, Gravity-Driven, Two-Layer Tubule-on-a-Chip (3D-MOTIVE Chip).

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) are known to have a therapeutic effect on nephrotoxicity. As animal models require significant time and resources to evaluate drug effects, there is a need for a new experimental technique that can accurately predict drug effects in humans. We evaluated the therapeutic effect of MSC-derived EVs in cisplatin nephrotoxicity using a three-dimensional, gravity-driven, two-layer tubule-on-a-chip (3D-MOTIVE chip). In the 3D-MOTIVE chip, 10 µM cisplatin decreased the number of attached cells compared to the vehicle. Conversely, annexin V and reactive oxygen species (ROS) were increased. Cell viability was increased 2.8-fold and 2.5-fold after treatment with EVs at 4 and 8 µg/mL, respectively, compared to the cisplatin-induced nephrotoxicity group. Cell attachment was increased 2.25-fold by treatment with 4 µg/mL EVs and 2.02-fold by 8 µg/mL EVs. Annexin V and ROS levels were decreased compared to those in the cisplatin-induced nephrotoxicity group. There were no significant differences in annexin V and ROS levels according to EV concentration. In sum, we created a cisplatin-induced nephrotoxicity model on a 3D-MOTIVE chip and found that MSC-derived EVs could restore cell viability. Thus, MSC-derived EVs may have the potential to ameliorate cisplatin-induced nephrotoxicity.

SIRT-1 regulates TGF-ß-induced dermal fibroblast migration via modulation of Cyr61 expression.

SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-ß further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-ß or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-ß or RSV. TGF-ß, RSV, or SIRT1 overexpression enhanced ß-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/ß-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

The miR-19a/b family positively regulates cardiomyocyte hypertrophy by targeting atrogin-1 and MuRF-1.

Progressive cardiac hypertrophy owing to pathological stimuli, such as pressure overload, is frequently associated with the development of heart failure, a major cause of morbidity and mortality worldwide. Growing evidence has shown that miRNAs are extensively involved in the pathogenesis of cardiac hypertrophy. In the present study, we examined the hypothesis that the miR-19a/b family acts as a key regulator of cardiac hypertrophy and apoptosis. Forced overexpression of miR-19a/b was sufficient to induce hypertrophy in rat neonatal cardiomyocytes. Luciferase assays revealed that miR-19a/b directly target the anti-hypertrophic genes atrogin-1 and MuRF-1 (muscle RING-finger protein-1). The endogenous expressions of the target genes were down-regulated by miR-19a/b. Pro-hypertrophic calcineurin/NFAT (nuclear factor of activated T-cells) signalling was elevated markedly in the presence of miR-19b, and the calcineurin inhibitor CsA (cyclosporin A) and the PKC (protein kinase C) inhibitor GF10923X significantly attenuated the miR-19b-mediated increase in cell size and expression of hypertrophic markers. Furthermore, miR-19b led to increased cell survival through up-regulation of the NFAT target gene encoding α-crystallin-B and repression of the pro-apoptotic gene Bim (Bcl-2-interacting mediator of cell death) under ER (endoplasmic reticulum) stress conditions. Taken together, the results of the present study demonstrate that the miR-19a/b family regulates phenotypes of cardiomyocytes via suppression of multiple direct target genes.

E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8.

The E2F1 transcription factor can promote proliferation or apoptosis when activated, and is a key downstream target of the retinoblastoma tumour suppressor protein (pRB). Here we show that E2F1 is a potent and specific inhibitor of beta-catenin/T-cell factor (TCF)-dependent transcription, and that this function contributes to E2F1-induced apoptosis. E2F1 deregulation suppresses beta-catenin activity in an adenomatous polyposis coli (APC)/glycogen synthase kinase-3 (GSK3)-independent manner, reducing the expression of key beta-catenin targets including c-MYC. This interaction explains why colorectal tumours, which depend on beta-catenin transcription for their abnormal proliferation, keep RB1 intact. Remarkably, E2F1 activity is also repressed by cyclin-dependent kinase-8 (CDK8), a colorectal oncoprotein. Elevated levels of CDK8 protect beta-catenin/TCF-dependent transcription from inhibition by E2F1. Thus, by retaining RB1 and amplifying CDK8, colorectal tumour cells select conditions that collectively suppress E2F1 and enhance the activity of beta-catenin.

Development of Drug Efficacy Testing Platform for Glomerulonephritis.

We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and endothelial cells with a bidirectional flow in the bottom channel. Using puromycin-induced GN, we observed decreased cell viability, increased albumin permeability, and reduced WT1 and nephrin compared to the normal GFB. Tacrolimus restored cell viability, reduced albumin permeability, and increased WT1 expression. Using serum from five membranous nephropathy (MN) patients, we created MN models using a GFB-mimicking chip. A notable decline in cell viability was observed in the serum-induced MN1 and MN2 models. However, tacrolimus restored it. Albumin permeability was reduced in the MN1, MN2, and MN5 models by tacrolimus treatment. MN1 displayed the best clinical response to tacrolimus, exhibiting increased expression of WT1 in chip-based evaluations after tacrolimus treatment. We successfully evaluated the efficacy of tacrolimus using puromycin-induced and serum-induced GN models on a chip that mimicked the structure and function of the GFB. The GFB-mimicking chip holds promise as a personalized platform for assessing drug efficacy using patient serum samples.

Comparison between characteristics of immunoglobulin M nephropathy and other glomerular diseases.

Background: Immunoglobulin M (IgM) nephropathy (IgMN) is characterized by the IgM deposition in the kidney's mesangium. We assessed the impact of electron-dense deposits (EDDs) on IgMN and compared it to other kidney diseases. Methods: We enrolled 63 adult patients with IgMN who underwent renal biopsy from May 2003 to June 2017. We compared clinicopathological features of IgMN based on EDD presence; compared characteristics to 91 minimal change disease (MCD), 103 focal segmental glomerulosclerosis (FSGS), and 469 immunoglobulin A nephropathy (IgAN) patients. Renal events were defined as a >50% decrease in estimated glomerular filtration rate (eGFR), eGFR of <15 mL/min/1.73 m2, or end-stage renal disease development. Results: IgMN patients with EDDs had increased mesangial cellularity, matrix accumulation, prominent immunofluorescent staining, and more diffuse podocyte effacement than those without EDD. Clinical characteristics and renal outcomes did not differ significantly based on EDD presence. During 79.5 ± 58.8 months of follow-up, renal events developed in 46.2% and 46.0% of IgMN cases with and without EDD. IgMN (46.0%) and FSGS cases (40.8%) had similar frequencies of renal events and higher frequency than MCD (18.7%) or IgAN cases (26.4%). IgMN cases had more severe manifestations than MCD and IgAN; higher blood pressure, lower proteinuria, and eGFR levels at biopsy than MCD cases; higher blood pressure, proteinuria, frequency of acute kidney injury, and lower eGFR levels. Conclusion: Clinical characteristics of IgMN did not differ based on EDD presence. Therefore, IgMN should be defined based on IF findings. IgMN shared clinical characteristics with FSGS but had more severe than MCD and IgAN.

Impact of metformin on cardiovascular and kidney outcome based on kidney function status in type 2 diabetic patients: a multicentric, retrospective cohort study.

Metformin is the primary treatment for type 2 diabetes mellitus (T2DM) due to its effectiveness in improving clinical outcomes in patients with preserved renal function, however, the evidence on the effectiveness of metformin in various renal functions is lacking. We performed a retrospective, multicenter, observational study used data of patients with T2DM obtained from three tertiary hospitals' databases. Patients given metformin within run-in periods and with at least one additional prescription formed the metformin cohort. A control cohort comprised those prescribed oral hypoglycemic agents other than metformin and never subsequently received a metformin prescription within observation period. For patients without diabetic nephropathy (DN), the outcomes included events of DN, major adverse cardiovascular events (MACE), and major adverse kidney events (MAKE). After 1:1 propensity matching, 1994 individuals each were selected for the metformin and control cohorts among T2DM patients without baseline DN. The incidence rate ratios (IRR) for DN, MACEs, and MAKEs between cohorts were 1.06 (95% CI 0.96-1.17), 0.76 (0.64-0.92), and 0.45 (0.33-0.62), respectively. In cohorts with renal function of CKD 3A, 3B, and 4, summarized IRRs of MACEs and MAKEs were 0.70 (0.57-0.87) and 0.39 (0.35-0.43) in CKD 3A, 0.83 (0.74-0.93) and 0.44 (0.40-0.48) in CKD 3B, and 0.71 (0.60-0.85) and 0.45 (0.39-0.51) in CKD 4. Our research indicates that metformin use in T2DM patients across various renal functions consistently correlates with a decreased risk of overt DN, MACE, and MAKE.

Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes.

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress (ERS), and triggers the unfolded protein response (UPR) that consequently reduces accumulation of unfolded proteins by increasing the quantity of ER chaperones. Calumenin, a Ca(2+)-binding protein with multiple EF hand motifs, which is located in the ER/SR, is highly expressed during the early developmental stage of the heart, similar to other ER-resident chaperones. The aim of this study was to investigate the functional role of calumenin during ERS in the heart. Like other chaperones (e.g., GRP94 and GRP78), calumenin expression was highly upregulated during ERS induced by 10 µg/ml tunicamycin, but attenuated in the presence of 500 µM PBA, the chemical chaperone in neonatal rat ventricular cardiomyocytes (NRVCs). Upon 7.5-fold overexpression of calumenin using a recombinant adenovirus system, the expression levels of ERS markers (GRP78, p-PERK, and p-elF2α) and ER-initiated apoptosis markers (CHOP and p-JNK) were reduced, whereas the survival protein BCL-2 was upregulated during ERS compared to the control. Evaluation of cell viability by TUNEL assay showed that apoptosis was also significantly reduced by calumenin overexpression in ERS-induced cells. Taken together, our results suggest that calumenin plays an essential role in the alleviation of ERS in neonatal rat cardiomyocytes.

Outcomes of minimal change disease without nephrotic range proteinuria.

Minimal change disease (MCD) is characterized by edema and nephrotic range proteinuria (NS). However, the fate of MCD without nephrotic proteinuria requires elucidation. We retrospectively reviewed 79 adults diagnosed with primary MCD at their initial renal biopsy at a tertiary hospital between May 2003 and June 2017. Clinicopathologic features were compared between patients with and without NS. The frequency of flaring to nephrotic proteinuria and renal outcomes were assessed during follow-up. There were 20 and 59 patients in the Non-NS and NS groups, respectively. The Non-NS group had a lower frequency of acute kidney injury (AKI) during the follow-up period [5.0% vs. 59.3%, p <0.001]. The response rate to steroid treatment was 100% in the Non-NS group and 92.3% in the NS group (p = 1.000). Except for one patient, the Non-NS group was treated with steroids when their proteinuria increased to a nephrotic level. There were no differences in the frequency of the first relapse or the number of relapses among patients with initial remission from nephrotic range proteinuria. At the final visit, the complete remission rate was 73.4%. The estimated glomerular filtration rate during follow-up was significantly better in the NS group than the Non-NS group, given the higher rates of AKI at renal biopsy. The rates of renal events, end-stage renal disease, and mortality did not differ between the groups. Adult MCD patients with nephrotic and non-nephrotic range proteinuria showed similar outcomes. Accordingly, this population must be carefully managed, regardless of the amount of proteinuria at renal biopsy.

Electronic alert outpatient protocol improves the quality of care for the risk of postcontrast acute kidney injury following computed tomography.

BACKGROUND: Prevention and diagnosis of postcontrast acute kidney injury (AKI) after contrast-enhanced computed tomography is burdensome in outpatient department. We investigated whether an electronic alert system could improve prevention and diagnosis of postcontrast AKI. METHODS: In March 2018, we launched an electronic alert system that automatically identifies patients with a baseline estimated glomerular filtration rate of <45 mL/min/1.73 m2, provides a prescription of fluid regimen, and recommends a follow-up for serum creatinine measurement. Participants prescribed contrast-enhanced computed tomography at outpatient department before and after the launch of the system were categorized as historical and alert group, respectively. Propensity for the surveillance of postcontrast AKI was compared using logistic regression. Risks of AKI, admission, mortality, and renal replacement therapy were analyzed. RESULTS: The historical and alert groups included 289 and 309 participants, respectively. The alert group was more likely to be men and take diuretics. The most frequent volume of prophylactic fluid in historical and alert group was 1,000 and 750 mL, respectively. Follow-up for AKI was more common in the alert group (adjusted odds ratio, 6.00; p < 0.001). Among them, incidence of postcontrast AKI was not statistically different. The two groups did not differ in risks of admission, mortality, or renal replacement therapy. CONCLUSION: The electronic alert system could assist in the detection of high-risk patients, prevention with reduced fluid volume, and proper diagnosis of postcontrast AKI, while limiting the prescribing clinicians' burden. Whether the system can improve long-term outcomes remains unclear.

The chemical chaperone 4-phenylbutyric acid attenuates pressure-overload cardiac hypertrophy by alleviating endoplasmic reticulum stress.

Evidence has shown that endoplasmic reticulum stress (ERS) is associated with the pathogenesis of cardiac hypertrophy. The aim of this study was to investigate whether direct alleviation of ER stress by 4-phenylbutyric acid (PBA), a known chemical chaperone drug, could attenuate pressure-overload cardiac hypertrophy in mice. The effects of orally administered PBA (100mg/kg body weight daily for a week) were examined using mice undergoing transverse aortic constriction (TAC-mice), an animal model to produce pressure overload. TAC application for 1 week led to a 1.8-fold increase in the ratio of the heart weight over body weight (HW/BW) and up-regulation of the hypertrophy markers ANF and BNF accompanied by up-regulation of ERS markers (GRP78, p-PERK, and p-elF2α). The oral administration of PBA to the TAC-mice reduced hypertrophy (19%) and severely downregulated the fibrosis-related genes (transforming growth factor-ß1, phospho-smad2, and pro-collagen isoforms). We conclude that ERS is induced as a consequence of remodeling during pathological hypertrophy and that PBA may help to relieve ERS and play a protective role against cardiac hypertrophy and possibly heart failure. We suggest PBA as a novel therapeutic agent for cardiac hypertrophy and fibrosis.

Drosophila E2F1 has context-specific pro- and antiapoptotic properties during development.

E2F transcription factors are generally believed to be positive regulators of apoptosis. In this study, we show that dE2F1 and dDP are important for the normal pattern of DNA damage-induced apoptosis in Drosophila wing discs. Unexpectedly, the role that E2F plays varies depending on the position of the cells within the disc. In irradiated wild-type discs, intervein cells show a high level of DNA damage-induced apoptosis, while cells within the D/V boundary are protected. In irradiated discs lacking E2F regulation, intervein cells are largely protected, but apoptotic cells are found at the D/V boundary. The protective effect of E2F at the D/V boundary is due to a spatially restricted role in the repression of hid. These loss-of-function experiments demonstrate that E2F cannot be classified simply as a pro- or antiapoptotic factor. Instead, the overall role of E2F in the damage response varies greatly and depends on the cellular context.

Transcriptional regulation of the Drosophila caudal homeobox gene by bHLH-PAS proteins.

Caudal-related homeobox transcription factors are involved in the definition of the anteroposterior axis and intestinal development. Recent reports indicate that dysregulation of CDX1 and CDX2, the human homologues of Drosophila caudal, are associated with several types of cancer. Very little is known, however, about the regulatory mechanisms that direct the caudal-related homeobox gene expression. In this study, we have identified the binding sites for bHLH-PAS proteins, referred to as CNS midline element (CME), in the 5'-flanking region of the Drosophila caudal gene. Analyses using transgenic flies carrying a caudal-lacZ fusion gene bearing a wild-type or mutant CME indicate that the CME sites are required for caudal gene expression in vivo. We also determined that the caudal promoter activity can be regulated by Trachealess (Trh)/Tango (Tgo) bHLH-PAS proteins, via the CME sites. Our results suggest that the Drosophila caudal gene is a target of the Trh/Tgo bHLH-PAS proteins.

Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity.

Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

Agmatine modulates melanogenesis via MITF signaling pathway.

Agmatine contained in soybean is also found in Manaca, an anti-aging plant, inhabited in Amazon and induces vasodilation by the promotion of NO synthesis in blood vessel. However, the research of agmatine on melanin synthesis related to hair greying is lacking. The aim of this study was to investigate the melanogenic effect of agmatine via regulation of MITF signaling pathway in B16F1 cells. It was determined whether agmatine regulates melanin synthesis at cellular level in addition to the effect of agmatine on mushroom tyrosinase in vitro in the presence of different concentrations of agmatine. Furthermore, the effect of agmatine on the protein expressions of tyrosinase, TRP-1, TRP-2, BMP-4, BMP-6, C-KIT, p-p38, MITF and C-FOS were examined by western blot analysis. In addition, immunofluorescence staining was carried out to visualize the location of MITF expression in cell. Agmatine at 256µM or more increased melanin synthesis as well as tyrosinase activity. Moreover, whereas agmatine increased the expression levels of TRP-1, BMP-6, p-p38 and MITF, it reduced the expression level of BMP-4. It was also found that agmatine enhanced the expression level of MITF in nucleus. These results suggest that agmatine could induce melanin synthesis though the regulation of MITF transcription factor via BMP-6/p38 signaling pathway.

PPARγ agonist rosiglitazone inhibits migration and invasion by downregulating Cyr61 in rheumatoid arthritis fibroblast-like synoviocytes.

AIM: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have anti-inflammatory properties that reduce inflammatory cytokine production in rheumatoid arthritis (RA). Cysteine-rich angiogenic inducer 61 (Cyr61) is associated with diseases related to chronic inflammation. The aim of this study was to investigate the mechanisms underlying the effects of PPARγ agonists on tumor necrosis factor (TNF)-α-induced fibroblast-like synoviocyte (FLS) invasion and migration, as well as Cyr61 production, in RA-FLS. METHODS: FLS were cultured with TNF-α and Cyr61 in the presence or absence of PPARγ agonists. Matrix metalloproteinase and Cyr61 expression levels in RA-FLS and culture supernatants were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The migration and invasion phenotypes of RA-FLS were determined by wound healing and Boyden chamber assays. RESULTS: Cyr61 protein was expressed in RA-FLS, and its intracellular expression and secretion levels were increased by TNF-α. Moreover, Cyr61 directly promoted RA-FLS migration and invasion. Rosiglitazone (RSG) significantly decreased TNF-α-induced Cyr61 expression. RSG decreased TNF-α-induced nuclear factor (NF)-κB activation and inhibitor of κBα degradation. Furthermore, RSG inhibited TNF-α-induced RA-FLS migration and invasion and decreased Cyr61 treatment-induced RA-FLS invasion. Finally, blocking Cyr61 significantly attenuated TNF-α-induced migration. CONCLUSIONS: Our results demonstrate for the first time that PPARγ agonists may have beneficial effects on the migration and invasion of RA-FLS via the downregulation of Cyr61. Therefore, PPARγ agonists could be potential treatment targets for RA.

Stability Evaluation of National Reference Standards for Blood Products in Korea.

National reference standards (NRSs) for biologics are established through potency estimation by a multicenter joint study of standard materials used in the approval process for national lot release and quality control of vaccines, blood products, and other biologics. In this study, a stability evaluation was conducted to determine whether the potency of NRSs for six blood products was being maintained at a consistent level in Korea. The present study conducted real-time stability tests via in-vivo/in-vitro bioassay on NRSs for blood coagulation factor VIII concentrate (2nd standard), antithrombin concentrate, prekallikrein activator, anti-hepatitis B immunoglobulin, blood coagulation factor IX concentrate, and anti-tetanus human immunoglobulin, as well as a trend analysis using cumulative annual results. The real-time stability test results showed that the mean potency of six NRSs was all within the control limit. In the trend analysis, the potency of NRS for blood coagulation factor VIII concentrate (2nd standard) showed a decreasing trend, while the potency of all other products had been stably maintained. The present study confirmed that the mean potency of NRSs for six blood products had been stably maintained in Korea. The findings of the present study establish a foundation that can ensure the quality of NRSs for biologics in Korea, and it is expected to make a major contribution to the supply of high-quality biologics.