Target-guided isolation and purification of cyclooxygenase-2 inhibitors from Meconopsis integrifolia (Maxim.) Franch. by high-speed counter-current chromatography combined with ultrafiltration liquid chromatography.

Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1→2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.

Electrohydrogenation of Nitriles with Amines by Cobalt Catalysis.

Catalytic hydrogenation of nitriles represents an efficient and sustainable one-step synthesis of valuable bulk and fine chemicals. We report herein a molecular cobalt electrocatalyst for selective hydrogenative coupling of nitriles with amines using protons as the hydrogen source. The key to success for this reductive reaction is the use of an electrocatalytic approach for efficient cobalt-hydride generation through a sequence of cathodic reduction and protonation. As only electrons (e- ) and protons (H+ ) as the redox equivalent and hydrogen source, this general electrohydrogenation protocol is showcased by highly selective and straightforward synthesis of various functionalized and structurally diverse amines, as well as deuterium isotope labeling applications. Mechanistic studies reveal that the electrogenerated cobalt-hydride transfer to nitrile process is the rate-determining step.

The effectiveness of consolidation chemotherapy in high-risk early-stage cervical cancer patients following concurrent chemoradiation after radical surgery.

OBJECTIVE: Studies determining which early-stage cervical cancer patients with high-risk factors benefit from consolidation chemotherapy after postoperative concurrent chemoradiotherapy (CCRT) are limited and inconsistent. The aim of this study was to evaluate the value of consolidation chemotherapy in early-stage cervical cancer. METHODS: From 2010 to 2019, a retrospective review was conducted among high-risk early-stage cervical cancer patients who were treated with postoperative CCRT or consolidation chemotherapy after postoperative CCRT. Disease-free survival (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: A total of 293 patients with early-stage cervical cancer were included in this study. A total of 188 patients were in the consolidation chemotherapy group, and 105 patients were in the postoperative CCRT alone group. The median follow-up was 48.3 months (range: 3-123 months). In the survival analyses, no significant differences in DFS (P = 0.21) or OS (P = 0.15) were observed between the groups. The grade 3-4 leukopenia and neutropenia rates in the consolidation group were higher than those in the concurrent chemoradiotherapy alone group (54.8% vs. 28.6%, P = 0.02; 49.4% vs. 10.5%, P = 0.001, respectively). For patients with ≥2 positive lymph nodes or ≥2 high-risk factors, consolidation chemotherapy significantly improved DFS (P = 0.013 and P = 0.002) and OS (P < 0.001 and P < 0.001) compared with CCRT alone. CONCLUSION: For early-stage cervical cancer, consolidation chemotherapy after postoperative CCRT improved survival outcomes in patients with ≥2 positive lymph nodes or ≥2 high-risk factors.

Target-Guided Isolation and Purification of Antioxidants from Urtica laetevirens Maxim. by HSCCC Combined with Online DPPH-HPLC Analysis.

Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-ß-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by 1H NMR spectroscopy.

Therapeutic apheresis in kidney diseases: an updated review.

Besides conventional medical therapies, therapeutic apheresis has become an important adjunctive or alternative therapeutic option to immunosuppressive agents for primary or secondary kidney diseases and kidney transplantation. The available therapeutic apheresis techniques used in kidney diseases, including plasma exchange, double-filtration plasmapheresis, immunoadsorption, and low-density lipoprotein apheresis. Plasma exchange is still the leading extracorporeal therapy. Recently, growing evidence supports the potential benefits of double-filtration plasmapheresis and immunoadsorption for more specific and effective clearance of pathogenic antibodies with fewer side effects. However, more randomized controlled trials are still needed. Low-density lipoprotein apheresis is also an important supplementary therapy used in patients with recurrent focal segmental glomerulosclerosis. This review collects the latest evidence from recent studies, focuses on the specific advantages and disadvantages of these techniques, and compares the discrepancy among them to determine the optimal therapeutic regimens for certain kidney diseases.

Recent Advances in the Emerging Therapeutic Strategies for Diabetic Kidney Diseases.

Diabetic kidney disease (DKD) is one of the most common causes of end-stage renal disease worldwide. The treatment of DKD is strongly associated with clinical outcomes in patients with diabetes mellitus. Traditional therapeutic strategies focus on the control of major risk factors, such as blood glucose, blood lipids, and blood pressure. Renin-angiotensin-aldosterone system inhibitors have been the main therapeutic measures in the past, but the emergence of sodium-glucose cotransporter 2 inhibitors, incretin mimetics, and endothelin-1 receptor antagonists has provided more options for the management of DKD. Simultaneously, with advances in research on the pathogenesis of DKD, some new therapies targeting renal inflammation, fibrosis, and oxidative stress have gradually entered clinical application. In addition, some recently discovered therapeutic targets and signaling pathways, mainly in preclinical and early clinical trial stages, are expected to provide benefits for patients with DKD in the future. This review summarizes the traditional treatments and emerging management options for DKD, demonstrating recent advances in the therapeutic strategies for DKD.

DPre: computational identification of differentiation bias and genes underlying cell type conversions.

SUMMARY: Cells are generally resistant to cell type conversions, but can be converted by the application of growth factors, chemical inhibitors and ectopic expression of genes. However, it remains difficult to accurately identify the destination cell type or differentiation bias when these techniques are used to alter cell type. Consequently, there is demand for computational techniques that can help researchers understand both the cell type and differentiation bias. While advanced tools identifying cell types exist for single cell data and the deconvolution of mixed cell populations, the problem of exploring partially differentiated cells of indeterminate transcriptional identity has not been addressed. To fill this gap, we developed driver-predictor, which relies on scoring per gene transcriptional similarity between RNA-Seq datasets to reveal directional bias of differentiation. By comparing against large cell type transcriptome libraries or a desired target expression profile, the tool enables the user to visualize both the changes in transcriptional identity as well as the genes accounting for the cell type changes. This software will be a powerful tool for researchers to explore in vitro experiments that involve cell type conversions. AVAILABILITY AND IMPLEMENTATION: Source code is open source under the MIT license and is freely available on https://github.com/LoaloaF/DPre. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Preoperative controlling nutritional status (CONUT) score is a prognostic factor for early-stage cervical cancer patients with high-risk factors.

OBJECTIVE: The goal of the study was to confirm whether preoperative controlling nutritional status (CONUT) was a prognostic factor in early-stage cervical cancer patients with high-risk factors after surgery and postoperative concurrent chemoradiotherapy (CCRT). METHODS: Between 2004 and 2015, a total of 698 patients who were treated with surgery and postoperative CCRT were included in this retrospective study. The prescribed dose for postoperative radiotherapy was 45-50.4 Gy in 25-28 fractions and the concurrent chemotherapy regimen contained cisplatin or paclitaxel. Based on the receiver operating characteristic (ROC) analysis, the patients were classified into low (<3) and high (≥3) CONUT groups. RESULTS: Of all study patients, 471 (67.5%) patients were included in the low CONUT group. The low CONUT group had significantly better 5-year disease-free survival (DFS) and overall survival (OS) than the high CONUT group (p<0.001 and p = 0.001, respectively). A high CONUT score was significantly associated with lymph node metastasis, parametrial invasion, and poorer nutritional status, including lower body mass index (BMI) and lower prognostic nutritional index (PNI) score (p<0.05, respectively). The CONUT score was an independent predictor of DFS and OS in multivariate analysis. Notably, the CONUT score still efficiently stratified DFS in the high PNI score group (P = 0.001). CONCLUSIONS: The preoperative high CONUT scores indicated poor prognosis for early-stage cervical cancer patients with high-risk factors treated with surgery and postoperative CCRT. In clinical practice, patients with high CONUT score should be considered to receive consolidation chemotherapy.

Post-operative small pelvic intensity-modulated radiation therapy for early-stage cervical cancer with intermediate-risk factors: efficacy and toxicity.

OBJECTIVE: The aim of the present study was to retrospectively evaluate the toxicity and efficacy of post-operative small pelvic intensity-modulated radiotherapy in early-stage cervical cancer patients with intermediate-risk factors. METHODS: Between 2012 and 2016, 151 patients who had cervical cancer (International Federation of Gynecology and Obstetrics stage I-IIA) with intermediate-risk factors were treated with post-operative small pelvic intensity-modulated radiotherapy. The median dose of 50.4 Gy in 28 fractions with small pelvic intensity-modulated radiotherapy was prescribed to the planning target volume. The intensity-modulated radiotherapy technique used was conventional fixed-field intensity-modulated radiotherapy or helical tomotherapy. RESULTS: The median follow-up was 37 months. The 3-year disease-free survival and overall survival rates were 89 and 96%, respectively. A total of 144 patients (95.3%) were alive at the last follow-up. In total, 6 patients (3.9%) had recurrence: locoregional recurrence in 3 patients (2%), distant metastasis in 2 (1.3%), and both in 1 (0.6%). Diarrhoea was the most common acute toxicity. There were no patients suffering from acute or late grade ≥ 3 toxicity. Only 4 patients (2.6%) had late grade 2 toxicities. CONCLUSIONS: For early-stage cervical cancer patients with intermediate-risk factors, post-operative small pelvic intensity-modulated radiotherapy was safe and well tolerated. The rates of acute and late toxicities were quite satisfactory.

Defects Engineering Leads to Enhanced Photocatalytic H2 Evolution on Graphitic Carbon Nitride-Covalent Organic Framework Nanosheet Composite.

Graphitic carbon nitride nanosheet (CNS) represents an attractive candidate for solar fuel production. However, the abundant defects in CNS lead to serious charge recombination and limit the photocatalytic performance. Herein, the synthesis of a CNS-covalent organic framework (CNS-COF) nanosheet composite is presented for the first time. CNS with significantly reduced defects is first obtained by rationally tuning the thermal exfoliation conditions of bulk carbon nitride. Subsequent modification of the CNS with trace COF nanosheet through chemical imine bonding can not only passivate the surface termination of carbon nitride in the boundary region, but also establish strong electronic coupling between these two components. As a consequence, enhanced charge separation and photocatalytic activity are realized on the resulting CNS-COF nanosheet composite. Under optimum conditions, hydrogen is evolved at a rate of 46.4 mmol g-1 h-1 . This corresponds to an apparent quantum efficiency of 31.8% at 425 nm, which is among the best values ever reported for carbon nitride-based materials.

Association between chronic kidney disease and Alzheimer's disease: an update.

It has been accepted that kidney function is connected with brain activity. In clinical studies, chronic kidney disease (CKD) patients have been found to be prone to suffering cognitive decline and Alzheimer's disease (AD). The cognitive function of CKD patients may improve after kidney transplantation. All these indicators show a possible link between kidney function and dementia. However, little is known about the mechanism behind the relation of CKD and AD. This review discusses the associations between CKD and AD from the perspective of the pathophysiology of the kidney and complications and/or concomitants of CKD that may lead to cognitive decline in the progression of CKD and AD. Potential preventive and therapeutic strategies for AD are also presented. Further studies are warranted in order to confirm whether the setting of CKD is a possible new determinant for cognitive impairment in AD.

Facile Secondary Deposition for Improving Quantum Dot Loading in Fabricating Quantum Dot Solar Cells.

Sufficient loading of presynthesized quantum dots (QDs) on mesoporous TiO2 electrodes is the prerequisite for the fabrication of high-performance QD-sensitized solar cells (QDSCs). Here, we provide a general approach for increasing QD loading on mesoporous TiO2 films by surface engineering. It was found that the zeta potential of presensitized TiO2 can be effectively adjusted by surfactant treatment, on the basis of which additional QDs are successfully introduced onto photoanodes during secondary deposition. The strategy developed, that is, the secondary deposition incorporating surfactant treatment, makes it possible to load various QDs onto photoanodes regardless of the nature of QDs. In standard AM 1.5G sunlight, a certified efficiency of 10.26% for the QDSC with Cu2S/brass counter electrodes was achieved by the secondary deposition of Zn-Cu-In-Se QDs.

Models of global gene expression define major domains of cell type and tissue identity.

The current classification of cells in an organism is largely based on their anatomic and developmental origin. Cells types and tissues are traditionally classified into those that arise from the three embryonic germ layers, the ectoderm, mesoderm and endoderm, but this model does not take into account the organization of cell type-specific patterns of gene expression. Here, we present computational models for cell type and tissue specification derived from a collection of 921 RNA-sequencing samples from 272 distinct mouse cell types or tissues. In an unbiased fashion, this analysis accurately predicts the three known germ layers. Unexpectedly, this analysis also suggests that in total there are eight major domains of cell type-specification, corresponding to the neurectoderm, neural crest, surface ectoderm, endoderm, mesoderm, blood mesoderm, germ cells and the embryonic domain. Further, we identify putative genes responsible for specifying the domain and the cell type. This model has implications for understanding trans-lineage differentiation for stem cells, developmental cell biology and regenerative medicine.

The classic signalling and trans-signalling of interleukin-6 are both injurious in podocyte under high glucose exposure.

Interleukin-6 (IL-6) is a multifunctional cytokine that employs IL-6 classic and trans-signalling pathways, and these two signal channels execute different or even opposite effects in certain diseases. As a cardinal event of diabetic kidney disease (DKD), whether the podocyte abnormalities are associated with IL-6 signalling, especially classic or trans-signalling respectively, remains unclear. In this study, we identified that the circulatory IL-6, soluble IL-6R (sIL-6R) and soluble glycoprotein 130 (sgp130) levels are elevated in patients with DKD. The expressions of membrane-bound IL-6R (mIL-6R), sIL-6R and gp130 are enhanced in kidney cortex of diabetic mice accompanying with activated STAT3 by tyrosine 705 residue phosphorylation, while not serine 727. Above data infer both classic signalling and trans-signalling of IL-6 are activated during DKD. In cultured podocyte, high glucose (HG) up-regulates the expression of mIL-6R and gp130, as well as STAT3 tyrosine 705 phosphorylation, in a time-dependent manner. Entirely blocking IL-6 signalling by gp130 shRNA, gp130 or IL-6 neutralizing antibodies attenuates HG-induced podocyte injury. Interestingly, either inhibiting IL-6 classic signalling by mIL-6R shRNA or suppressing its trans-signalling using sgp130 protein dramatically alleviates HG-induced podocyte injury, suggesting both IL-6 classic signalling and trans-signalling play a detrimental role in HG-induced podocyte injury. Additionally, activation of IL-6 classic or trans-signalling aggravates podocyte damage in vitro. In summary, our observations demonstrate that the activation of either IL-6 classic or trans-signalling advances podocyte harming under hyperglycaemia. Thus, suppressing IL-6 classic and trans-signalling simultaneously may be more beneficial in podocyte protection and presents a novel therapeutic target for DKD.

IL-6 increases podocyte motility via MLC-mediated focal adhesion impairment and cytoskeleton disassembly.

The disturbance of podocyte motility is an essential pathogenic mechanisms of foot process effacement during proteinuric diseases, and myosin light chain (MLC) is a pivotal component in regulating the motility of podocytes. Inflammatory cytokine interleukin-6 (IL-6) has been reported to induce podocyte abnormalities by various mechanisms, however, whether aberrant cell motility contributes to the IL-6-induced podocyte injury remains unknown. Here, by wound healing, transwell, and cell migration assays, we confirmed that IL-6 accelerates the motility of podocyte. Simultaneously, the phosphorylation of MLC is elevated along with perturbed focal adhesion (FAs) and cytoskeleton. Next, via genetic and pharmacologic interruption of MLC or its phosphorylation we revealed that the activation of MLC is implicated in IL-6-mediated podocyte hypermotility as well as the disassembly of FAs and F-actin. By using stattic, an inhibitor for STAT3 phosphorylation, we uncovered that STAT3 activation is the upstream event for MLC phosphorylation and the following aberrant motility of podocytes. Additionally, we found that calcitriol markedly attenuates podocyte hypermotility via blocking STAT3-MLC. In conclusion, our study demonstrated that IL-6 interrupts FAs dynamic, cytoskeleton organization, and eventually leads to podocyte hypermotility via STAT3/MLC, whereas calcitriol exerts its protective role by inhibiting this pathway. These findings enrich the mechanisms accounting for IL-6-mediated podocyte injury from the standpoint of cell motility and provide a novel therapeutic target for podocyte disorders.

A New Pathogenesis of Albuminuria: Role of Transcytosis.

Transcytosis is an important intracellular transport process by which multicellular organisms selectively move cargoes from apical to basolateral membranes without disrupting cellular homeostasis. In kidney, macromolecular components in the serum, such as albumin, low-density lipoprotein and immunoglobulins, pass through the glomerular filtration barrier (GFB) and proximal tubular cells (PTCs) by transcytosis. Protein transcytosis plays a vital role in the pathology of albuminuria, which causes progressive destruction of the GFB structure and function. However, the pathophysiological consequences of protein transcytosis in the kidney remain largely unknown. This article summarizes recent researches on the regulation of albumin transcytosis across the GFB and PTCs in both physiological and pathological conditions. Understanding the mechanism of albumin transcytosis may reveal potential therapeutic targets for prevention or alleviation of the pathological consequences of albuminuria.

Inhibition of SIRT2 Alleviates Fibroblast Activation and Renal Tubulointerstitial Fibrosis via MDM2.

BACKGROUND/AIMS: Renal tubular epithelial cells and fibroblasts are the main sources of myofibroblasts, and these cells produce the extracellular matrix during tubulointerstitial fibrosis (TIF). Histone deacetylases (HDAC) inhibitors exert an antifibrogenic effect in the skin, liver and lung. Sirtuin 2 (SIRT2), which is a class III HDAC, is an important member of NAD+-dependent protein deacetylases. The current study evaluated the role of SIRT2 in renal TIF. METHODS: Immunohistochemical staining and Western blot were performed to evaluate SIRT2 expression in TIF patients and unilateral urethral obstruction (UUO) mice. Western blot was used to assess the protein levels of SIRT2, α-SMA, collagen III, fibronectin, and MDM2 in tubular epithelial cells and fibroblasts. The specific inhibitor AGK2 was used to inhibit SIRT2 activity, and targeted siRNA was used to suppress SIRT2 expression. RESULTS: SIRT2 expression increased in the tubulointerstitium of TIF patients and UUO mice. SIRT2 inhibition ameliorated TIF in UUO mice. SIRT2 expression in tubular cells was unchanged after exposure to TGF-ß1. The SIRT2-specifc inhibitor AGK2 did not attenuate TGF-ß1-induced tubular epithelial-mesenchymal transition. However, SIRT2 was upregulated in fibroblasts, and fibroblasts were activated after TGF-ß1 treatment. Genetic knockdown and chemical inhibition of SIRT2 attenuated TGF-ß1-induced fibroblast activation. We also explored the downstream signaling of SIRT2 during fibroblast activation. Genetic knockdown and chemical inhibition of SIRT2 suppressed TGF-ß1-induced increase in MDM2 expression, and inhibition of the MDM2-p53 interaction using Nutlin-3 did not suppress SIRT2 upregulation. CONCLUSION: Our results suggest that SIRT2 participates in the activation of fibroblasts and TIF, which is mediated via regulation of the MDM2 pathway, and the downregulation of SIRT2 may be a therapeutic strategy for renal fibrosis.

Microencapsulated Paraffin Phase-Change Material with Calcium Carbonate Shell for Thermal Energy Storage and Solar-Thermal Conversion.

A series of microencapsulated phase-change materials (MEPCMs) based on paraffin core and calcium carbonate (CaCO3) shell were synthesized, and the effect of emulsifier type and pH value on morphology, structure, and properties of paraffin@CaCO3 MEPCMs were investigated. The results showed that CaCO3 shell was formed in vaterite and calcite crystalline phase when emulsifier was sodium dodecyl benzene sulfonate and styrene-maleic anhydride (SMA), respectively. When sodium dodecyl sulfate was used as an emulsifier, both vaterite and calcite CaCO3 were formed. The forming mechanism of emulsifier type on CaCO3 crystalline phase was studied. Furthermore, phase-change enthalpy and leakage rate of MEPCMs were related with the type of emulsifier and the pH value of the emulsion. With optimum condition of SMA as emulsifier and pH value of 7, paraffin@CaCO3 MEPCMs had an encapsulation ratio at 56.6% and leakage rate at 2.88%, illustrating its considerable heat storage capability and leakage-prevention property. The 50 heating-cooling cycles test indicated that the MEPCMs owned excellent thermal reliability. The thermal conductivity of MEPCMs was significantly improved due to the existence of CaCO3 shell. In addition to excellent thermal storage ability, the paraffin@CaCO3 MEPCMs also owned good mechanical property and light-to-heat energy conversion efficiency. The characteristics of MEPCMs indicated its potential application in solar energy resource.

MDM2 is implicated in high-glucose-induced podocyte mitotic catastrophe via Notch1 signalling.

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in 'post-mitosis' state and unable to regenerate. Re-entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi-nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up-regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p-H3. Genetic deletion of MDM2 partly reversed HG-induced mitotic phase re-entering of podocytes. Moreover, HG-induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin-3a, an inhibitor of MDM2-p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG-mediated podocyte MC. In conclusion, high glucose up-regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG-induced MC in podocytes.

MDM2 mediates fibroblast activation and renal tubulointerstitial fibrosis via a p53-independent pathway.

It is well recognized that murine double minute gene 2 (MDM2) plays a critical role in cell proliferation and inflammatory processes during tumorigenesis. It is also reported that MDM2 is expressed in glomeruli and involved in podocyte injury. However, whether MDM2 is implicated in renal fibrosis remains unclear. Here we investigated the role of MDM2 in tubulointerstitial fibrosis (TIF). By immunohistochemical staining and Western blotting we confirmed that MDM2 is upregulated in the tubulointerstitial compartment in patients with TIF and unilateral urethral obstruction (UUO) mice, which mainly originates from myofibroblasts. Consistently, in vitro MDM2 is increased in TGF-ß1-treated fibroblasts, one of the major sources of collagen-producing myofibroblasts during TIF, along with fibroblast activation. Importantly, genetic deletion of MDM2 significantly attenuates fibroblast activation. We then analyzed the possible downstream signaling of MDM2 during fibroblast activation. p53-dependent pathway is the classic downstream signaling of MDM2, and Nutlin-3 is a small molecular inhibitor of MDM2-p53 interaction. To our surprise, Nutlin-3 could not ameliorate fibroblast activation in vitro and TIF in UUO mice. However, we found that Notch1 signaling is attenuated during fibroblast activation, which could be markedly rescued by MDM2 knockdown. Overexpression of intracellular domain of Notch1 (NICD) by plasmid could obviously minimize fibroblast activation induced by TGF-ß1. In addition, the degradation of NICD is strikingly suppressed by PYR-41, an inhibitor of ubiquitin-activating enzyme E1, and proteasome inhibitor MG132. Taken together, our findings provide the first evidence that MDM2 is involved in fibroblast activation and TIF, which associates with Notch1 ubiquitination and proteasome degradation.