Role of Human Primary Renal Fibroblast in TGF-β1-Mediated Fibrosis-Mimicking Devices

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-β (TGF-β) and TGF-β inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-β1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-β-treated group and TGF-β-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-β-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-β1, TGF-β2, and TGF-β3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.     

Urine-Derived Stem Cell-Secreted Klotho Plays a Crucial Role in the HK-2 Fibrosis Model by Inhibiting the TGF-β Signaling Pathway

Renal fibrosis is an irreversible and progressive process that causes severe dysfunction in chronic kidney disease (CKD). The progression of CKD stages is highly associated with a gradual reduction in serum Klotho levels. We focused on Klotho protein as a key therapeutic factor against CKD. Urine-derived stem cells (UDSCs) have been identified as a novel stem cell source for kidney regeneration and CKD treatment because of their kidney tissue-specific origin. However, the relationship between UDSCs and Klotho in the kidneys is not yet known. In this study, we discovered that UDSCs were stem cells that expressed Klotho protein more strongly than other mesenchymal stem cells (MSCs). UDSCs also suppressed fibrosis by inhibiting transforming growth factor (TGF)-β in HK-2 human renal proximal tubule cells in an in vitro model. Klotho siRNA silencing reduced the TGF-inhibiting ability of UDSCs. Here, we suggest an alternative cell source that can overcome the limitations of MSCs through the synergetic effect of the origin specificity of UDSCs and the anti-fibrotic effect of Klotho.     

Empagliflozin Ameliorates Free Fatty Acid Induced-Lipotoxicity in Renal Proximal Tubular Cells via the PPARγ/CD36 Pathway in Obese Mice

High serum levels of free fatty acids (FFAs) could contribute to obesity-induced nephropathy. CD36, a class B scavenger receptor, is a major receptor mediating FFA uptake in renal proximal tubular cells. Empagliflozin, a new anti-diabetic agent, is a specific inhibitor of sodium-glucose co-transporter 2 channels presented on renal proximal tubular cells and inhibits glucose reabsorption. In addition, empagliflozin has shown renoprotective effects. However, the mechanism through which empagliflozin regulates CD36 expression and attenuates FFA-induced lipotoxicity remains unclear. Herein, we aimed to elucidate the crosstalk between empagliflozin and CD36 in FFA-induced renal injury. C57BL/6 mice fed a high-fat diet (HFD) and palmitic acid-treated HK-2 renal tubular cells were used for in vivo and in vitro assessments. Empagliflozin attenuated HFD-induced body weight gain, insulin resistance, and inflammation in mice. In HFD-fed mice, CD36 was upregulated in the tubular area of the kidney, whereas empagliflozin attenuated CD36 expression. Furthermore, empagliflozin downregulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ. Treatment with a PPARγ inhibitor (GW9662) did not further decrease PPARγ expression, whereas a PPARγ antagonist reversed this effect; this suggested that empagliflozin may, at least partly, decrease CD36 by modulating PPARγ. In conclusion, empagliflozin can ameliorate FFA-induced renal tubular injury via the PPARγ/CD36 pathway.     

Knockdown of a β-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a β-adrenergic-like octopamine receptor (TcOctβ2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctβ2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.     

Modulation of Nitrosative Stress via Glutathione-Dependent Formaldehyde Dehydrogenase and S-Nitrosoglutathione Reductase

Glutathione-dependent formaldehyde dehydrogenase (GFD) from Taiwanofungus camphorata plays important roles in formaldehyde detoxification and antioxidation. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) against nitrosative stress. We investigated the modulation of HEK (human embryonic kidney) 293T cells under nitrosative stress by transfecting a codon optimized GFD cDNA from Taiwanofungus camphorata (Tc-GFD-O) to these cells. The parental and transfected HEK 293T cells were then subjected to S-nitrosoglutathione treatment to induce nitrosative stress. The results showed that in Tc-GFD-O-transfected 293T cells, the expression and activity of GFD increased. Additionally, these cells under the nitrosative stress induced by S-nitrosoglutathione showed both higher viability and less apoptosis than the parental 293T cells. This finding suggests that the Tc-GFD-O in HEK 293T cells may provide a protective function under nitrosative stress.     

Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.     

Dicer Nuclease‐Promoted Production of Let7a‐1 MicroRNA Is Enhanced in the Presence of Tryptophan‐Containing Amphiphilic Peptides

A library of Trp‐containing amphiphilic peptides was synthesized and screened for the ability to bind to pre‐miRNA targets. Two members of this family, peptides Ac‐WKKLLKWLKKLLKLAG‐NH₂ ( 2 b ) and Ac‐WKKLLKWLKKLLDabLAG‐NH₂ ( 4 b ) were found to have nanomolar binding affinities to pre‐let7a‐1. Peptides 2 b and 4 b caused an increase in the in vitro Dicer cleavage of pre‐let7a‐1. This observation was confirmed by a cell‐based assay, the results of which show an up to 50 % increase in Dicer activity. Enhanced expression of let7a‐1 promoted by the peptides results in specific reductions of target mRNAs. The results of a microarray study show that lower amount of fluctuating genes are generated in the presence of 2 b or 4 b , relative to that from exogenous delivery of let7a‐1. Because peptides 2 b and 4 b promote enhanced formation of mature let7a‐1 only at the endogenous miRNA level, this specifically controls genes related to let7a‐1.     

The application of synchrotron-based spectroscopic techniques to the study of chromate conversion coatings

This work utilized synchrotron spectroscopic techniques such as extended X-ray absorption fine structure (EXAFS), X-ray absorption near edge structure (XANES) and synchrotron infrared microspectroscopy (SIRMS) to understand the chemistry, formation, structure and aging of protective chromate conversion coatings (CCC). We shall discuss issues related CCC formed on aluminum-copper alloys. SIRMS combined with argon ion sputtering demonstrated spatial and depth chemical variations of the CCC formed on AA2024-T3. XANES indicated that in the first stages of CCC formation the thickness depends on the Cu content of the substrate and that thinner CCC appears to contain less Cr⁶⁺ for protection. Micro-XANES shows that even monolayer coverage of chromate can protect the alloy surface. EXAFS and XANES measurements were used to monitor the structural changes of the CCC with time. It was observed that a slight decrease in the Cr(VI)Cr(III) ratio occurred during the first 24 h following coating. The structure of the CCC is in close agreement with a Cr mixed oxide. EXAFS data appeared to indicate an increase in ordering in the CCC structure with time.     

MiRNA-548ah,a Potential Molecule Associated with Transition from Immune Tolerance to Immune Activation of Chronic Hepatitis B

Objective: The present study aims to identify the differently expressed microRNA (miRNA) molecules and target genes of miRNA in the immune tolerance (IT) and immune activation (IA) stages of chronic hepatitis B (CHB). Methods: miRNA expression profiles of peripheral blood mononuclear cells (PBMCs) at the IT and IA stages of CHB were screened using miRNA microarrays and authenticated using a quantitative real-time polymerase chain reaction (RT-PCR). Gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) were used to analyze the significant functions and pathways of possible target genes of miRNAs. Assays of the gain and loss of function of the miRNAs were performed to verify the target genes in THP-1 cell lines. The luciferase reporter test was used on 293T cells as direct targets. Results: Significantly upregulated miR-548 and miR-4804 were observed in the miRNA microarrays and confirmed by RT-PCR in PBMCs at the IT and IA stages of CHB. GO and KEGG analysis revealed that MiR-548 and miR-4804 could be involved in numerous signaling pathways and protein binding activity. IFNγR1 was predicted as a target gene and validated as the direct gene of MiR-548. Significant negative correlation was found between the miR-548ah and mRNA levels of IFN-γR1 in CHB patients. Conclusions: The abnormal expression profiles of miRNA in PBMCs could be closely associated with immune activation of chronic HBV infection. miR-548, by targeting IFN-γR1, may represent a mechanism that can facilitate viral pathogenesis and help determine new therapeutic molecular targets.     

A Novel Protein Is Lower Expressed in Renal Cell Carcinoma

Engrailed-2 (EN2) has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731) and anti-EN2 antibody (MAB2600) were used for immunohistochemistry (IHC) respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p < 0.05), so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma.     

In Vivo and In Vitro Evaluation of Urinary Biomarkers in Ischemia/Reperfusion-Induced Kidney Injury

Oxidative stress plays an important role in the pathophysiology of acute kidney injury (AKI). Previously, we reported that vanin-1, which is involved in oxidative stress, is associated with renal tubular injury. This study was aimed to determine whether urinary vanin-1 is a biomarker for the early diagnosis of AKI in two experimental models: in vivo and in vitro. In a rat model of AKI, ischemic AKI was induced in uninephrectomized rats by clamping the left renal artery for 45 min and then reperfusing the kidney. On Day 1 after renal ischemia/reperfusion (I/R), serum creatinine (SCr) in I/R rats was higher than in sham-operated rats, but this did not reach significance. Urinary N-acetyl-β-D-glucosaminidase (NAG) exhibited a significant increase but decreased on Day 2 in I/R rats. In contrast, urinary vanin-1 significantly increased on Day 1 and remained at a significant high level on Day 2 in I/R rats. Renal vanin-1 protein decreased on Days 1 and 3. In line with these findings, immunofluorescence staining demonstrated that vanin-1 was attenuated in the renal proximal tubules of I/R rats. Our in vitro results confirmed that the supernatant from HK-2 cells under hypoxia/reoxygenation included significantly higher levels of vanin-1 as well as KIM-1 and NGAL. In conclusion, our results suggest that urinary vanin-1 might be a potential novel biomarker of AKI induced by I/R.     

Proteus mirabilis Urease: Unsuspected Non-Enzymatic Properties Relevant to Pathogenicity

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures’ supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1β and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca²⁺ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1β. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.     

Role of DNA-Dependent Protein Kinase in Mediating Cyst Growth in Autosomal Dominant Polycystic Kidney Disease

DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein involved in DNA damage response (DDR) signaling that may mediate kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD) due to its pleiotropic effects on proliferation and survival. To test this hypothesis, the expression of DNA-PK in human ADPKD and the in vitro effects of DNA-PK inhibition in a three-dimensional model of Madin-Darby Canine Kidney (MDCK) cyst growth and human ADPKD cells were assessed. In human ADPKD, the mRNA expression for all three subunits of the DNA-PK complex was increased, and using immunohistochemistry, the catalytic subunit (DNA-PKcs) was detected in the cyst lining epithelia of human ADPKD, in a focal manner. In vitro, NU7441 (a DNA-PK kinase inhibitor) reduced MDCK cyst growth by up to 52% after long-term treatment over 6–12 days. Although human ADPKD cell lines (WT9-7/WT9-12) did not exhibit synthetic lethality in response to DNA-PK kinase inhibition compared to normal human kidney cells (HK-2), the combination of low-dose NU7441 enhanced the anti-proliferative effects of sirolimus in WT9-7 and WT9-12 cells by 17 ± 10% and 11 ± 7%, respectively. In conclusion, these preliminary data suggest that DNA-PK mediates kidney cyst growth in vivo without a synthetically lethal interaction, conferring cell-specificity in human ADPKD cells. NU7441 enhanced the anti-proliferative effects of rapamycin complex 1 inhibitors, but the effect was modest.