Primary Tubular Epithelial Cells Research Articles

#2949 Diverse effect of STAT3 inhibition on murine primary tubular epithelial cells

Abstract Background and Aims The incidence of chronic kidney diseases increases worldwide, and kidney fibrosis is the final common pathway regardless of the etiology. TGF-β plays a crucial role in the pathogenesis, affecting pro-fibrotic genes and transcription factors. STAT3 (signal transducer and activator of transcription 3) acts via the JAK/STAT signaling. Inhibition of STAT3 in animal studies was shown to attenuate the progression of kidney fibrosis. Yet, the effect of STAT3 inhibition on renal tubular epithelial cells has not been elucidated. We aimed to investigate in vitro the impact of pharmacological STAT3 inhibition with NSC74859 (NSC) and STATTIC on the expression of fibrosis associated genes of primary tubular epithelial cells. Method Primary tubular epithelial cells from kidneys of C57Bl6/J mice were isolated and used in our studies. Cells (P10-P15) were seeded on gelatin-coated 6-well culture plates (100000 cells/well) and subjected to treatment with 10 ng/ml TGF-β, 100 µM NSC, 10 µM STATTIC or vehicle (n = 3/group) as follows: Control; NSC; STATTIC; TGF-β; TGF-β+NSC; TGF-β+STATTIC. After 24 h treatment, mRNA and protein expressions were analyzed. Statistical evaluation was performed using Kruskal-Wallis test. Results In cells not treated with TGF-β, neither NSC nor STATTIC treatment affected the expression of Tgfb1 (TGF-β) and Acta2 (SMA) genes compared to controls (100%), but reduced Col1a1 expression to 58% and 54%, respectively (p < 0.05). NSC reduced to 54%, while STATTIC tended to increase (169%) the gene expression of Egr2 transcription factor. TGF-β treatment induced STAT3 phosphorylation by 45% accompanied by a fibrotic response and epithelial-to-mesenchymal transition (EMT) as indicated by significantly increased Tgfb1, Acta2, Egr2 and Col1a1 (p < 0.05). NSC treatment did not inhibit the TGF-β-induced Tgfb1 upregulation (178%), but markedly, although not significantly, reduced the expression of Acta2 (100%), Egr2 (98%) and Col1a1 (114%) in the TGF-β+NSC group. Interestingly, although STATTIC treatment dramatically inhibited STAT3 expression (38%, p < 0.05) it significantly enhanced the TGF-β-induced overexpression of Tgfb1 (210%, p < 0.01) and Egr2 (307%, p < 0.05), while markedly reduced Acta2 (64%, p < 0.001) and Col1a1 (81%, p < 0.01) in the TGF-β+STATTIC group. Conclusion Our results indicate that in vitro inhibition of STAT3 using two inhibitors with same mode of action lead to diverse responses in primary murine tubular epithelial cells, partially suppressing EMT markers but inducing pro-fibrotic EGR2. Further studies are needed to clarify the underlying mechanisms.

TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway.

Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage. Notably, the knockout of transient receptor potential channel 6 (TRPC6) has shown promise in alleviating renal fibrosis. However, the regulatory impact of TRPC6 on renal fibrosis remains unclear. In vivo, TRPC6 knockout (TRPC6-/-) mice and age-matched 129 SvEv (WT) mice underwent unilateral renal ischemia-reperfusion (uIR) injury surgery on the left renal pedicle or sham operation. Kidneys and serum were collected on days 7, 14, 21, and 28 after euthanasia. In vitro, primary tubular epithelial cells (PTECs) were isolated from TRPC6-/- and WT mice, followed by treatment with transforming growth factor β1 (TGFβ1) for 72 h. The anti-fibrotic effect of TRPC6-/- and the underlying mechanisms were assessed through hematoxylin-eosin staining, Masson staining, immunostaining, qRT-PCR, and Western blotting. Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFβ1. TRPC6-/- alleviated renal fibrosis by reducing the expression of fibrotic markers (Col-1, α-SMA, and vimentin), as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro. Additionally, we found that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3 beta (GSK3β) signaling pathway, a pivotal player in renal fibrosis, was down-regulated following TRPC6 deletion. These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3β pathway. Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

Lactobacillus acidophilus KBL409 protects against kidney injury via improving mitochondrial function with chronic kidney disease.

Recent advances have led to greater recognition of the role of mitochondrial dysfunction in the pathogenesis of chronic kidney disease (CKD). There has been evidence that CKD is also associated with dysbiosis. Here, we aimed to evaluate whether probiotic supplements can have protective effects against kidney injury via improving mitochondrial function. An animal model of CKD was induced by feeding C57BL/6 mice a diet containing 0.2% adenine. KBL409, a strain of Lactobacillus acidophilus, was administered via oral gavage at a dose of 1 × 109CFU daily. To clarify the underlying mechanisms by which probiotics exert protective effects on mitochondria in CKD, primary mouse tubular epithelial cells stimulated with TGF-β and p-cresyl sulfate were administered with butyrate. In CKD mice, PGC-1α and AMPK, key mitochondrial energy metabolism regulators, were down-regulated. In addition, mitochondrial dynamics shifted toward fission, the number of fragmented cristae increased, and mitochondrial mass decreased. These alterations were restored by KBL409 administration. KBL409 supplementation also improved defects in fatty acid oxidation and glycolysis and restored the suppressed enzyme levels involved in TCA cycle. Accordingly, there was a concomitant improvement in mitochondrial respiration and ATP production assessed by mitochondrial function assay. These favorable effects of KBL409 on mitochondria ultimately decreased kidney fibrosis in CKD mice. In vitro analyses with butyrate recapitulated the findings of animal study. This study demonstrates that administration of the probiotic Lactobacillus acidophilus KBL409 protects against kidney injury via improving mitochondrial function.

Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31.

The synthetic tetra-peptide SS-31 shows promise in alleviating mitochondrial dysfunction associated with common diseases. However, the precise pharmacological basis of its mitoprotective effects remains unknown. To uncover the biological targets of SS-31, we performed a genome-scale CRISPR screen in HK-2 cells, a cell culture model where SS-31 mitigates cisplatin-associated cell death and mitochondrial dysfunction. The identified hit candidate gene was functionally validated using knockout cell lines, siRNA mediated downregulation, and tubular epithelial specific conditional knockout mice. Biochemical interaction studies were also performed to examine the interaction of SS-31 with the identified target protein. Our primary screen and validation studies in HK-2 and primary murine tubular epithelial cells showed that phospholipid scramblase 3 (PLSCR3), an understudied inner mitochondrial membrane protein, is essential for the protective effects of SS-31. For in vivo validation, we generated tubular epithelial-specific knockout mice and found that Plscr3 gene ablation did not influence kidney function under normal conditions or affect the severity of cisplatin and rhabdomyolysis-associated AKI. However, Plscr3 gene deletion completely abrogated the protective effects of SS-31 during cisplatin and rhabdomyolysis-associated AKI. Biochemical studies showed that SS-31 directly binds to a previously uncharacterized N-terminal domain and stimulates PLSCR3 scramblase activity. Finally, PLSCR3 protein expression was found to be increased in the kidneys of AKI patients. PLSCR3 is identified as the essential biological target that facilitates the mitoprotective effects of SS-31 in vitro and in vivo. PLSCR3 agonists can alleviate mitochondrial dysfunction linked to AKI.

Kaempferitrin attenuates unilateral ureteral obstruction-induced renal inflammation and fibrosis in mice by inhibiting NOX4-mediated tubular ferroptosis.

Tubular ferroptosis significantly contributes to renal inflammation and fibrosis, critical factors in chronic kidney disease (CKD). This study aims to investigate Kaempferitrin, a potent flavonoid glycoside from Bauhinia forficata leaves, renowned for its anti-inflammatory and antitumor effects, and to elucidate its potential mechanisms in mitigating inflammation and fibrosis induced by tubular ferroptosis. The study investigated Kaempferitrin's impact on tubular ferroptosis using a unilateral ureteral obstruction (UUO) model-induced renal inflammation and fibrosis. In vitro, erastin-induced ferroptosis in primary tubular epithelial cells (TECs) was utilized to further explore Kaempferitrin's effects. Additionally, NADPH oxidase 4 (NOX4) transfection in TECs and cellular thermal shift assay (CETSA) were conducted to identify Kaempferitrin's target protein. Kaempferitrin effectively improved renal function, indicated by reduced serum creatinine and blood urea nitrogen levels. In the UUO model, it significantly reduced tubular necrosis, inflammation, and fibrosis. Its renoprotective effects were linked to ferroptosis inhibition, evidenced by decreased iron, 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) levels, and increased glutathione (GSH). Kaempferitrin also normalized glutathione peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11(SLC7A11) expression, critical ferroptosis mediators. In vitro, it protected TECs from ferroptosis and consistently suppressed NOX4 expression. NOX4 transfection negated Kaempferitrin's antiferroptosis effects, while CETSA confirmed Kaempferitrin-NOX4 interaction. Kaempferitrin shows promise as a nephroprotective agent by inhibiting NOX4-mediated ferroptosis in tubular cells, offering potential therapeutic value for CKD.

Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

C/EBPα aggravates renal fibrosis in CKD through the NOX4-ROS-apoptosis pathway in tubular epithelial cells

BackgroundChronic kidney disease (CKD) is a prevalent renal disorder with various risk factors. Emerging evidence indicates that the transcriptional factor CCAAT/enhancer binding protein alpha (C/EBPα) may be associated with renal fibrosis. However, the precise role of C/EBPα in CKD progression remains unexplored. MethodsWe investigated the involvement of C/EBPα in CKD using two distinct mouse models induced by folic acid (FA) and unilateral ureteral obstruction (UUO). Additionally, we used RNA sequencing and KEGG analysis to identify potential downstream pathways governed by C/EBPα. FindingsCebpa knockout significantly shielded mice from renal fibrosis and reduced reactive oxygen species (ROS) levels in both the FA and UUO models. Primary tubular epithelial cells (PTECs) lacking Cebpa exhibited reduced apoptosis and ROS accumulation following treatment with TGF-β. RNA sequencing analysis suggested that apoptosis is among the primary pathways regulated by C/EBPα, and identified NADPH oxidoreductase 4 (NOX4) as a key protein upregulated upon C/EBPα induction (ICCB280). Treatment with l-Theanine, a potential NOX4 inhibitor, mitigated renal fibrosis and inflammation in both the FA and UUO mouse models. InterpretationOur study unveils a role for C/EBPα in suppressing renal fibrosis, mitigating ROS accumulation, and reducing cell apoptosis. Furthermore, we investigate whether these protective effects are mediated by C/EBPα's regulation of NOX4 expression. These findings present a promising therapeutic target for modulating ROS and apoptosis in renal tubular cells, potentially offering an approach to treating CKD and other fibrotic diseases.

3D Printed Tubulointerstitium Chip as an In Vitro Testing Platform.

Chronic kidney disease (CKD) ranks as the twelfth leading cause of death worldwide with limited treatment options. The development of in vitro models replicating defined segments of the kidney functional units, the nephrons, in a physiologically relevant and reproducible manner can facilitate drug testing. The aim of this study was to produce an in vitro organ-on-a-chip platform with extrusion-based three-dimensional (3D)printing. The manufacturing of the tubular platform was produced by printing sacrificial fibers with varying diameters, providing a suitable structure for cell adhesion and proliferation. The chip platform was seeded with primary murine tubular epithelial cells and human umbilical vein endothelial cells. The effect of channel geometry, its reproducibility, coatings for cell adhesion, and specific cell markers were investigated. The developed chip presents single and dual channels, mimicking segments of a renal tubule and the capillary network, together with an extracellular matrix gel analogue placed in the middle of the two channels, envisioning the renal tubulointerstitium in vitro. The 3D printed platform enables perfusable circular cross-section channels with fully automated, rapid, and reproducible manufacturing processes at low costs. This kidney tubulointerstitium on-a-chip provides the first step toward the production of more complex in vitro models for drug testing.

Galectin-3 Mediates Endotoxin Internalization and Caspase-4/11 Activation in Tubular Epithelials and Macrophages During Sepsis and Sepsis-Associated Acute Kidney Injury.

Besides being recognized by membrane receptor TLR4, lipopolysaccharide (LPS) can also be internalized into the cytosol and activate Caspase-4/11 pyroptotic pathways to further amplify inflammation in sepsis. The objective of this study was to investigate whether Galectin-3 (Gal3) could promote the uptake of LPS by governing RAGE or administering endocytosis, consequently activating Caspase 4/11 and mediating pyroptosis in sepsis-associated acute kidney injury (SA-AKI). By pinpointing Gal3, LPS, and EEA1 (endosome-marker) or LAMP1 (lysosome-marker) respectively, immunofluorescence discovered that Gal3 and LPS were mainly aggregated in early endosomes initially and translocated into lysosomes afterwards. In cells and animal models, Gal3 and the Caspase-4/11 pathways were simultaneously activated, and the overexpression of Gal3 could exacerbate pyroptosis, whereas inhibition of Gal3 or the knockdown of its expression could ameliorate pyroptosis, reduce the pathological changes of SA-AKI and improve the survival of the animals with SA-AKI. Silencing RAGE reduced pyroptosis in primary tubular epithelial cells (PTCs) activated by Gal3 and LPS but not in cells activated by Gal3 and outer membrane vesicles (with LPS inside), whereas pyroptosis in both was reduced by blockade of Gal3, indicating Gal3 promoted pyroptosis through both RAGE-dependent and RAGE-independent pathways. Our investigation further revealed a positive correlation between serum Gal3 and pyroptotic biomarkers IL-1 beta and IL-18 in patients with sepsis, and that serum Gal3 was an independent risk factor for mortality. Through our collective exploration, we unraveled the significant role of Gal3 in the internalization of LPS and the provocation of more intense pyroptosis, thus making it a vital pathogenic factor in SA-AKI and a possible therapeutic target.Gal3 enabled the internalization of endotoxin into endosomes and lysosomes via both RAGE-dependent (A) and RAGE-independent (B) pathways, leading to pyroptosis. The suppression of Gal3 curbed Caspase4/11 noncanonical inflammasomes and diminished sepsis and SA-AKI.

Exaggerated Angiotensin II-Induced NF-kB p65 Signaling and Oxidative Stress in GSTM1-Deficient Kidney Primary Tubular Epithelial Cells Is Ameliorated by Hydrogen Sulfide

Exaggerated Angiotensin II-Induced NF-kB p65 Signaling and Oxidative Stress in GSTM1-Deficient Kidney Primary Tubular Epithelial Cells Is Ameliorated by Hydrogen Sulfide

Pioglitazone Protects Tubular Epithelial Cells during Kidney Fibrosis by Attenuating miRNA Dysregulation and Autophagy Dysfunction Induced by TGF-β.

Excessive renal TGF-β production and pro-fibrotic miRNAs are important drivers of kidney fibrosis that lack any efficient treatment. Dysfunctional autophagy might play an important role in the pathogenesis. We aimed to study the yet unknown effects of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone (Pio) on renal autophagy and miRNA dysregulation during fibrosis. Mouse primary tubular epithelial cells (PTEC) were isolated, pre-treated with 5 µM pioglitazone, and then stimulated with 10 ng/mL TGF-β1 for 24 h. Male 10-week-old C57Bl6 control (CTL) and TGF-β overexpressing mice were fed with regular chow (TGF) or Pio-containing chow (20 mg/kg/day) for 5 weeks (TGF + Pio). PTEC and kidneys were evaluated for mRNA and protein expression. In PTEC, pioglitazone attenuated (p < 0.05) the TGF-β-induced up-regulation of Col1a1 (1.4-fold), Tgfb1 (2.2-fold), Ctgf (1.5-fold), Egr2 (2.5-fold) mRNAs, miR-130a (1.6-fold), and miR-199a (1.5-fold), inhibited epithelial-to-mesenchymal transition, and rescued autophagy function. In TGF mice, pioglitazone greatly improved kidney fibrosis and related dysfunctional autophagy (increased LC3-II/I ratio and reduced SQSTM1 protein content (p < 0.05)). These were accompanied by 5-fold, 3-fold, 12-fold, and 2-fold suppression (p < 0.05) of renal Ccl2, Il6, C3, and Lgals3 mRNA expression, respectively. Our results implicate that pioglitazone counteracts multiple pro-fibrotic processes in the kidney, including autophagy dysfunction and miRNA dysregulation.

The aging kidney is characterized by tubuloinflammaging, a phenotype associated with MHC-II gene expression.

Even during physiologic aging, the kidney experiences a loss of mass and a progressive functional decline. This is clinically relevant as it leads to an increased risk of acute and chronic kidney disease. The kidney tubular system plays an important role in the underlying aging process, but the involved cellular mechanisms remain largely elusive. Kidneys of 3-, 12- and 24-month-old male C57BL/6J mice were used for RNA sequencing, histological examination, immunostaining and RNA-in-situ-hybridization. Single cell RNA sequencing data of differentially aged murine and human kidneys was analyzed to identify age-dependent expression patterns in tubular epithelial cells. Senescent and non-senescent primary tubular epithelial cells from mouse kidney were used for in vitro experiments. During normal kidney aging, tubular cells adopt an inflammatory phenotype, characterized by the expression of MHC class II related genes. In our analysis of bulk and single cell transcriptional data we found that subsets of tubular cells show an age-related expression of Cd74, H2-Eb1 and H2-Ab1 in mice and CD74, HLA-DQB1 and HLADRB1 in humans. Expression of MHC class II related genes was associated with a phenotype of tubular cell senescence, and the selective elimination of senescent cells reversed the phenotype. Exposure to the Cd74 ligand MIF promoted a prosenescent phenotype in tubular cell cultures. Together, these data suggest that during normal renal aging tubular cells activate a program of 'tubuloinflammaging', which might contribute to age-related phenotypical changes and to increased disease susceptibility.

Differences in the Susceptibility of Human Tubular Epithelial Cells for Infection with Orthohantaviruses.

Diseases induced by infection with pathogenic orthohantaviruses are characterized by a pronounced organ-specific manifestation. Pathogenic Eurasian orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS) with often massive proteinuria. Therefore, the use of a relevant kidney cell culture would be favorable to analyze the underlying cellular mechanisms of orthohantavirus-induced acute kidney injury (AKI). We tested different human tubular epithelial cell lines for their suitability as an in vitro infection model. Permissiveness and replication kinetics of highly pathogenic Hantaan virus (HTNV) and non-/low-pathogenic Tula virus (TULV) were analyzed in tubular epithelial cell lines and compared to human primary tubular epithelial cells. Ana-lysis of the cell line HK-2 revealed the same results for viral replication, morphological and functional effects as observed for HTNV in primary cells. In contrast, the cell lines RPTEC/TERT1 and TH1 demonstrated only poor infection rates after inoculation with HTNV and are unusable as an infection model. While pathogenic HNTV infects primary tubular and HK-2 cells, non-/low-pathogenic TULV infects neither primary tubular cells nor the cell line HK-2. Our results show that permissiveness of renal cells varies between orthohantaviruses with differences in pathogenicity and that HK-2 cells demonstrate a suitable in vitro model to study viral tropism and pathogenesis of orthohantavirus-induced AKI.

#4419 THE IMPACT OF TRANSGLUTAMINASE 2 INHIBITION ON HYPERTENSION-INDUCED FIBROSIS IN HYPERTENSIVE NEPHROPATHY

Abstract Background and Aims Hypertensive nephropathy is a chronic disease that requires the novel treatment. Transglutaminase 2 (TG2), linked to various diseases including cardiovascular and kidney diseases, has been suggested to play a role in the development of hypertensive nephropathy through its contribution to fibrous tissue formation, oxidative stress regulation, and inflammation. This study aimed to explore the role of TG2 in the development of hypertensive nephropathy and evaluate its potential as a therapeutic target. Method An in vitro hypertensive model was established using a device that mimics hypertension by applying rotational force. An in vivo hypertensive model was established in rats through subtotal 5/6 nephrectomy to induce fibrosis. The relationship between TG2 expression and the progression of hypertension-induced fibrosis was analyzed, and the impact of TG2 inhibition on fibrosis was evaluated using the TG2 inhibitor, cysteamine. Results In the in vitro study, rotational force was applied to induce hypertension using a hypertensive mimic device, which resulted in fibrosis in primary human glomerular endothelial cells and tubular epithelial cells and increased expression of TG2. The administration of cysteamine at concentrations of 0.5 mM, 1 mM, and 2 mM decreased fibrosis and phosphorylated P65 in a dose-dependent manner. The in vivo hypertensive model demonstrated a significant increase in blood pressure (P&amp;lt;0.001) and kidney fibrosis (P&amp;lt;0.001) over time at 4 and 8 weeks after surgery. A significant increase in TG2 expression was observed as hypertension and fibrosis progressed (P&amp;lt;0.001). Conclusion The progression of hypertension-induced fibrosis was accompanied by an increase in TG2 expression. Inhibition of TG2 showed improvement in fibrosis, suggesting further study of its mechanism may provide new therapeutic options for hypertensive nephropathy.

#4966 CELLULAR PRION PROTEIN ACTIVATES THE TBK1-IRF3 SIGNALING PATHWAY TO AGGRAVATE RENAL FIBROSIS

Abstract Background and Aims Chronic kidney disease (CKD) is an irreversible degenerative disease characterized by gradual loss of renal function, contributing to high morbidity and mortality as well as heavy economic cost to society. Clinically, there are few available strategies to slow CKD progression. Because the uremic phenotypes include many features of aging, CKD is considered as a premature aging syndrome. Neurodegenerative diseases, such as Alzheimer's disease, and Parkinson's disease are extensively studied aging-related diseases, in which cytotoxic proteins misfolding and aggregation in neural cells is the main characteristic. The misfolding and aggregation of the prion protein is tightly linked to the development of prion diseases. Interestingly, a remarkable elevation of cellular prion protein (PrPC) in the urine and plasma of CKD patients was reported. However, the role of PrPC in the pathogenesis of kidney diseases remains largely unknown. The aim of the current study is to investigate the role of PrPC in renal fibrosis. Methods Immunohistochemistry staining was conducted to evaluate the expression of PrPC in renal biopsies from CKD patients. Enzyme linked immunosorbent assay (ELISA) was performed to detect urinary PrPC of CKD patients. Animal model of renal fibrosis was induced by unilateral ureteral occlusion (UUO). Western blot, real time PCR and immunofluorescence staining were performed to evaluate the expression and distribution of PrPC. Proximal tubule specific Prnp knockout mice (PEPCK-Prnp-KO) was constructed to explore the pathogenic role of PrPC in renal fibrosis. Primary proximal tubular epithelial cells (PTCs) overexpressed PrPC were collected for RNA-seq analysis. TBK1 inhibitor amlexanox, and IRF3 siRNA were used to block TBK1-IRF3 pathway in rat tubular epithelial cells (NRK-52E), respectively. Amlexanox was administered orally to UUO mice. H&amp;E, Masson and Sirius red staining were used to estimate renal pathology. Results The protein expression levels of PrPC both in renal biopsies and urine from CKD patients were elevated and significantly correlated with the severity of interstitial fibrosis and the decline of eGFR. PrPC proteins were significantly increased, and aggregated in proximal renal tubules in fibrotic renal tissues induced by UUO. Compared with wild-type littermates, PEPCK-Prnp-KO mice showed reduced extracellular matrix accumulation and interstitial inflammation at day 7 after UUO. PrPC provokes profibrotic response of renal tubular cells via the TBK1-IRF3 pathway. TBK1 inhibitor amlexanox or silencing endogenous IRF3 significantly inhibited PrPC-induced profibrogenic phenotypic transformation in NRK-52 cells. Amlexanox attenuated interstitial fibrosis induced by UUO. Conclusion Our experimental results indicate that PrPC-TBK1-IRF3 pathway plays a detrimental role in profibrogenic transformation of renal tubular cells and thus contributes to interstitial fibrosis. Blocking TBK1 activation is a plausible strategy for therapeutic intervention of chronic kidney disorders.

#5526 COMPLEMENT C3 EXERTS DIRECT PRO-FIBROTIC EFFECTS ON KIDNEY TUBULES

Abstract Background and Aims Renal complement expression has been observed in both experimental and human kidney diseases. We have previously reported massive tubular C3 expression in fibrotic TGF-beta1 transgenic mice (Nephrol Dial Transpl 2015, 30; S3:FP301). Still, little is known whether this intra-renal complement is only an inflammatory marker of renal fibrosis or also exerts direct pro-fibrotic effects in the pathogenesis. In the present study we aimed to investigate the effect of C3a receptor activation in murine primary tubular epithelial cells. Method Primary tubular epithelial cells (TEC) were isolated from kidneys of four weeks-old male C57Bl6 mouse. The cells were grown in DMEM/12 medium supplemented with 2% FBS and recombinant 5 ng/ml EGF, and characterized for epithelial and mesenchymal markers. Verified TEC between P4 and P10 passages were used for the experiments. TEC cells were seeded on 6-well plates and treated for 24 hours with PBS (CTL, n=3), 100 nM C3a receptor agonist peptide (C3aR, n=3) or 10 ng/ml recombinant TGF-beta1 (TGFb, n=3). Then, mRNA and protein expressions were assessed and evaluated using Kruskal-Wallis test (p&amp;lt;0.05). Results TGF-beta induced a 2-fold Col1a1 and 1,6-fold Tgfb1 mRNA expression, accompanied by 4-fold Egr2 but did not affect Ccl2 or C3 mRNA expression. In contrast, C3aR group depicted 1,6-fold C3 and 1,8-fold Ccl2 mRNA expression, an only 1,6-fold Egr2 but a 2-fold Col1a1 and 1,5-fold Tgfb1 mRNA expression. Despite the similar Tgfb1 mRNA overexpression in TGFb and C3aR groups, marked TGFB1 protein overexpression (15-fold) was only observed in TGF-beta treated cells but not in the C3aR group. Conclusion Our study data indicate that renal complement C3 production can exert autocrine / paracrine direct pro-fibrotic effects on tubular epithelial cells unrelated to TGF-beta protein levels. Therefore, local C3 overexpression might play a significant pathogenetic role in kidney fibrosis.

Regulatory roles of SP-A and exosomes in pneumonia-induced acute lung and kidney injuries.

Pneumonia-induced sepsis can cause multiple organ dysfunction including acute lung and kidney injury (ALI and AKI). Surfactant protein A (SP-A), a critical innate immune molecule, is expressed in the lung and kidney. Extracellular vesicles like exosomes are involved in the processes of pathophysiology. Here we tested one hypothesis that SP-A regulates pneumonia-induced AKI through the modulation of exosomes and cell death. Wild-type (WT), SP-A knockout (KO), and humanized SP-A transgenic (hTG, lung-specific SP-A expression) mice were used in this study. After intratracheal infection with Pseudomonas aeruginosa, KO mice showed increased mortality, higher injury scores, more severe inflammation in the lung and kidney, and increased serum TNF-α, IL-1β, and IL-6 levels compared to WT and hTG mice. Infected hTG mice exhibited similar lung injury but more severe kidney injury than infected WT mice. Increased renal tubular apoptosis and pyroptosis in the kidney of KO mice were found when compared with WT and hTG mice. We found that serum exosomes from septic mice cause ALI and AKI through mediating apoptosis and proptosis when mice were injected intravenously. Furthermore, primary proximal tubular epithelial cells isolated from KO mice showed more sensitivity than those from WT mice after exposure to septic serum exosomes. Collectively, SP-A attenuates pneumonia-induced ALI and AKI by regulating inflammation, apoptosis and pyroptosis; serum exosomes are important mediators in the pathogenesis of AKI.

Exposure to Nanoplastics Induces an Inflammatory Response in Healthy and Type 2 Diabetic Primary Human Proximal Tubular Epithelial Cells

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Hypoxia induces polycystin-1 expression in the renal epithelium.

Mutations in polycystin-1 which is encoded by the PKD1 gene are the main causes for the development of autosomal dominant polycystic kidney disease. However, only little is known about the physiological function of polycystin-1 and even less about the regulation of its expression. Here, we show that expression of PKD1 is induced by hypoxia and compounds that stabilize the hypoxia-inducible transcription factor (HIF) 1α in primary human tubular epithelial cells. Knockdown of HIF subunits confirms HIF-1α-dependent regulation of polycystin-1 expression. Furthermore, HIF ChIP-seq reveals that HIF interacts with a regulatory DNA element within the PKD1 gene in renal tubule-derived cells. HIF-dependent expression of polycystin-1 can also be demonstrated in vivo in kidneys of mice treated with substances that stabilize HIF. Polycystin-1 and HIF-1α have been shown to promote epithelial branching during kidney development. In line with these findings, we show that expression of polycystin-1 within mouse embryonic ureteric bud branches is regulated by HIF. Our finding links expression of one of the main regulators of accurate renal development with the hypoxia signalling pathway and provides additional insight into the pathophysiology of polycystic kidney disease.