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Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology.

ABSTRACT: Besides the glomerulus, the tubulointerstitium is often concomitantly affected in certain diseases, e.g., diabetic nephropathy, and activation of the renin-angiotensin system, to a certain extent, worsens its outcome because of perturbations in hemodynamics and possibly tubuloglomerular feedback. Certain studies suggest that pathobiology of the tubulointerstitium is influenced by small GTPases, e.g., Rap1. We investigated the effect of ANG II on inflammatory cytokines, while at the same time focusing on upstream effector of Rap1, i.e., Epac1, and some of the downstream tubular transport molecules, i.e., Na/H exchanger 3 (NHE3). ANG II treatment of LLC-PK1 cells decreased Rap1a GTPase activity in a time- and dose-dependent manner. ANG II treatment led to an increased membrane translocation of NHE3, which was reduced with Epac1 and PKA activators. ANG II-induced NHE3 translocation was notably reduced with the transfection of Rap1a dominant positive mutants, i.e., Rap1a-G12V or Rap1a-T35A. Transfection of cells with dominant negative Rap1a mutants, i.e., Rap1a-S17A, or Epac1 mutant, i.e., EPAC-?cAMP, normalized ANG II-induced translocation of NHE3. In addition, ANG II treatment led to an increased expression of inflammatory cytokines, i.e., IL-1?, IL-6, IL-8, and TNF-?, which was reduced with Rap1a-G12V or Rap1a-T35A transfection, while it reverted to previous comparable levels following transfection of Rap1a-S17A or EPAC-?cAMP. ANG II-induced expression of cytokines was reduced with the treatment with NHE3 inhibitor S3226 or with Epac1 and PKA activators. These data suggest that this novel Epac1-Rap1a-NHE3 pathway conceivably modulates ANG II-induced expression of inflammatory cytokines, and this information may yield the impetus for developing strategies to reduce tubulointertstitial inflammation in various renal diseases.

SUBMITTER: Xie P

PROVIDER: S-EPMC4042107 | biostudies-other | 2014 Jun

REPOSITORIES: biostudies-other

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Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology.

Xie Ping P Joladarashi Darukeshwara D Dudeja Pradeep P Sun Lin L Kanwar Yashpal S YS

American journal of physiology. Renal physiology 20140219 11

Besides the glomerulus, the tubulointerstitium is often concomitantly affected in certain diseases, e.g., diabetic nephropathy, and activation of the renin-angiotensin system, to a certain extent, worsens its outcome because of perturbations in hemodynamics and possibly tubuloglomerular feedback. Certain studies suggest that pathobiology of the tubulointerstitium is influenced by small GTPases, e.g., Rap1. We investigated the effect of ANG II on inflammatory cytokines, while at the same time foc ...[more]

PMID: 24553435

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Project description:Background: Gegen Qinlian decoction (GGQLD) is a typical traditional Chinese medicine (TCM) prescription documented in Shang Han Lun. Clinically, GGQLD has been utilized to manage the inflammatory symptoms of metabolic diseases and to protect against renal damage in China. In the present study, a hypothesis was proposed that the multi-target solution of GGQLD produced anti-inflammatory effects on ameliorating hyperuricemia (HUA). Methods: A total of 30 primary HUA patients receiving GGQLD treatment (two doses daily) for 4 weeks were selected. Then, differences in uric acid (UA) levels and expression of peripheral blood mononuclear cells (PBMCs) and urinary exosomes before and after treatment were analyzed. The therapeutic indexes for the active ingredients in GGQLD against HUA were confirmed through pharmacological subnetwork analysis. Besides, the HUA rat model was established through oral gavage of potassium oxonate and treated with oral GGQLD. In addition, proximal tubular epithelial cells (PTECs) were stimulated by UA and intervened with GGQLD for 48 h. Subsequently, RNA-seq, flow cytometry, and confocal immunofluorescence microscopy were further conducted to characterize the differences in UA-mediated inflammation and apoptosis of human renal tubular epithelial cells pre- and post-administration of GGQLD. In the meanwhile, quantitative real-time PCR (qPCR) was carried out to determine gene expression, whereas a western blotting (WB) assay was conducted to measure protein expression. Results: Our network analysis revealed that GGQLD treated HUA via the anti-inflammatory and antiapoptotic pathways. Additionally, NLPR3 expression significantly decreased in PBMCs and urinary exosomes of HUA patients after GGQLD treatment. In vivo, GGQLD treatment alleviated HUA-induced renal inflammation, which was associated with decreased expression of NLRP3 inflammasomes and apoptosis-related mRNAs. Moreover, GGQLD promoted renal UA excretion by inhibiting the activation of GSDMD-dependent pyroptosis induced by NLRP3 inflammasomes and by reducing apoptosis via the mitochondrial apoptosis signaling pathway in vitro. Conclusion: This study indicates that GGQLD efficiently reduces inflammatory responses while promoting UA excretion in HUA. Our findings also provide compelling evidence supporting the idea that GGQLD protects against the UA-mediated renal tubular epithelial cell inflammation through the mitochondrial apoptosis signaling pathways. Taken together, these findings have demonstrated a novel therapeutic method for the treatment of HUA.

Project description:Background: Qingda granules (QDG) exhibit antihypertension and multiple-target-organ protection. However, the therapeutic potential of QDG on hypertensive renal injury remains unknown. Therefore, the main objective of the current study is to explore the effects and underlying mechanisms of QDG treatment on renal injury in angiotensin (Ang) II-infused mice. Methods and results: Mice were infused with Ang II (500 ng/kg/min) or saline for 4 weeks with subcutaneously implanted osmotic pumps. After infusion, mice in the Ang II + QDG group were intragastrically administrated with QDG daily (1.145 g/kg/day), whereas the control group and Ang II group were intragastrically administrated with the same amount of double-distilled water. Blood pressure of the mice monitored using the CODA™ noninvasive blood pressure system revealed that QDG treatment significantly attenuated elevated blood pressure. Moreover, hematoxylin-eosin staining indicated that QDG treatment ameliorated Ang II-induced renal morphological changes, including glomerular sclerosis and atrophy, epithelial cell atrophy, and tubular dilatation. RNA-sequencing (RNA-seq) identified 662 differentially expressed transcripts (DETs) in renal tissues of Ang II-infused mice, which were reversed after QDG treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis based on DETs in both comparisons of Ang II vs. Control and Ang II + QDG vs. Ang II identified multiple enriched pathways, including apoptosis and p53 pathways. Consistently, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining and Annexin V staining revealed that QDG treatment significantly attenuated Ang II-induced cell apoptosis in renal tissues and cultured renal tubular epithelial cell lines (NRK-52E). Furthermore, western blot analysis indicated that Ang II infusion significantly upregulated the protein expression of p53, BCL2-associated X (BAX), cle-caspase-9, and cle-caspase-3, while downregulating the protein expression of BCL-2 in renal tissues, which were attenuated after QDG treatment. Conclusion: Collectively, QDG treatment significantly attenuated hypertensive renal injury, partially by attenuating renal apoptosis and suppressing p53 pathways, which might be the underlying mechanisms.

Dataset Information

Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology.

Publications

Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology.

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