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Proximal Tubular Cell Angiotensinogen Upregulation by High Glucose is Mediated by Reactive Oxygen Species and not Dependent on AT1 Receptor Activation
Author(s) -
Cypress Michael,
Satou Ryousuke,
Hopfer Ulrich,
Navar Luis Gabriel
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.740.9
Subject(s) - olmesartan , downregulation and upregulation , endocrinology , medicine , chemistry , angiotensin ii , reactive oxygen species , angiotensin ii receptor type 1 , renal sodium reabsorption , receptor , mannitol , renin–angiotensin system , western blot , reabsorption , sodium , biochemistry , biology , organic chemistry , blood pressure , gene
Enhanced proximal tubular angiotensinogen (AGT) levels have been shown in diabetes mellitus, which can be a key factor in intrarenal RAS activation. AGT is upregulated in proximal tubular cells (PTC) by high glucose (HG), but the detailed mechanisms are unclear. Sodium‐glucose cotransporter 2 (SGLT2) is expressed in PTC and is responsible for glucose reabsorption and consequent ROS generation. Therefore, changes in SGLT2 levels may alter the regulation of AGT in PTC under diabetic conditions. The goal of this study was to determine the effects of HG‐induced ROS and angiotensin II type 1 receptor (AT1R) activation, which also generates ROS, on AGT augmentation by HG in PTC. Immortalized mouse PTC derived from the early proximal tubular segment were treated with 5 mM (normal), 15 mM, or 25 mM D‐glucose for up to 24 hours. D‐Mannitol was used as an osmotic control. PTC were treated with sodium pyruvate (1 mM) to determine if enhanced glycolysis stimulates AGT. Moreover, 2.5 mM tempol, an antioxidant, was used to evaluate if HG‐induced AGT augmentation is ROS‐dependent. To determine the role of AT1R activation in AGT upregulation by HG, 1 mM olmesartan, an AT1R blocker, was used. Furthermore, PTC were treated with 10 −10 –10 −7 M angiotensin II (Ang II) and/or HG. In addition, the effect of HG and Ang II on SGLT2 expression was investigated. AGT and SGLT2 protein levels were quantified by Western blot analysis, and AGT mRNA was measured by qRT‐PCR. AGT protein levels were increased by 15 mM (4.4±0.2‐fold compared to control) and 25 mM (4.6±0.2‐fold) glucose. AGT mRNA augmentation was also observed (31.1±3.5‐fold) by HG. On the other hand, AGT levels were not increased by mannitol, indicating that elevated osmolality does not change AGT levels. Pyruvate stimulated AGT expression (10.7±1.0‐fold) under normal glucose conditions, supporting a role for glycolysis in AGT augmentation. Pretreatment with tempol attenuated AGT augmentation in HG‐treated cells (77% suppression compared to HG without tempol), suggesting that ROS mediates HG‐induced AGT upregulation. AT1R blockade and Ang II did not alter AGT responses to HG. Moreover, SGLT2 protein levels were unchanged by HG or Ang II. These findings indicate that AGT upregulation by HG occurs independently of AT1R activation, but is mediated by glycolysis and ROS in PTC. In addition, SGLT2 upregulation is not required for HG‐induced AGT augmentation. Support or Funding Information This study was supported by the National Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337) and the American Heart Association Predoctoral Fellowship (15PRE25090156).