Abstract P129: Angiotensin II Does Not Directly Stimulate The TLR4-MD2-MyD88 Inflammatory Pathway

Current evidence suggests that the deleterious actions of Angiotensin II (Ang II) reflect activation of both innate and adaptive inflammatory pathways. Indeed, recent studies find that Ang II stimulates the TLR-4 signaling cascade to promote cytokine release and renal injury by directly binding to the TLR4 accessory protein MD2 to induce TLR-4 dimerization and internalization that is independent of the AT 1 receptor (AT 1 R). Moreover, knockdown of TLR4 or MD2 attenuates renal injury and expression of pro-inflammatory cytokines in a chronic Ang II infusion model and in rat proximal tubule NRK-52e cells treated with Ang II. In the present study, we evaluated the proposed Ang II-TLR4-MD2 pathway in the NRK-52e cells regarding the stimulation of the chemokine MCP-1 (CCL2) and activation of the TLR4-MyD88 complex. NRK-52e cells, maintained in antibiotic-free media, were transferred to serum-free media prior to stimulation with the TLR4 agonists LPS and palmitate or Ang II for 24 hrs; reported data are the means ± SEM, n=4. The NRK-52e cells were sensitive to a low dose of LPS (1 ng/ml) stimulating CCL2 release 20-fold [Basal: 24.3 ± 1.0 pg/ml vs. 504 ± 30.4 pg/ml, p<0.001]. The LPS induced release of CCL2 was abolished by the specific TLR4 inhibitor Tak-242 [TAK: 23.9 ± 1.3 pg/ml] and was significantly reduced by the MD2 inhibitor L48H37 [135 ± 24.2 pg/ml]. Palmitate [100 μM] also stimulated CCL2 release [482 ± 21.0 pg/ml; p<0.001] that was abolished by TAK [32.3 ± 6.3 pg/ml]. The overall effects are consistent with previously reported responses to LPS and palmitate in these cells, as well as TLR4 protein expression and TAK inhibition. However, Ang II treatment over a dose range of 0.1 to 10 μM for 24 hrs failed to elicit CCL2 release [24.3 ± 1.0, 22.6 ± 0.5, and 27.6 ± 2.3 pg/ml, respectively; P>0.1 vs. Basal]. Ang II [1 μM] also failed to augment the CCL2 response to LPS [496 ± 6.5 pg/ml] or palmitate [511 ± 35.0 pg/ml]. Finally, pre-treatment of the NRK-52e cells with the AT 1 R antagonist candesartan [5 μM] failed to attenuate CCL2 release to LPS [512 ± 21 pg/ml]. We conclude that Ang II does not directly stimulate the TLR4-MD2 complex to induce CCL2 and that the inflammatory events associated with Ang II in vivo may reflect the release of other factors (LPS, AGEs, HMGB1) that serve as TLR-4 agonists.

Direct fibrogenic effects of aldosterone on normotensive kidney: an effect modified by 11β-HSD activity

Aldosterone (Aldo) can be a profibrotic factor in cardiovascular and renal tissues. This study tests the hypothesis that prolonged Aldo exposure is able to directly induce fibrotic changes in the kidney of a normal nonhypertensive animal. Immortalized rat proximal tubule cells (IRPTC) containing 11β-hydroxysteroid dehydrogenase (11β-HSD1) but no mineralocorticoid receptors (MR) and mouse inner medullary collecting duct cells (IMCD) containing 11β-HSD2 and MR were examined. IRPTC exposed to Aldo or corticosterone (10 nM) for 48 h demonstrated no change in collagen production as assessed by Sirius red staining. In contrast, IMCD treated with Aldo exhibited a marked increase in the expression of collagen, fibronectin, and connective tissue growth factor (CTGF), whereas corticosterone alone had no effect. The Aldo-induced overexperession of collagen, fibronectin, and CTGF was substantially attenuated by the MR antagonist RU-318 and by the 11β-HSD end product 11-dehydrocorticosterone, but not by the glucocorticoid receptor antagonist RU-486. In vivo, early fibrotic changes with elevated collagen, fibronectin, and CTGF expression were observed in kidneys isolated from normotensive adrenalectomized mice receiving a continuous infusion of Aldo (8 μg·kg−1·day−1) for 1 wk. These changes were not present in corticosterone-treated mice. Aldo-induced changes were attenuated in adrenally intact mice and in mice treated with RU-318 or 11-dehydrocorticosterone. Thus, extended Aldo exposure produces fibrotic changes in cells containing MR and in normal kidneys. MR antagonists and the end products of 11β-HSD attenuate these fibrogenic effects.

Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro

We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after ∼140 min) into the right jugular vein, and the fluorescence signal (at 650–670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30–40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 ± 0.26, mean ± SE, n = 10), indicating a “priming,” or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 ± 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand β2-glycoprotein.

Low salt intake increases adenosine type 1 receptor expression and function in the rat proximal tubule

Adenosine mediates Na+ reabsorption in the proximal tubule (PT) and other segments by activating adenosine type 1 receptors (A1-AR). We tested the hypothesis that A1-AR in the PT is regulated by salt intake and participates in the kidney adaptation to changes in salt intake. Absolute fluid reabsorption ( Jv) was measured by direct in vivo microperfusion and recollection in rats maintained on low (LS; 0.03% Na, wt/wt)-, normal (NS; 0.3% Na)-, and high-salt (HS; 3.0% Na) diets for 1 wk. The effect of microperfusion of BG9719 a highly selective inhibitor of A1-ARs or adenosine deaminase (AD), which metabolizes adenosine, was measured in each group. Jv was higher in PT from LS rats (LA: 2.8 ± 0.2 vs. NS: 2.1 ± 0.2 nl·min−1·mm−1, P < 0.001). Jv in HS rats was not different from NS. BG9719 reduced Jv in LS rats by 66 ± 6% (LS: 2.8 ± 0.2 vs LS+CVT: 1.3 ± 0.3 nl·min−1·mm−1, P < 0.001), which was greater than its effect in NS (45 ± 4%) or HS (41 ± 4%) rats. AD reduced Jv similarly, suggesting that A1-ARs are activated by local production of adenosine. Expression of A1-AR mRNA and protein was higher ( P < 0.01) in microdissected PTs in LS rats compared with NS and HS. We conclude that A1-ARs in the PT are increased by low salt intake and that A1-AR participates in the increased PT reabsorption of solute and fluid in response to low salt intake.