Renal TNF-α Expression Research Articles

Glutathione improves the prognosis of Intrauterine Growth Restriction via downregulated hepatic and renal TNFα expression in Wistar rats

Objective: Oxidative stress is requisite for the development of IUGR, with subsequent manifestation in the reduced levels of antioxidants enzymes. Glutathione protects against free radical-mediated injury, therefore we elucidated on its role in protecting against oxidative stress-induced consequences of IUGR. In order to improve the prognosis of IUGR in affected infants, we investigated the protective role of glutathione in rats exposed to IUGR. Method: Ten female Sprague-Dawley rats were mated overnight. The pregnant rats were divided into 2 groups of 5 rats each. From gestational day 9 until parturition, group A received normal saline while group B received 50 mg/kg daily of L-NAME. Pups from group A were allowed free access to food and water, while group B pups were randomly assigned into 3 groups; G1 pups were left untreated; 1.5g/kg of gluthatione was administered to G2 pups from PND 4-10 and G3 pups from PND 25-31. We measured the body weight of rats; immunolocalized and further quantified TNFα expression in the hepatic and renal tissues. Results: Postnatal GSH administration increased body weight in treated groups exposed to IUGR more significantly from days 4-10 as opposed to days 25-31. IUGR resulted in a significant increase in the TNFα immunoreactivity in the hepatic and renal tissues of the untreated group of rats when compared with the control and treated groups. GSH significantly reduced TNFα immunoreactivity in the kidney and liver of the treated groups, especially the days 4-10. Conclusions: Oral GSH administration regulates the inflammatory response in IUGR at the early neonatal period.

Differential Effects of Specific Deletion of the Dopamine D2 Receptor in Renal Proximal Tubules of Male and Female Mice

Previous work from our laboratory showed that the dopamine D2 receptor (D2R) in the kidney has a direct and significant role in normal blood pressure (BP) control and negative regulation of renal inflammation and injury, indicating that the D2R has protective effects in the kidney by limiting the inflammatory and fibrotic reactions. In mice, renal‐restricted silencing of D2R expression results in renal fibrosis and end organ damage that is reversed by renal‐restricted rescue of D2R expression. The D2R is expressed in renal proximal and distal convoluted tubules. To delineate the role of the proximal tubule in the D2R‐mediated regulation of renal inflammation and injury, and blood pressure we generated Drd2 fl/fl, PSGLT2::Cre+ mice (D2R PT−/−) that lack D2R only in the renal proximal tubule and Drd2 fl/fl, PSGLT2::Cre‐ (D2R PT+/+) mice that do not have the deletion. Mice were genotyped for Drd2 fl/fl and a smaller amplicon representing the Cre deletion mutant. Immunohistochemistry for D2R and a brush border membrane marker confirmed the absence of D2R mainly in the S1 and S2 segments of the renal proximal tubule of D2R PT−/−. BP measured under anesthesia showed that male D2R PT−/− had higher BP than male D2R PT+/+ mice (113±1 vs 102±1 mmHg, n=4/group; P<0.05). The renal expression of the inflammatory markers TNFα (2.6±0.09 vs 1.0±0.03 fold; P<0.05) and TGFβ (2.2±0.19 vs 1.0±0.06; P<0.05), as well as the extracellular matrix proteins Col1a1 (2.0±0.15 vs 1.0±0.06; P<0.05) and Fn1 (2.0±0.11 vs 1.0±0.06; P<0.05), were all increased in male D2R PT−/−, relative to male D2R PT+/+ mice. Sirius Red staining of kidney sections also showed increased (0.52±0.01 vs 0.35±0.01 %; P<0.05) areas of renal cortical fibrosis in male D2R PT−/− mice. Moreover, the renal expression of the inflammatory and pro‐apoptotic marker NGAL (5.9±0.54 vs 1.0±0.03; P<0.05) and the cell proliferation marker Ki‐67 (1.6±0.10 vs 1.0±0.03; P<0.05) were increased in male D2R PT−/− mice. In contrast to the increased BP of male D2R PT−/− mice, BPs in female D2R PT−/− and female D2R PT+/+ mice (103±5 vs 105±3; n=4/group) were similar. The renal expressions of TNFα and TGFβ were increased to the same extent (about 2‐fold) in female D2R PT−/− and male D2R PT−/− mice. The renal expression of Fn1 was increased to a lesser extent (1.4‐fold) in female D2R PT−/− than male D2R PT−/− mice while the renal expression of Col1a1 was not increased in female D2R PT−/− mice. NGAL was increased to a lesser extent (3.1‐fold) while Ki‐67 was increased to a greater extent (9.3‐fold) in female than male D2R PT−/− mice. Our results show that the proximal tubular deletion of the D2R increases BP and markers of extracellular matrix and injury in male D2R PT−/− mice but to a lesser extent or not at all in female D2R PT−/− mice. However, D2R PT deletion increased the markers of inflammation to similar levels in male and female D2R PT−/− mice. Female D2R PT−/− mice had a marked increase in renal Ki‐67, suggesting that the milder effect of the renal D2R deletion in female mice may be related to an increase in cell proliferation.Support or Funding InformationUS National Institutes of Health, R01DK090918, P01HL074940, P01HL068686, R01HL092196, R37HL023081, and R01DK039308This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prorenin receptor mediates inflammation in renal ischemia.

We hypothesized that PRR contributes to renal inflammation in the 2-kidney, 1-clip (2K1C) renal ischaemia model. Male Sprague-Dawley rats were fed normal sodium diet. Blood pressure (BP) was obtained on days 0 and 28 after left renal artery clipping that reduced renal blood flow by 40%. Renal expression of TNF-α, COX-2, NF-κB, IL-1β, MCP-1 and collagen type I were assessed in sham and 2K1C rats with or without left renal administration of scramble or PRR shRNA. At baseline, there were no differences in BP. Compared to sham, MAP significantly increased in clipped animals (sham 102±1.9 vs 2K1C 131.8±3.09mmHg, P<.05) and was not influenced by scramble or PRR shRNA treatment. Compared to sham and contra lateral (non-clipped) kidney, there was upregulation in mRNA and protein expression of PRR (99% and 45%, P<.01), TNF-α (72% and 50%, P<.05), COX-2 (72% and 39%, P<.05), p-NF-κB (92%, P<.05), MCP-1 (87%, P<.05) and immunostaining of collagen type I in the clipped kidney. These increases were not influenced by scramble shRNA. Compared to 2K1C and scramble shRNA, PRR shRNA treatment in the clipped kidney significantly reduced the expression of PRR (62% and 57%, P<.01), TNF-α (51% and 50%, P<.05), COX-2 (50% and 56%, P<.05), p-NF-κB by 68% (P<.05), MCP-1 by 73% (P<.05) and collagen type I respectively. Ang II was increased in both kidneys and did not change in response to scramble or PRR shRNA treatments. We conclude that PRR mediates renal inflammation in renal ischaemia independent of blood pressure and Ang II.

Elsholtzia ciliata (Thunb.) Hylander attenuates renal inflammation and interstitial fibrosis via regulation of TGF-ß and Smad3 expression on unilateral ureteral obstruction rat model

BackgroundRenal interstitial fibrosis is characterized by excessive accumulation of extracellular matrix, which leads to end-stage renal failure. PurposeThe aim of this study was to explore the effect of Elsholtzia ciliata (Thunb.) Hylander ethanol extract (ECE) on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Study designAfter quantitative analysis of ECE using the high performance liquid chromatography-photodiode array (HPLC-PDA) method, an in vitro study was performed to assess the anti-inflammatory and anti-fibrotic effects of ECE, using lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß), respectively. MethodsFor in vivo study, all male Sprague Dawley (SD) rats (n=10/group), except for those in the control group, underwent UUO. The rats were orally treated with water (control), captopril (positive control, 200mg/kg), and ECE (300 and 500mg/kg) for 14 days. ResultsIn ECE, luteolin and rosmarinic acid were relatively abundant among the other flavonoids and phenolic acids. ECE treatment ameliorated LPS-induced overexpression of nuclear factor-κB, tumor necrosis factor (TNF-α), and interleukin-6 and improved oxidative stress in RAW 264.7 cells. Furthermore, ECE treatment suppressed TGF-ß-induced α-smooth muscle actin and matrix metalloproteinase 9 expression in human renal mesangial cells. In the UUO model, 14 consecutive days of ECE treatment improved UUO-induced renal damage and attenuated histopathological alterations and interstitial fibrosis. Moreover, the renal expression of TNF-α, TGF-ß, and Smad 3 were inhibited by ECE treatment. ConclusionTaken together, the effects of ECE may be mediated by blocking the activation of TGF-ß and inflammatory cytokines, leading subsequently to degradation of the ECM accumulation pathway. Based on these findings, ECE might serve as an improved treatment strategy for renal fibrotic disease.

A Nonpeptide Angiotensin II Type 2 Receptor Agonist Prevents Renal Inflammation in Early Diabetes

We hypothesized that direct AT2R stimulation improves albuminuria in diabetes by preventing renal inflammation and improving oxidative stress. Normoglycemic controls (NCs) and streptozotocin-induced diabetes Sprague-Dawley rats (DM) were treated for 4 weeks with vehicle (V) or the AT2R agonist Compound 21 (C21). At the end of study, we evaluated blood pressure, urinary albumin to creatinine ratio (UACR), renal interstitial fluid (RIF) levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), nitric oxide (NO), cGMP, and 8-isoprostane, and renal expression of TNF-α, IL-6, and AT2R. There were no significant differences in blood pressure between different treatments. DM rats demonstrated increased UACR, RIF TNF-α, IL-6 and 8-isoprostane, and messenger RNA (mRNA) for TNF-α and IL-6. DM rats also had reduced RIF NO and cGMP. C21 treatment of DM rats limited the increase in UACR, normalized RIF TNF-α, IL-6 and 8-isoprostane, and in mRNA for TNF-α and IL-6, and increased RIF NO and cGMP. In NC rats, C21 treatment did not change these parameters. AT2R mRNA and protein expressions increased in DM rats compared with NC but were not influenced by C21 treatment. We conclude that direct AT2R stimulation in diabetic rats improves diabetic albuminuria through the prevention of renal inflammation and improved production of NO and cGMP.

Valsartan slows the progression of diabetic nephropathy in db/db mice via a reduction in podocyte injury, and renal oxidative stress and inflammation

Higher doses of AngII (angiotensin II) blockers are intended to optimize albuminuria reduction rather than for blood pressure control in chronic kidney disease. However, the long-term renoprotection of high-dose AngII blockers has yet to be defined. The present study sought to determine whether doses of ARB (AngII receptor blocker) that maximally reduce proteinuria could slow the progression of glomerulosclerosis in the uninephrectomized db/db mouse, a model of Type2 diabetes. Untreated uninephrectomized db/db mice had normal blood pressure, but developed progressive albuminuria and mesangial matrix expansion between 18 and 22weeks of age, which was associated with increased renal expression of TGFβ1 (transforming growth factor β1), PAI-1 (plasminogen-activator inhibitor-1), typeIV collagen and FN (fibronectin). Treatment with valsartan in the drinking water of db/db mice from 18 to 22weeks of age, at a dose that was determined previously to maximally reduce proteinuria, prevented the increases in albuminuria and the markers of renal fibrosis seen in untreated db/db mice. In addition, WT-1 (Wilms tumour protein-1)-immunopositive podocyte numbers were found to be lower in the untreated glomeruli of mice with diabetes. The expression of podocin and nephrin were continually decreased in mice with diabetes between 18 and 22weeks of age. These changes are indicative of podocyte injury and the administration of valsartan ameliorated them substantially. Renal expression of TNFα (tumour necrosis factor α), MCP-1 (monocyte chemoattractant protein-1), Nox2 (NADPH oxidase 2), p22phox and p47phox and urine TBARS (thiobarbituric acid-reacting substance) levels, the markers of renal inflammation and oxidative stress, were increased during disease progression in mice with diabetes. Valsartan treatment was shown to reduce these markers. Thus high doses of valsartan not only reduce albuminuria maximally, but also halt the progression of the glomerulosclerosis resulting from Type2 diabetes via a reduction in podocyte injury and renal oxidative stress and inflammation.

Inflammatory factor-specific sumoylation regulates NF-κB signalling in glomerular cells from diabetic rats

The activation of nuclear factor (NF)-κB by cytokines under hyperglycaemic conditions is a potential mechanism for complications in diabetes. We investigated whether small ubiquitin-like modifier 4 (SUMO4) regulates renal NF-κB signalling in diabetic rats. Histological changes in kidney were analysed in diabetic GK rats. The expressions of tumour necrosis factor (TNF)-α, NF-κB (p65), IκBα and SUMO4 in renal tissues were examined by immunohistochemistry and Western blotting. Primary cultured glomerular endothelial cells from rats were stimulated by TNF-α or interleukin (IL)-2. The renal expression of TNF-α, NF-κB (p65), IκBα and SUMO4 was significantly higher in diabetic GK rats than in control rats. In control rats, no nuclear translocation was observed for IκBα or NF-κB (p65). However, in diabetic GK rats, translocation of NF-κB (p65) and IκBα into the nucleus was observed, and the expression of SUMO4 and IκBα was up-regulated in the glomerular endothelial cells. SUMO4 was localised in both the cytoplasm and nucleus, while IκBα was predominantly located in the nucleus after stimulation with TNF-α. In contrast, SUMO4 was localised in the nucleus, and increased cytoplasm SUMO4 localisation was found after stimulation with IL-2. SUMO4 plays a role in regulating NF-κB signalling in glomerular cells. Cytokines have a unique effect in regulating the sumoylation of NF-κB.

A Novel Role for Tm7sf2 Gene in Regulating TNFα Expression

We have explored the role of Tm7sf2 gene, which codifies for 3β-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2+/+ and Tm7sf2−/− mice, to determine the in vitro effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an in vivo model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2−/− mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.

Combined Aliskiren and Amlodipine Reduce Albuminuria via Reduction in Renal Inflammation in Diabetic Rats

We hypothesized that compared with hydrochlorothiazide (HCTZ), the renin inhibitor aliskiren (ALISK) or amlodipine (AMLO) and their combination reduce albuminuria via reduction in renal inflammation, independent of blood pressure (BP) changes. We studied normal and streptozotocin-induced diabetic (DM) Sprague-Dawley rats treated for 6 weeks with vehicle, ALISK, HCTZ, or AMLO individually and combined and evaluated the effects of treatments on BP, urine albumin to creatinine ratio, renal interstitial fluid levels of angiotensin II, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) and renal expression of TNF-α, IL-6, transforming growth factor beta 1, and nuclear factor kappa B. There were no differences in BP between treatments. Only ALISK and its combinations reduced renal interstitial fluid angiotensin II. Urine albumin to creatinine ratio increased in DM rats and decreased with ALISK alone or combined with HCTZ or AMLO. HCTZ or AMLO individually and combined did not influence urine albumin to creatinine ratio. Renal interstitial fluid TNF-α and IL-6, and the renal expression of TNF-α, IL-6, transforming growth factor beta 1, and nuclear factor kappa B were increased in DM rats. These renal inflammatory markers were reduced only with ALISK or AMLO individually or combined with other treatments. We conclude that ALISK alone and combined with HCTZ or AMLO reduced albuminuria in diabetes via reduction in renal inflammation, independent of BP changes.

(Pro)renin receptor contributes to diabetic nephropathy by enhancing renal inflammation

1. (Pro)renin receptor (PRR) binding to renin or prorenin mediates angiotensin (Ang) II-dependent and -independent effects. Expression of the PRR is increased in kidneys of diabetic rats, but its role in diabetic nephropathy is unknown. In the present study, we investigated the contribution of the PRR to the development of diabetic nephropathy through enhancement of renal production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta. 2. Normoglycaemic control and streptozotocin-diabetic Sprague-Dawley rats were used in the study. The urine albumin : creatinine ratio (UACR), renal interstitial fluid (RIF) levels of AngII, TNF-alpha and IL-1beta and renal expression of TNF-alpha and IL-1beta were evaluated in control, untreated diabetic and diabetic rats treated with either a PRR blocker (PRRB; 0.2 mg/kg per day NH3-RILLKKMPSV-COOH), the AT(1) receptor antagonist valsartan (2 mg/kg per day) or combined therapy, administered directly into the renal cortical interstitium for 14 days via osmotic minipumps. 3. Compared with values in normoglycaemic control rats, UACR and RIF AngII, TNF-alpha and IL-1beta were significantly higher in untreated diabetic rats. Treatment of diabetic rats with the PRRB or valsartan alone and in combination significantly reduced UACR and RIF TNF-alpha and IL-1beta levels. Renal expression of TNF-alpha and IL-1beta was higher in untreated diabetic rats than in control rats, but was reduced significantly following treatment with PRRB or valsartan alone and in combination. Renal PRR expression was increased in untreated and PRRB-treated diabetic rats and reduced in rats receiving valsartan alone or combination therapy. The PRRB had no effect on RIF AngII levels, whereas valsartan alone and in combination with the PRRB significantly increased AngII levels. 4. In conclusion, the PRR is involved in the development and progression of kidney disease in diabetes by enhancing renal production of the inflammatory cytokines TNF-alpha and IL-1beta, independent of renal AngII effects.

Autonomic Renal Denervation Ameliorates Experimental Glomerulonephritis

Increasing evidence indicates that inflammation of visceral organs is significantly affected by the autonomic nervous system. Such neuroimmune interactions have not been studied in the kidney. Here, we show that the rat kidney is innervated by both tyrosine hydroxylase-positive sympathetic efferent nerve fibers and calcitonin gene-related peptide-positive primary afferent nerve fibers, both of which are found in proximity to macrophages and dendritic cells. Complete surgical bilateral renal denervation was performed 2 d before glomerulonephritis was induced by injecting the monoclonal anti-Thy-1.1 antibody OX-7. Denervation significantly reduced albuminuria, mesangiolysis, formation of microaneurysms, deposition of glomerular collagen IV, and expression of TGF-beta compared with sham-operated controls. Accordingly, inflammation, identified by accumulation of interstitial macrophages and renal expression of TNF-alpha, and mesangial cell proliferation were significantly reduced. These findings indicate that autonomic renal denervation ameliorates and, by inference, innervation exacerbates acute inflammation in the kidney; therefore, neurotransmitters or neuropeptides and their receptors might represent novel targets for the treatment of acute glomerulonephritis.