Endoplasmic reticulum stress inhibition blunts the development of essential hypertension in the spontaneously hypertensive rat.

Essential hypertension is the leading cause of premature death worldwide. However, hypertension's cause remains uncertain. endoplasmic reticulum (ER) stress has recently been associated with hypertension, but it is unclear whether ER stress causes hypertension. To clarify this question, we examined if ER stress occurs in blood vessels before the development of hypertension and if ER stress inhibition would prevent hypertension development. We used the spontaneously hypertensive rat (SHR) as a model of human essential hypertension and the Wistar-Kyoto (WKY) rat as its normotensive control. Resistance arteries collected from young rats determined that ER stress was present in SHR vessels before the onset of hypertension. To assess the effect of ER stress inhibition on hypertension development, another subset of rats were treated with 4-phenylbutyric acid (4-PBA; 1 g·kg-1·day-1) for 8 wk from 5 wk of age. Blood pressure was measured via radiotelemetry and compared with untreated SHR and WKY rats. Mesenteric resistance arteries were collected and assessed for structural and functional changes associated with hypertension. Systolic and diastolic blood pressures were significantly lower in the 4-PBA-treated SHR groups than in untreated SHRs. Additionally, 4-PBA significantly decreased the media-to-lumen ratio and ER stress marker expression, improved vasodilatory response, and reduced contractile responses in resistance arteries from SHRs. Overall, ER stress inhibition blunted the development of hypertension in the SHR. These data add evidence to the hypothesis that a component of hypertension in the SHR is caused by ER stress. NEW & NOTEWORTHY In this study, 4-phenylbutyric acid's (4-PBA's) molecular chaperone capability was used to inhibit endoplasmic reticulum (ER) stress in the small arteries of young spontaneously hypertensive rats (SHRs) and reduce their hypertension. These effects are likely mediated through 4-PBA's effects to reduce resistant artery contractility and increase nitric oxide-mediated endothelial vasodilation through a process preventing endothelial dysfunction. Overall, ER stress inhibition blunted the development of hypertension in this young SHR model. This suggests that a component of the increase in blood pressure found in SHRs is due to ER stress. However, it is important to note that inhibition of ER stress was not able to fully restore the blood pressure to normal, suggesting that a component of hypertension may not be due to ER stress. This study points to the inhibition of ER stress as an important new physiological pathway to lower blood pressure, where other known approaches may not achieve blood pressure-lowering targets.

IL-6 mediates ER expansion during hyperpolarization of alternatively activated macrophages.

Although recent evidence has shown that IL-6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL-6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1-XBP1 expansion pathway, IL-4/IL-13 and IL-4/IL-13/IL-6-mediated alternative programming of murine bone marrow-derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase-1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1-XBP1 arm was achieved using STF-083010 and was verified by RT-PCR. The addition of IL-6 to the conventional alternative programming cocktail IL-4/IL-13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response-mediated induction of molecular chaperones. IRE1-XBP1 inhibition substantially reduced the IL-6-mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL-6 enhances ER expansion and the profibrotic capacity of IL-4/IL-13-mediated activation of macrophages. Therapeutic strategies targeting IL-6 or the IRE1-XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.

Endoplasmic reticulum stress inhibition limits the progression of chronic kidney disease in the Dahl salt-sensitive rat.

Proteinuria is one of the primary risk factors for the progression of chronic kidney disease (CKD) and has been implicated in the induction of endoplasmic reticulum (ER) stress. We hypothesized that the suppression of ER stress with a low molecular weight chemical chaperone, 4-phenylbutyric acid (4-PBA), would reduce the severity of CKD and proteinuria in the Dahl salt-sensitive (SS) hypertensive rat. To induce hypertension and CKD, 12-wk-old male rats were placed on a high-salt (HS) diet for 4 wk with or without 4-PBA treatment. We assessed blood pressure and markers of CKD, including proteinuria, albuminuria, and renal pathology. Furthermore, we determined if HS feeding resulted in an impaired myogenic response, subsequent to ER stress. 4-PBA treatment reduced salt-induced hypertension, proteinuria, and albuminuria and preserved myogenic constriction. Furthermore, renal pathology was reduced with 4-PBA treatment, as indicated by lowered expression of profibrotic markers and fewer intratubular protein casts. In addition, ER stress in the glomerulus was reduced, and the integrity of the glomerular filtration barrier was preserved. These results suggest that 4-PBA treatment protects against proteinuria in the SS rat by preserving the myogenic response and by preventing ER stress, which led to a breakdown in the glomerular filtration barrier. As such, alleviating ER stress serves as a viable therapeutic strategy to preserve kidney function and to delay the progression of CKD in the animal model under study.

Analysis of the potency of various low molecular weight chemical chaperones to prevent protein aggregation.

Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin-induced pulmonary fibrosis.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SREBP-1 Mediates Angiotensin II-Induced TGF-ß1 Upregulation and Glomerular Fibrosis.

Angiotensin II is an important mediator of CKD of diverse etiology. A common pathologic feature of CKD is glomerular fibrosis, a central mediator of which is the profibrotic cytokine TGF-ß. The mechanisms underlying the induction of TGF-ß and matrix by angiotensin II are not completely understood. Recent studies showed that overexpression of the transcription factor SREBP-1 induces glomerular sclerosis and that angiotensin II can activate SREBP-1 in tubular cells. We thus studied whether SREBP-1 is activated by angiotensin II and mediates angiotensin II-induced profibrogenic responses in primary rat mesangial cells. Treatment of cells with angiotensin II induced the upregulation and activation of SREBP-1. Angiotensin II-induced activation of SREBP-1 required signaling through the angiotensin II type I receptor and activation of PI3K/Akt in addition to the chaperone SCAP and protease S1P. Notably, angiotensin II-induced endoplasmic reticulum stress was identified as a key mediator of Akt-SREBP-1 activation, and inhibition of endoplasmic reticulum stress or SREBP-1 prevented angiotensin II-induced SREBP-1 binding to the TGF-ß promoter, TGF-ß upregulation, and downstream fibronectin upregulation. Endoplasmic reticulum stress alone, however, did not induce TGF-ß upregulation despite activating SREBP-1. Although not required for SREBP-1 activation by angiotensin II, EGF receptor signaling was necessary for activation of the SREBP-1 cotranscription factor Sp1, which provided a required second signal for TGF-ß upregulation. In vivo, endoplasmic reticulum stress and SREBP-1-dependent effects were induced in glomeruli of angiotensin II-infused mice, and administration of the SREBP inhibitor fatostatin prevented angiotensin II-induced TGF-ß upregulation and matrix accumulation. SREBP-1 and endoplasmic reticulum stress thus provide potential novel therapeutic targets for the treatment of CKD.

Decreased endogenous production of hydrogen sulfide accelerates atherosclerosis.

BACKGROUND: Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H2S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H2S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H2S in atherosclerosis development. METHODS AND RESULTS: Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N-acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. CONCLUSIONS: Endogenously synthesized H2S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H2S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H2S pathway is an important therapeutic target for protection against atherosclerosis.

Integrated stress response modulates cellular redox state via induction of cystathionine γ-lyase: cross-talk between integrated stress response and thiol metabolism.

The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine ß-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4(-/-)) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4(-/-) MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4(-/-) MEFs could be rescued from l-Hcy-induced apoptosis by ß-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine ß-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4(+/+) MEFs. Consistent with ATF4(-/-) MEFs, CSE(-/-) MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE(+/+) MEFs. Liver and kidney GSH levels were also reduced in CSE(-/-) mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.

Interrelationship between cardiac hypertrophy, heart failure, and chronic kidney disease: endoplasmic reticulum stress as a mediator of pathogenesis.

Synthesis of transmembrane and secretory proteins occurs within the endoplasmic reticulum (ER) and is extremely important in the normal functioning of both the heart and kidney. The dysregulation of protein synthesis/processing within the ER causes the accumulation of unfolded proteins, thereby leading to ER stress and the activation of the unfolded protein response. Sarcoplasmic reticulum/ER Ca2+ disequilibrium can lead to cardiac hypertrophy via cytosolic Ca2+ elevation and stimulation of the Ca2+/calmodulin, calcineurin, NF-AT3 pathway. Although cardiac hypertrophy may be initially adaptive, prolonged or severe ER stress resulting from the increased protein synthesis associated with cardiac hypertrophy can lead to apoptosis of cardiac myocytes and result in reduced cardiac output and chronic heart failure. The failing heart has a dramatic effect on renal function because of inadequate perfusion and stimulates the release of many neurohumoral factors that may lead to further ER stress within the heart, including angiotensin II and arginine-vasopressin. Renal failure attributable to proteinuria and uremia also induces ER stress within the kidney, which contributes to the transformation of tubular epithelial cells to a fibroblast-like phenotype, fibrosis, and tubular cell apoptosis, further diminishing renal function. As a consequence, cardiorenal syndrome may develop into a vicious circle with poor prognosis. New therapeutic modalities to alleviate ER stress through stimulation of the cytoprotective components of the unfolded protein response, including GRP78 upregulation and eukaryotic initiation factor 2α phosphorylation, may hold promise to reduce the high morbidity and mortality associated with cardiorenal syndrome.

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death.

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)ß), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

TDAG51 mediates epithelial-to-mesenchymal transition in human proximal tubular epithelium.

Epithelial-to-mesenchymal transition (EMT) contributes to renal fibrosis in chronic kidney disease. Endoplasmic reticulum (ER) stress, a feature of many forms of kidney disease, results from the accumulation of misfolded proteins in the ER and leads to the unfolded protein response (UPR). We hypothesized that ER stress mediates EMT in human renal proximal tubules. ER stress is induced by a variety of stressors differing in their mechanism of action, including tunicamycin, thapsigargin, and the calcineurin inhibitor cyclosporine A. These ER stressors increased the UPR markers GRP78, GRP94, and phospho-eIF2α in human proximal tubular cells. Thapsigargin and cyclosporine A also increased cytosolic Ca(2+) concentration and T cell death-associated gene 51 (TDAG51) expression, whereas tunicamycin did not. Thapsigargin was also shown to increase levels of active transforming growth factor (TGF)-ß1 in the media of cultured human proximal tubular cells. Thapsigargin induced cytoskeletal rearrangement, ß-catenin nuclear translocation, and α-smooth muscle actin and vinculin expression in proximal tubular cells, indicating an EMT response. Subconfluent primary human proximal tubular cells were induced to undergo EMT by TGF-ß1 treatment. In contrast, tunicamycin treatment did not produce an EMT response. Plasmid-mediated overexpression of TDAG51 resulted in cell shape change and ß-catenin nuclear translocation. These results allowed us to develop a two-hit model of ER stress-induced EMT, where Ca(2+) dysregulation-mediated TDAG51 upregulation primes the cell for mesenchymal transformation via Wnt signaling and then TGF-ß1 activation leads to a complete EMT response. Thus the release of Ca(2+) from ER stores mediates EMT in human proximal tubular epithelium via the induction of TDAG51.

Induction of the unfolded protein response after monocyte to macrophage differentiation augments cell survival in early atherosclerotic lesions.

Endoplasmic reticulum (ER) stress causes macrophage cell death within advanced atherosclerotic lesions, thereby contributing to necrotic core formation and increasing the risk of atherothrombotic disease. However, unlike in advanced lesions, the appearance of dead/apoptotic macrophages in early lesions is less prominent. Given that activation of the unfolded protein response (UPR) is detected in early lesion-resident macrophages and can enhance cell survival against ER stress, we investigated whether UPR activation occurs after monocyte to macrophage differentiation and confers a cytoprotective advantage to the macrophage. Human peripheral blood monocytes were treated with monocyte colony-stimulating factor to induce macrophage differentiation, as assessed by changes in ultrastructure and scavenger receptor expression. UPR markers, including GRP78, GRP94, and spliced XBP-1, were induced after macrophage differentiation and occurred after a significant increase in de novo protein synthesis. UPR activation after differentiation reduced macrophage cell death by ER stress-inducing agents. Further, GRP78 overexpression in macrophages was sufficient to reduce ER stress-induced cell death. Consistent with these in vitro findings, UPR activation was observed in viable lesion-resident macrophages from human carotid arteries and from the aortas of apoE(-/-) mice. However, no evidence of apoptosis was observed in early lesion-resident macrophages from the aortas of apoE(-/-) mice. Thus, our findings that UPR activation occurs during macrophage differentiation and is cytoprotective against ER stress-inducing agents suggest an important cellular mechanism for macrophage survival within early atherosclerotic lesions.

Inhibitory Antibodies against PCSK9 Reduce Surface CD36 and Mitigate Diet-Induced Renal Lipotoxicity.

Background: PCSK9 modulates the uptake of circulating lipids through a range of receptors, including the low-density lipoprotein receptor (LDLR) and CD36. In the kidney, CD36 is known to contribute to renal injury through pro-inflammatory and -fibrotic pathways. In this study, we sought to investigate the role of PCSK9 in modulating renal lipid accumulation and injury through CD36 using a high fat diet (HFD)-induced murine model. Methods: The effect of PCSK9 on the expression of CD36 and intracellular accumulation of lipid was examined in cultured renal cells and in the kidneys of male C57BL/6J mice. The effect of these findings was subsequently explored in a model of HFD-induced renal injury in Pcsk9 -/- and Pcsk9 +/+ littermate control mice on a C57BL/6J background. Results: In the absence of PCSK9, we observed heightened CD36 expression levels, which increased free fatty acid (FFA) uptake in cultured renal tubular cells. As a result, PCSK9 deficiency was associated with an increase in long-chain saturated FFA-induced ER stress. Consistent with these observations, Pcsk9-/- mice fed a HFD displayed elevated ER stress, inflammation, fibrosis, and renal injury relative to HFD-fed control mice. In contrast to Pcsk9-/- mice, pretreatment of WT C57BL/6J mice with evolocumab, an anti-PCSK9 monoclonal antibody (mAb) that binds to and inhibits the function of circulating PCSK9, protected against HFD-induced renal injury in association with reducing cell surface CD36 expression on renal epithelia. Conclusions: We report that circulating PCSK9 modulates renal lipid uptake in a manner dependent on renal CD36. In the context of increased dietary fat consumption, the absence of circulating PCSK9 may promote renal lipid accumulation and subsequent renal injury. However, although the administration of evolocumab blocks the interaction of PCSK9 with the LDLR, this evolocumab/PCSK9 complex can still bind CD36, thereby protecting against HFD-induced renal lipotoxicity.

Binding of anti-GRP78 autoantibodies to cell surface GRP78 increases tissue factor procoagulant activity via the release of calcium from endoplasmic reticulum stores.

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.

Inhibition of histone deacetylation with vorinostat does not prevent tunicamycin-mediated acute kidney injury.

Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory drugs, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic that induces ER stress and is a commonly used model of AKI. 4-phenylbutyrate (4-PBA) is a chemical chaperone and histone deacetylase (HDAC) inhibitor and has been shown to protect the kidney from ER stress, apoptosis, and structural damage in a tunicamycin model of AKI. The renal protection provided by 4-PBA is attributed to its ability to prevent misfolded protein aggregation and inhibit ER stress; however, the HDAC inhibitor effects of 4-PBA have not been examined in the TM-induced model of AKI. As such, the main objective of this study was to determine if histone hyperacetylation provides any protective effects against TM-mediated AKI. The FDA-approved HDAC inhibitor vorinostat was used, as it has no ER stress inhibitory effects and therefore the histone hyperacetylation properties alone could be investigated. In vitro work demonstrated that vorinostat inhibited histone deacetylation in cultured proximal tubular cells but did not prevent ER stress or protein aggregation induced by TM. Vorinostat induced a significant increase in cell death, and exacerbated TM-mediated total cell death and apoptotic cell death. Wild type male mice were treated with TM (0.5 mg/kg, intraperitoneal injection), with or without vorinostat (50 mg/kg/day) or 4-PBA (1 g/kg/day). Mice treated with 4-PBA or vorinostat exhibited similar levels of histone hyperacetylation. Expression of the pro-apoptotic protein CHOP was induced with TM, and not inhibited by vorinostat. Further, vorinostat did not prevent any renal damage or decline in renal function caused by tunicamycin. These data suggest that the protective mechanisms found by 4-PBA are primarily due to its molecular chaperone properties, and the HDAC inhibitors used did not provide any protection against renal injury caused by ER stress.

TDAG51 induces renal interstitial fibrosis through modulation of TGF-ß receptor 1 in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by the gradual loss of renal function and is a major public health concern. Risk factors for CKD include hypertension and proteinuria, both of which are associated with endoplasmic reticulum (ER) stress. ER stress-induced TDAG51 protein expression is increased at an early time point in mice with CKD. Based on these findings, wild-type and TDAG51 knock-out (TDKO) mice were used in an angiotensin II/deoxycorticosterone acetate/salt model of CKD. Both wild-type and TDKO mice developed hypertension, increased proteinuria and albuminuria, glomerular injury, and tubular damage. However, TDKO mice were protected from apoptosis and renal interstitial fibrosis. Human proximal tubular cells were used to demonstrate that TDAG51 expression induces apoptosis through a CHOP-dependent mechanism. Further, a mouse model of intrinsic acute kidney injury demonstrated that CHOP is required for ER stress-mediated apoptosis. Renal fibroblasts were used to demonstrate that TGF-ß induces collagen production through an IRE1-dependent mechanism; cells treated with a TGF-ß receptor 1 inhibitor prevented XBP1 splicing, a downstream consequence of IRE1 activation. Interestingly, TDKO mice express significantly less TGF-ß receptor 1, thus, preventing TGF-ß-mediated XBP1 splicing. In conclusion, TDAG51 induces apoptosis in the kidney through a CHOP-dependent mechanism, while contributing to renal interstitial fibrosis through a TGF-ß-IRE1-XBP1 pathway.

Development of a continuous assay for the measurement of tissue factor procoagulant activity on intact cells.

Tissue factor (TF) is the major physiological initiator of the coagulation cascade and has an important function in the morbidity and mortality associated with many disease states, including cancer-associated thrombosis and atherosclerosis. TF normally exists in a partially encrypted state and its de-encryption on circulating monocytes, platelets or endothelial cells by inflammatory mediators can lead to thrombosis. Furthermore, many cancer cells express large amounts of TF and these cells communicate readily with the circulation through the fenestrated tumor endothelium. To assess agents or conditions that modulate the encryption state of TF, we developed a continuous assay for the determination of TF procoagulant activity (PCA) in a cell-based system. We have shown the use of this assay at detecting agents that de-encrypt TF thereby leading to an increase in TF PCA in three cancer cell lines, namely, T24/83 bladder carcinoma cells and PC-3 and DU145 prostate cancer cells. Further, through use of this assay, we have shown that the endoplasmic reticulum calcium pump inhibitor, thapsigargin, stimulates the de-encryption of TF. The continuous assay for the determination of TF PCA proved to have inherently less intra- and inter-assay variability than the widely used discontinuous assay and is considerably less labor intensive. Further, the continuous assay produced progress curves that were compatible with curve fitting to allow for the determination of the nature of reaction as well as rate constants for the underlying enzymes, TF/FVIIa and FXa. The continuous assay for the assessment of TF PCA on intact cells is applicable for high-throughput screening to allow for the determination of compounds that modulate TF PCA.

Enhanced Myogenic Constriction in the SHR Preglomerular Vessels Is Mediated by Thromboxane A2 Synthesis.

BACKGROUND: Spontaneously Hypertensive Rats (SHR) have chronically elevated blood pressures at 30 weeks of age (systolic: 191.0 ± 1.0, diastolic: 128.8 ± 0.9). However, despite this chronic malignant hypertension, SHR kidneys remain relatively free of pathology due to having an augmented myogenic constriction (MC). We hypothesized that the enhanced MC in the SHR preglomerular vessels was due to increased prostaglandin and decreased nitric oxide (NO) synthesis, providing renal protection. METHODS: SHR and Wistar Kyoto (WKY) arcuate and mesenteric arteries were treated with indomethacin (prostaglandin synthesis inhibitor), N omega-nitro-L-arginine (L-NNA, NO synthase inhibitor), and nifedipine (L-type calcium channel blocker); and MC was measured in these vessels. The role of endothelium in MC was examined by removing endothelium from WKY and SHR preglomerular and mesenteric arteries using human hair, and measuring MC. We also studied the source of prostaglandin in the SHR by treating endothelium-removed arcuate arteries with indomethacin and furegrelate (thromboxane synthase inhibitor). RESULTS: MC was enhanced in the SHR preglomerular vessels but not the mesenteric arteries. Indomethacin and LNNA removed the enhanced MC in the SHR. Nifedipine also inhibited MC in both WKY and SHR arcuate and mesenteric arteries. Removing endothelium did not change MC in either arcuate or mesenteric arteries of WKY and SHR rats; and did not remove the augmented MC in the SHR arcuate arteries. Indomethacin and furegrelate decreased MC in endothelium-removed SHR arcuate arteries and obliterated the enhanced MC in the SHR. CONCLUSION: The enhanced MC in the SHR arcuate arteries was due to thromboxane A2 synthesis from the tunica media and/or adventitia layers. MC was not dependent on endothelium, but was dependent on L-type calcium channels. Nevertheless, SHR arcuate arteries displayed differential intracellular calcium signaling compared to the WKYs.

Protein misfolding in endoplasmic reticulum stress with applications to renal diseases.

Protein misfolding may be the result of a variety of different processes that disrupt the ability of a protein to form a thermodynamically stable tertiary structure that allows it to perform its proper function. In this chapter, we explore the nature of a protein's form that allows it to have a stable tertiary structure, and examine specific mutation that are known to occur in the coding regions of DNA that disrupt a protein's ability to be folded into a thermodynamically stable tertiary structure. We examine the consequences of these protein misfoldings in terms of the endoplasmic reticulum stress response and resulting unfolded protein response. These conditions are specifically related to renal diseases. Further, we explore novel therapeutics, pharmacological chaperones, that are being developed to alleviate the disease burden associated with protein misfolding caused by mutations. These interventions aim to stabilize protein folding intermediates and allow proper folding to occur as well as prevent protein aggregation and the resulting pathophysiological consequences.