Mild Electrical Stimulation with Heat Shock Ameliorates Insulin Resistance via Enhanced Insulin Signaling

Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42°C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor β subunit (IRβ) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12–15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway.     

Indomethacin reduces glomerular and tubular damage markers but not renal inflammation in chronic kidney disease patients: a post-hoc analysis

Under specific conditions non-steroidal anti-inflammatory drugs (NSAIDs) may be used to lower therapy-resistant proteinuria. The potentially beneficial anti-proteinuric, tubulo-protective, and anti-inflammatory effects of NSAIDs may be offset by an increased risk of (renal) side effects. We investigated the effect of indomethacin on urinary markers of glomerular and tubular damage and renal inflammation. We performed a post-hoc analysis of a prospective open-label crossover study in chronic kidney disease patients (n?=?12) with mild renal function impairment and stable residual proteinuria of 4.7±4.1 g/d. After a wash-out period of six wks without any RAAS blocking agents or other therapy to lower proteinuria (untreated proteinuria (UP)), patients subsequently received indomethacin 75 mg BID for 4 wks (NSAID). Healthy subjects (n?=?10) screened for kidney donation served as controls. Urine and plasma levels of total IgG, IgG4, KIM-1, beta-2-microglobulin, H-FABP, MCP-1 and NGAL were determined using ELISA. Following NSAID treatment, 24 h -urinary excretion of glomerular and proximal tubular damage markers was reduced in comparison with the period without anti-proteinuric treatment (total IgG: UP 131[38-513] vs NSAID 38[17-218] mg/24 h, p<0.01; IgG4: 50[16-68] vs 10[1-38] mg/24 h, p<0.001; beta-2-microglobulin: 200[55-404] vs 50[28-110] ug/24 h, p?=?0.03; KIM-1: 9[5]-[14] vs 5[2]-[9] ug/24 h, p?=?0.01). Fractional excretions of these damage markers were also reduced by NSAID. The distal tubular marker H-FABP showed a trend to reduction following NSAID treatment. Surprisingly, NSAID treatment did not reduce urinary excretion of the inflammation markers MCP-1 and NGAL, but did reduce plasma MCP-1 levels, resulting in an increased fractional MCP-1 excretion. In conclusion, the anti-proteinuric effect of indomethacin is associated with reduced urinary excretion of glomerular and tubular damage markers, but not with reduced excretion of renal inflammation markers. Future studies should address whether the short term glomerulo- and tubulo-protective effects as observed outweigh the possible side-effects of NSAID treatment on the long term.     

The anti-inflammatory effects of heat shock protein 72 involve inhibition of high-mobility-group box 1 release and proinflammatory function in macrophages

High-mobility-group box 1 (HMGB1), a nuclear protein, has recently been identified as an important mediator of local and systemic inflammatory diseases when released into the extracellular milieu. Anti-inflammatory regulation by the stress response is an effective autoprotective mechanism when the host encounters harmful stimuli, but the mechanism of action remains incompletely delineated. In this study, we demonstrate that increases in levels of a major stress-inducible protein, heat shock protein 72 (Hsp72) by gene transfection attenuated LPS- or TNF-alpha-induced HMGB1 cytoplasmic translocation and release. The mechanisms involved inhibition of the chromosome region maintenance 1 (CRM1)-dependent nuclear export pathway. Overexpression of Hsp72 inhibited CRM1 translocation and interaction between HMGB1 and CRM1 in macrophages post-LPS and TNF-alpha treatment. In addition, overexpression of Hsp72 strongly inhibited HMGB1-induced cytokine (TNF-alpha, IL-1beta) expression and release, which correlated closely with: 1) inhibition of the MAP kinases (p38, JNK, and ERK); and 2) inhibition of the NF-kappaB pathway. Taken together, these experiments suggest that the anti-inflammatory activity of Hsp72 is achieved by interfering with both the release and proinflammatory function of HMGB1. Our experimental data provide important insights into the anti-inflammatory mechanisms of heat shock protein protection.     

The function of HSP72 in suppression of c-Jun N-terminal kinase activation can be dissociated from its role in prevention of protein damage.

Activation of the c-Jun N-terminal kinase (JNK) by a variety of stimuli is critical for regulation of many cellular processes including apoptosis. The major inducible heat shock protein Hsp72 has previously been demonstrated to inhibit activation of JNK in cells exposed to heat shock and other protein-damaging agents, thus suppressing apoptosis. Hsp72 can protect proteins from stress-induced damage. To test if this protective function of Hsp72 is involved in JNK suppression, we investigated whether Hsp72 can avert activation of JNK by stimuli that do not cause protein damage. We show that Hsp72 suppresses activation of JNK induced by non-protein-damaging stimuli, interleukin-1 and UV irradiation, as well as by constitutively active components of the JNK signaling cascade Cdc42 and MEKK1. Furthermore, Hsp72 strongly reduced activation of JNK by phosphatase inhibitors. We also demonstrate that an Hsp72 mutant that lacks the ATPase domain is still capable of JNK suppression, thus indicating that the protein refolding activity of Hsp72 is not critical for inhibition of JNK activation. Taken together these data suggest that Hsp72 plays a regulatory role in JNK signaling and that the function of Hsp72 in protein protection or refolding is not involved in JNK regulation.     

The Accumulation of VEGFA in the Glomerular Basement Membrane and Its Relationship with Podocyte Injury and Proteinuria in Alport Syndrome

The pathogenesis of proteinuria in Alport syndrome (AS) remains unclear. Vascular endothelial growth factor A (VEGFA) is a key regulator of the glomerular filtration barrier (GFB). This study explored the expression of VEGFA in the glomeruli and its accumulation in the glomerular basement membrane (GBM) and their relationship with podocyte injury and proteinuria in Alport syndrome (AS). Clinical data and renal tissues of control patients (11 cases) and AS patients (25 cases) were included. AS patients were further divided into 2 groups according to the quantities of their urinary protein: mild to moderate proteinuria group (proteinuria <50 mg/kg/d, 15 cases) and heavy proteinuria group (proteinuria ≥50 mg/kg/d, 10 cases). The expression and distribution of VEGFA and VEGF receptor 2 (VEGFR2) in the GFB, the phosphorylation of VEGFR2 (p-VEGFR2) and nephrin (p-nephrin), and the expression of synaptopodin and nephrin in the glomeruli were detected by immune electron microscopy and/or immunofluorescence, and their relationships to proteinuria in AS patients were analyzed. The accumulation of VEGFA in the GBM was increased in AS patients. The expression of VEGFA and the levels of p-VEGFR2 and p-nephrin in glomeruli were increased and were positively correlated with the degree of proteinuria in AS patients. The expression of synaptopodin and nephrin were decreased and were negatively correlated with the degree of proteinuria in AS patients. The over expressed VEGFA in the glomeruli and its accumulation in the GBM may activate the VEGFA-VEGFR2 and nephrin signaling pathways and lead to podocyte injury and occurrence of proteinuria in AS.     

Alport 综合征1 例报道及文献复习

目的:探讨Alport综合征的临床表现,病理学特征及研究进展。方法:分析1例此病患者的临床资料。结果:本例患者临床表现为慢性视力下降。尿常规检查提示蛋白尿,血尿。肾肾穿刺活检的光镜、电镜检查均支持诊断。结论:Alport综合征患者中眼部异常的表现有独特性;了解眼部病变特征并结合全身病史,病理学检查有助于疾病的诊断和随诊。     

Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.     

ASK1 inhibitor treatment suppresses p38/JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis

Activation of p38 mitogen‐activated protein kinase (MAPK) and c‐Jun amino terminal kinase (JNK) is prominent in human crescentic glomerulonephritis. p38 and JNK inhibitors suppress crescentic disease in animal models; however, the upstream mechanisms inducing activation of these kinases in crescentic glomerulonephritis are unknown. We investigated the hypothesis that apoptosis signal‐regulating kinase 1 (ASK1/MAP3K5) promote p38/JNK activation and renal injury in models of nephrotoxic serum nephritis (NTN); acute glomerular injury in SD rats, and crescentic disease in WKY rats. Treatment with the selective ASK1 inhibitor, GS‐444217 or vehicle began 1 hour before nephrotoxic serum injection and continued until animals were killed on day 1 (SD rats) or 14 (WKY rats). NTN resulted in phosphorylation (activation) of p38 and c‐Jun in both models which was substantially reduced by ASK1 inhibitor treatment. In SD rats, GS‐444217 prevented proteinuria and glomerular thrombosis with suppression of macrophage activation on day 1 NTN. In WKY rats, GS‐444217 reduced crescent formation, prevented renal impairment and reduced proteinuria on day 14 NTN. Macrophage activation, T‐cell infiltration and renal fibrosis were also reduced by GS‐444217. In conclusion, GS‐444217 treatment inhibited p38/JNK activation and development of renal injury in rat NTN. ASK1 inhibitors may have therapeutic potential in rapidly progressive glomerulonephritis.     

Regression of Glomerular and Tubulointerstitial Injuries by Dietary Salt Reduction with Combination Therapy of Angiotensin II Receptor Blocker and Calcium Channel Blocker in Dahl Salt-Sensitive Rats

A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.     

Glomerular eicosanoid production in acute serum sickness nephritis

To determine if the induction of immune-mediated glomerular injury influences the formation of glomerular cyclooxygenase products, we measured thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) production by isolated glomeruli of rabbits induced with acute serum sickness nephritis by the administration of bovine serum ablumin (BSA). Animals were randomly assigned to one of three experimental groups: animals injected with BSA (BSA group; n = 11); animals injected with normal saline (control group; n = 11); and animals injected with BSA which were treated with the thromboxane synthetase inhibitor, OKY-046 (BSA + OKY-046; n = 6). Animals in the BSA and BSA + OKY groups developed severe proteinuria and glomerular histologic lesions of nephritis. No differences in proteinuria, serum creatinine and severity of histologic nephritis were observed between the two groups. Examination of glomerular eicosanoid production at the end of the experiment showed a marked reduction of glomerular PGE2 and 6-keto-PGF1 alpha production with a smaller reduction of glomerular TXB2 production in the BSA group. In the BSA + OKY-046 group, the production of TXB2 was significantly less than that in the BSA group; despite this, no effect on proteinuria could be discerned.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome

Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and/or proteinuria with structural defects of the glomerular basement membrane. It can be associated with extrarenal manifestations (high-tone sensorineural hearing loss and ocular abnormalities). Somatic mutations in COL4A5 (X-linked), COL4A3 and COL4A4 genes (both autosomal recessive and autosomal dominant) cause Alport syndrome. Somatic mosaicism in Alport patients is very rare. The reason for this may be due to the difficulty of detection.     

Suppression of stress kinase JNK is involved in HSP72-mediated protection of myogenic cells from transient energy deprivation. HSP72 alleviates the stewss-induced inhibition of JNK dephosphorylation

Since protection of cells from stress-induced apoptosis by the heat shock protein Hsp72 involves suppression of stress kinase JNK, we suggested that Hsp72-mediated JNK inhibition might also be critical for myocardial protection from ischemia/reperfusion. Transient energy deprivation of H9c2 myogenic cells, used as an in vitro model of myocardial ischemia, led to cell death that had morphological features of apoptosis and necrosis and was independent of caspases. Surprisingly, this unusual type of cell death was regulated by JNK and ERK kinases. In fact, specific inhibition of JNK increased cell survival; specific inhibition of ERKs enhanced deleterious consequences of energy deprivation, whereas inhibition of p38 kinase had no effect. Hsp72 suppressed activation of JNK and did not increase ERK activity, suggesting that inhibition of JNK is the important component of Hsp72-mediated protection. Upon transient energy deprivation, activation of JNK proceeds via two distinct pathways, stimulation of JNK phosphorylation by a protein kinase SEK1 and inhibition of JNK dephosphorylation. Remarkably, in cells exposed to transient energy deprivation, Hsp72 enhanced the rate of JNK dephosphorylation but did not affect SEK1 activity. Therefore, it appears that Hsp72 specifically down-regulates JNK by accelerating its dephosphorylation, which reduces the susceptibility of cardiac cells to simulated ischemia/reperfusion.     

Molecular and functional defects in kidneys of mice lacking collagen alpha 3(IV): implications for Alport syndrome

Collagen IV is a major structural component of all basal laminae (BLs). Six collagen IV alpha chains are present in mammals; alpha 1 and alpha 2(IV) are broadly expressed in embryos and adults, whereas alpha 3- 6(IV) are restricted to a defined subset of BLs. In the glomerular BL of the kidney, the alpha 1 and alpha 2(IV) chains are replaced by the alpha 3-5(IV) chains as development proceeds. In humans, mutation of the collagen alpha 3, alpha 4, or alpha 5(IV) chain genes results in a delayed onset renal disease called Alport syndrome. We show here that mice lacking collagen alpha 3(IV) display a renal phenotype strikingly similar to Alport syndrome: decreased glomerular filtration (leading to uremia), compromised glomerular integrity (leading to proteinuria), structural changes in glomerular BL, and glomerulonephritis. Interestingly, numerous changes in the molecular composition of glomerular BL precede the onset of renal dysfunction; these include loss of collagens alpha 4 and alpha 5(IV), retention of collagen alpha 1/2(IV), appearance of fibronectin and collagen VI, and increased levels of perlecan. We suggest that these alterations contribute, along with loss of collagen IV isoforms per se, to renal pathology.     

Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase

Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43 degrees C for 20 min) suppressed UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibited JNK-dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK.     

Complement activates the c-Jun N-terminal kinase/stress-activated protein kinase in glomerular epithelial cells

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A(2) (cPLA(2)), and products of cPLA(2)-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA(2) in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and alpha-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H(2)O(2) activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA(2) and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.     

Heat shock treatment suppresses angiotensin II-induced activation of NF-kappaB pathway and heart inflammation: a role for IKK depletion by heat shock?

Heat shock (HS) proteins (Hsps) function in tissue protection through their chaperone activity and by interacting with cell signaling pathways to suppress apoptosis. Here, we investigated the effect of HS treatment on the nuclear factor (NF)-kappaB signaling pathway in the angiotensin II (ANG II) model of inflammation. Male Sprague-Dawley rats were divided into sham and HS-, ANG II-, and HS + ANG II-treated groups. HS treatment was administered 24 h before the initiation of ANG II infusion. HS treatment (42 degrees C for 15 min) decreased 7-day ANG II-induced hypertension from 191 +/- 4 to 147 +/- 3 mmHg (P < 0.01). Histological staining of hearts showed that HS treatment reduced ANG II-induced leukocyte infiltration, perivascular and interstitial inflammation, and fibrosis. Heart NF-kappaB nuclear translocation and activity, examined by Western blot analysis and electrophoretic mobility shift assay, was suppressed by HS treatment. HS treatment depleted IkappaB kinase-alpha (IKK-alpha) and phosphorylated IKK-alpha and suppressed the depletion of IkappaB-alpha and the accumulation of phosphorylated IkappaB-alpha. HS treatment blocked ANG II induced expression of IL-6 and ICAM-1 in the heart. ANG II and HS treatment induced high-level expression of Hsp27 and Hsp70 and their phosphorylation. Phosphorylated isoforms of Hsp27 and Hsp70 may play an important role in protecting the heart against ANG II-induced inflammation.     

Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis

The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72 Delta EEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72 Delta EEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and Delta Hsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and Delta Hsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.