Jianlin Yin

TLR4 activation mediates kidney ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4(-/-) and MyD88(-/-) mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4(-/-) and MyD88(-/-) TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4(-/-) mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4(-/-) BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.

Real‐time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real‐time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA‐binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real‐time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low‐level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

Apoptosis of vascular smooth muscle cells induced by cholesterol and its oxides in vitro and in vivo

The ability of cholesterol and its oxides to induce apoptosis in vascular smooth muscle cells in tissue culture and in a rabbit model of atherosclerosis was evaluated. Apoptosis was detected using DNA laddering and in situ end-labelling of fragmented DNA. Cholesterol oxides, but not cholesterol, were found to inhibit proliferation and induce apoptosis of vascular smooth muscle cells in tissue culture. 7-ketocholesterol was found to be the most potent inhibitor of proliferation, while 25-hydroxycholesterol was found to be the most potent inducer of apoptosis. These data suggest that the inhibition of proliferation and the induction of apoptosis by cholesterol oxides within vascular smooth muscle cells use different pathways, suggesting a differential role for these cholesterol oxides within the arterial wall. Cholesterol feeding after balloon injury in a rabbit model of atherosclerosis is known to result in the accumulation of cholesterol oxides. However, we found that cholesterol feeding had no effect on the level of apoptosis in the rabbit aortic wall after balloon injury, suggesting that the major factor determining apoptosis in our model was the balloon injury.

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding

The redistribution of purinergic P2X receptor subunits (P2X1 to P2X7) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X1 receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X4 clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X5 clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X3, P2X6 and P2X7 cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X4 receptors may contribute to neointimal proliferation.

Growth arrest-specific gene 6 expression in proliferating rabbit vascular smooth muscle cells in vitro and in vivo.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in the processes of atherosclerosis and restenosis. The protein product of the growth arrest‐specific gene 6 (Gas‐6) has recently been identified as a ligand for the Axl/Rse/Mer tyrosine kinase receptor family, which may be involved in proliferation and migration of VSMCs. Here we show that Gas‐6 gene expression is increased in proliferating VSMCs in tissue culture (2.5‐fold increase by Northern blot) and following neointimal proliferation in a rabbit balloon‐injury model (3‐fold increase by Western blot). Neither platelet‐derived growth factor (PDGF) nor thrombin stimulate the expression of Gas‐6 in cultured VSMCs despite the ability of the PDGF, but not thrombin, to stimulate proliferation in growth‐arrested cells. These data suggest a role for the Gas‐6 regulatory system in VSMC proliferation, which may be a target for therapeutic interventions in the atherosclerotic process and restenosis after angioplasty.

Chronic angiotensin-converting enzyme inhibition up-regulates mouse kidney growth arrest specific-6 protein and the AXL subfamily of receptor tyrosine kinases

Introduction. Growth arrest specific-6 (GAS-6), a vitamin K-dependent protein, is a potential mediator in progressive and chronic renal disease, specifically as a mediator of abnormal mesangial cell proliferation. Nitric oxide and angiotensin II affect mesangial cell proliferation. However, an association between nitric oxide synthase or angiotensin II on GAS-6 expression in the kidney has not previously been examined. Thus, our aim was to examine the effects of antihypertensive angiotensin-converting enzyme inhibitors and chronic nitric oxide synthase inhibition on the kidney expression of GAS-6 and its receptors AXL, MER and RSE. Methods. Four groups of adult male C57BL/6J mice were studied: group 1, untreated controls (tap water for six weeks); group 2, treated orally with a nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 0.325 mg/ml for six weeks); group 3, treated orally with captopril (0.6875 mg/ml for six weeks); group 4, co-treated orally with L-NAME and captopril (same doses for six weeks).At the end of the study, kidneys were placed in fixative and processed to paraffin for immunohistochemical staining. Results. GAS-6 and its receptors were not present in control and L-NAME-treated mice. Positive GAS-6 staining was detectable only in those mice receiving some form of chronic dosing with captopril, whether they were treated with captopril only or with captopril and L-NAME. Immunohistochemical detection across cases for MER and RSE was rare, whereas AXL-positive staining in the kidney mirrored GAS-6 staining/expression.The staining of GAS6 and AXL was predominantly localised to the renal tubular cells. Conclusions. These findings suggest that GAS-6 may not be a final common pathway for nitric oxide synthase inhibition-induced renal disease. Renal tubular GAS-6 expression following captopril treatment was unexpected and could be beneficial in preventing tubular atrophy following the onset of persistent systemic hypertension.

Stimulation of mesangial cells by angiotensin II and lipopolysaccharide increases expression of interleukin-18, but not IL-18 receptor.

Background/Aims: Mesangial cell (MC) hyperplasia is associated with several kidney diseases. Experimental studies confirm upregulation of IL-18 in glomerular disease and renal allograft rejection. We evaluated whether MCs express IL-18 and IL-18 receptor-α (IL-18Rα) with and without stimulation by LPS, AngII and PDGF. Methods: Glomeruli were isolated using Dynabeads perfusion. MCs were cultured by glomerular explantation. IL-18 and IL-18Rα expression were detected by RT-PCR, ELISA and flow cytometry. Results: Significantly higher levels of IL-18 expression were detected in isolated glomeruli, compared to cortical tissue devoid of glomeruli, and in MCs, compared to tubular cells (both p < 0.01). Increased IL-18 expression was detected in MCs, but not podocytes, endothelial cells or tubular cells in response to LPS stimulation. IL-18 mRNA and protein expression were significantly upregulated by AngII (p < 0.05) and LPS (p < 0.01), but not PDGF-BB, in primary MCs and a MC line (MES13). IL-18Rα mRNA was almost undetectable in MCs treated with or without LPS, AngII and PDGF-BB. IL-18Rα protein was not detected by flow cytometry. Conclusions: MCs express IL-18, which was significantly increased after LPS and AngII stimulation, but do not express appreciable levels of IL-18Rα. MC-derived IL-18 is unlikely to be an autocrine mediator in glomerular disease given the lack of IL-18Rα.

Delay in opening the infarct related coronary artery increases plasma atrial natriuretic peptide levels.

Plasma atrial natriuretic peptide (ANP) levels were measured in rabbits during the late healing phase of myocardial infarcts. Significant differences in plasma ANP levels (P < 0.02) were found between rabbits that had undergone very late (6 h) or early reperfusion (20 and 45 min of ischemia) of the infarct related coronary artery. Differences in ANP levels were independent of infarct size, ventricular remodeling and infarct expansion. We conclude late reperfusion of infarct related artery, independent of myocardial salvage, is associated with increased circulating ANP plasma levels.

A method to determine the fractal dimension of the cross-sectional jaggedness of the infarct scar edge

Abstract This study describes the use of a shareware software package available from the National Institutes of Health for computing the fractal dimension. Specifically, when fractal analysis is used in its correct context it provides for a quantitative description of the space filling properties of two-dimensional objects. A rabbit model of post myocardial infarction is described where the cross-sectional infarct edge is reconstructed and its jaggedness determined by calculating its fractal dimension via the pixel dilation method. The fractal dimensions of the anterior and posterior lateral infarct edges were calculated to have a mean of 1.16 and 1.29, respectively. In conclusion, the fractal technique can be used to describe the complex jaggedness of the infarct edge. This case study also illustrates the fact that the complexity of an infarcted area is not uniform across the scar. For example, we found that the space filling properties of the anterior and posterior borders of a myocardial infarct can differ by more than 2-fold.