George Tse

Heat shock protein 90 inhibition abrogates TLR4-mediated NF-κB activity and reduces renal ischemia-reperfusion injury

Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury. Toll-like receptor 4 (TLR4) mediates sterile inflammation following renal IRI. Heat shock protein 90 (Hsp90) inhibition is a potential strategy to reduce IRI, and AT13387 is a novel Hsp90 inhibitor with low toxicity. This study assessed if pre-treatment with AT13387 could reduce renal IRI and established if the mechanism of protection involved a reduction in inflammatory signalling. Mice were pre-treated with AT13387 prior to renal IRI. 24 h later, renal function was determined by serum creatinine, kidney damage by tubular necrosis score, renal TLR4 expression by PCR and inflammation by cytokine array. In vitro, human embryonic kidney cells were co-transfected to express TLR4 and a secreted alkaline phosphatase NF-κB reporter. Cells were pre-treated with AT13387 and exposed to endotoxin-free hyaluronan to stimulate sterile TLR4-specific NF-κB inflammatory activation. Following renal IRI, AT13387 significantly reduced serum creatinine, tubular necrosis, TLR4 expression and NF-κB-dependent chemokines. In vitro, AT13387-treatment resulted in breakdown of IκB kinase, which abolished TLR4-mediated NF-κB activation by hyaluronan. AT13387 is a new agent with translational potential that reduces renal IRI. The mechanism of protection may involve breakdown of IκB kinase and repression of TLR4-mediated NF-κB inflammatory activity.

Vascular and plaque imaging with ultrasmall superparamagnetic particles of iron oxide

Cardiovascular Magnetic Resonance (CMR) has become a primary tool for non-invasive assessment of cardiovascular anatomy, pathology and function. Existing contrast agents have been utilised for the identification of infarction, fibrosis, perfusion deficits and for angiography. Novel ultrasmall superparamagnetic particles of iron oxide (USPIO) contrast agents that are taken up by inflammatory cells can detect cellular inflammation non-invasively using CMR, potentially aiding the diagnosis of inflammatory medical conditions, guiding their treatment and giving insight into their pathophysiology. In this review we describe the utilization of USPIO as a novel contrast agent in vascular disease.

Systematic review of mouse kidney transplantation

A mouse model of kidney transplantation was first described in 1973 by Skoskiewicz et al. Although the mouse model is technically difficult, it is attractive for several reasons: the mouse genome has been characterized and in many aspects is similar to man and there is a greater diversity of experimental reagents and techniques available for mouse studies than other experimental models. We reviewed the literature on all studies of mouse kidney transplantation to report the donor and recipient strain combinations that have been investigated and the resultant survival and histological outcomes. Some models of kidney transplantation have used the transplanted kidney as a life‐supporting organ, however, in many studies the recipient mouses native kidney has been left in situ. Several different combinations of inbred mouse strains have been reported, with varying degrees of injury, survival or tolerance because of haplotype differences. This model has been exceptionally useful as an investigational tool to understand multiple aspects of transplantation including acute rejection, cellular and humoral rejection mechanisms and their treatment. Furthermore, this model has been used to investigate disease mechanisms beyond transplant rejection including intrinsic renal disease and infection‐associated pathology.

A Comparative Study of 2 Computer-assisted Methods of Quantifying Brightfield Microscopy Images

Immunohistochemistry continues to be a powerful tool for the detection of antigens. There are several commercially available software packages that allow image analysis; however, these can be complex, require relatively high level of computer skills, and can be expensive. We compared 2 commonly available software packages, Adobe Photoshop CS6 and ImageJ, in their ability to quantify percentage positive area after picrosirius red (PSR) staining and 3,3′-diaminobenzidine (DAB) staining. On analysis of DAB-stained B cells in the mouse spleen, with a biotinylated primary rat anti-mouse-B220 antibody, there was no significant difference on converting images from brightfield microscopy to binary images to measure black and white pixels using ImageJ compared with measuring a range of brown pixels with Photoshop (Student t test, P=0.243, correlation r=0.985). When analyzing mouse kidney allografts stained with PSR, Photoshop achieved a greater interquartile range while maintaining a lower 10th percentile value compared with analysis with ImageJ. A lower 10% percentile reflects that Photoshop analysis is better at analyzing tissues with low levels of positive pixels; particularly relevant for control tissues or negative controls, whereas after ImageJ analysis the same images would result in spuriously high levels of positivity. Furthermore comparing the 2 methods by Bland-Altman plot revealed that these 2 methodologies did not agree when measuring images with a higher percentage of positive staining and correlation was poor (r=0.804). We conclude that for computer-assisted analysis of images of DAB-stained tissue there is no difference between using Photoshop or ImageJ. However, for analysis of color images where differentiation into a binary pattern is not easy, such as with PSR, Photoshop is superior at identifying higher levels of positivity while maintaining differentiation of low levels of positive staining.

Macrophages and transplant rejection: a novel future target?

In this issue of Transplantation, Ma et al. describe the protective effect of administering the c-fms kinase inhibitor upon cellular rejection, suggesting an important pathogenic role for macrophages. In contrast, no effect upon the development of humerol rejection was evident. The role of macrophages in rejection is discussed.

Mouse kidney transplantation: models of allograft rejection.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Nanoparticle Enhanced MRI Scanning to Detect Cellular Inflammation in Experimental Chronic Renal Allograft Rejection

Objectives. We investigated whether ultrasmall paramagnetic particles of iron oxide- (USPIO-) enhanced magnetic resonance imaging (MRI) can detect experimental chronic allograft damage in a murine renal allograft model. Materials and Methods. Two cohorts of mice underwent renal transplantation with either a syngeneic isograft or allograft kidney. MRI scanning was performed prior to and 48 hours after USPIO infusion using T2∗-weighted protocols. R2∗ values were calculated to indicate the degree of USPIO uptake. Native kidneys and skeletal muscle were imaged as reference tissues and renal explants analysed by histology and electron microscopy. Results. R2∗ values in the allograft group were higher compared to the isograft group when indexed to native kidney (median 1.24 (interquartile range: 1.12 to 1.36) versus 0.96 (0.92 to 1.04), P < 0.01). R2∗ values were also higher in the allograft transplant when indexed to skeletal muscle (6.24 (5.63 to 13.51)) compared to native kidney (2.91 (1.11 to 6.46) P < 0.05). Increased R2∗ signal in kidney allograft was associated with macrophage and iron staining on histology. USPIO were identified within tissue resident macrophages on electron microscopy. Conclusion. USPIO-enhanced MRI identifies macrophage.

Investigation of the role of B lymphocytes and tertiary lymphoid tissue in a murine model of renal chronic allograft damage

Abstract Background Nodular B-lymphocyte rich infiltrates have been identified in chronically rejected renal allografts and biopsies of acute transplant rejection, and this has been associated with the development of tertiary lymphoid tissue (TLT). However their significance is unclear, with conflicting published data. We aim to investigate the role and significance of B-lymphocyte infiltrates and the development of TLT in a murine model of renal chronic allograft damage. Methods We used congenic strains with donor C57bl/6-BM12 mice kidneys transplanted into C57bl/6 recipients, this being a single MHC-II mismatch. Using immunohistochemistry we investigated the presence of B lymphocytes within the allograft. In addition we investigated other markers of chronic allograft damage in this model including lymphatic expansion, microvessel rarefication, and fibrosis. Findings Nodular aggregates of B cells appearing to be TLT developed over 12 weeks; however, in some allografts we observed a scattered B-cell pattern. The B-cell infiltrate of the allograft cortex increased progressively with a significant increased density by 12 weeks compared with 5 days after transplantation. Microvessels were counted with a 25-point graticule, and there was a significant difference between allograft and native kidney cortex (p Interpretation We have shown that this strain combination closely models that of chronic rejection of the renal allograft. Furthermore, we have identified the progressive infiltration and expansion of the B-lymphocytes compartment within the allograft cortex. This work has provided the basis for further investigation of B-lymphocyte depletion and the prospects of identifying a regulatory B lymphocyte. Funding Kidney Research UK.

Provision of Internet Information on Day Surgery and Inguinal Hernia Repair in UK Hospitals

The internet has become vital to information dissemination. Patients are increasingly using the internet for enquires regarding healthcare. A study in the UK of more than 1000 patients found that 40% had access to the internet and that 11% sought information prior to an outpatient clinic consultation. In a consecutive series of 105 patients undergoing elective laparoscopic surgery, 31% with access to the internet used it to acquire information on their operation. A similar picture exists in Germany with a reported 18% of patients using the internet prior to surgery.