Medullary Blood Oxygen Level-Dependent MRI Index (R2*) is Associated with Annual Loss of Kidney Function in Moderate CKD.

BACKGROUND: The estimated glomerular filtration rate (eGFR) is frequently used to monitor progression of kidney disease. Multiple values have to be obtained, sometimes over years to determine the rate of decline in kidney function. Recent data suggest that functional MRI (fMRI) methods may be able to predict loss of eGFR. In a prior study, baseline data with multi-parametric MRI in individuals with diabetes and moderate CKD was reported. This report extends our prior observations in order to evaluate the temporal variability of the fMRI measurements over 36 months and their association with annual change in eGFR. METHODS: Twenty-four subjects with moderate CKD completed 3 sets of MRI scans over a 36-month period. Blood oxygenation level-dependent (BOLD), arterial spin labeling perfusion, and diffusion MRI images were acquired using a 3 T scanner. Coefficients of variation was used to evaluate variability between subjects at each time point and temporal variability within each subject. We have conducted mixed effects models to examine the trajectory change in GFR over time using time and MRI variables as fixed effects and baseline intercept as random effect. Associations of MRI image markers with annual change in eGFR were evaluated. RESULTS: Multi-parametric functional renal MRI techniques in individuals with moderate CKD showed higher temporal variability in R2* of medulla compared to healthy individuals. This was consistent with the significant lower R2* in medulla observed at 36 months compared to baseline values. The results of linear mixed model showing that R2*_Medulla was the only predictor associated with change in eGFR over time. Furthermore, a significant association of medullary R2* with annual loss of eGFR was observed at all the 3 time points. CONCLUSIONS: The lower R2* values and the higher temporal variability in the renal medulla over time suggest the ability to monitor progressive CKD. These were confirmed by the fact that reduced medullary R2* was associated with higher annual loss in eGFR. These data collectively emphasize the need for inclusion of medulla in the analysis of renal BOLD MRI studies.

Renal BOLD MRI in patients with chronic kidney disease: comparison of the semi-automated twelve layer concentric objects (TLCO) and manual ROI methods.

OBJECTIVE: Blood oxygenation level dependent (BOLD) MRI technique is used to evaluate changes in intra-renal oxygenation in chronic kidney disease (CKD). The purpose of this study was to evaluate if the novel twelve layer concentric objects (TLCO) method has advantages over the manually defined regions of interest (ROI) analysis. METHODS AND MATERIALS: Existing renal BOLD MRI data acquired before and after furosemide on a 3 T scanner from 41 CKD patients and 13 age matched healthy controls were analyzed using TLCO method and compared with previously reported ROI analysis. RESULTS: Regional R2* measurements were strongly correlated between the two methods, while ΔR2* was moderately correlated. Medullary R2* by ROI analysis showed higher values compared to R2*_Inner by TLCO, probably due to the contributions from the cortex to R2*_Inner. R2*_Slope and Δ(R2*_Slope), unique parameters based on the TLCO method provided the most significant differences between stage 3a CKD patients and controls and were correlated with eGFR. DISCUSSION: There was a high degree of agreement between the two methods in terms of regional R2* measurements and both methods did not show differences between moderate CKD patients and controls. However, R2*_Slope and Δ(R2*_Slope) showed the largest sensitivity in distinguishing CKD from controls.

Cortical Perfusion and Tubular Function as Evaluated by Magnetic Resonance Imaging Correlates with Annual Loss in Renal Function in Moderate Chronic Kidney Disease.

BACKGROUND: Chronic hypoxia is a well-recognized factor in the pathogenesis of chronic kidney disease (CKD). Loss of microcirculation is thought to lead to enhanced renal hypoxia, which in turn results in the development of fibrosis, a hallmark of progressive CKD. To evaluate the role of functional magnetic resonance imaging (MRI), we performed perfusion, oxygenation, and diffusion MRI measurements in individuals with diabetes and stage 3 CKD. METHODS: Fifty-four subjects (41 individuals with diabetes and stage 3 CKD and 13 healthy controls) participated in this study. Data with blood oxygenation level dependent (BOLD), arterial spin labeling perfusion and diffusion MRI were acquired using a 3T scanner. RESULTS: Renal cortical perfusion was reduced in CKD compared to the controls (109.54 ± 25.38 vs. 203.17 ± 27.47 mL/min/100 g; p < 0.001). Cortical apparent diffusion coefficient showed no significant reduction in CKD compared to controls (1,596.10 ± 196.64 vs. 1,668.72 ± 77.29 × 10-6 mm2/s; p = 0.45) but was significantly associated with perfusion. Cortical R2* values were modestly increased in CKD (20.76 ± 4.08 vs. 18.74 ± 2.37 s-1; p = 0.12). Within the CKD group, R2*_Medulla and R2*_Kidney were moderately and negatively associated with estimated glomerular filtration rate. There was a significant association between cortical perfusion and medullary response to furosemide with annual loss of renal function, used as an estimate of CKD progression. CONCLUSIONS: Subjects with a moderate degree of CKD had significantly lower renal perfusion. Diffusion and BOLD MRI showed more modest differences between the groups. Individuals with progressive CKD had lower perfusion and response to furosemide.

TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis.

Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration permselectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower, whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse.

Longitudinal changes in MRI markers in a reversible unilateral ureteral obstruction mouse model: preliminary experience.

PURPOSE: To evaluate longitudinal changes in renal oxygenation and diffusion measurements in a model of reversible unilateral ureteral obstruction (rUUO) which has been shown to induce chronic renal functional deficits in a strain dependent way. C57BL/6 mice show higher degree of functional deficit compared with BALB/c mice. Because hypoxia and development of fibrosis are associated with chronic kidney diseases and are responsible for progression, we hypothesized that MRI measurements would be able to monitor the longitudinal changes in this model and will show strain dependent differences in response. Here blood oxygenation level dependent (BOLD) and diffusion MRI measurements were performed at three time points over a 30 day period in mice with rUUO. MATERIALS AND METHODS: The studies were performed on a 4.7T scanner with the mice anesthetized with isoflurane before UUO, 2 and 28 days postrelease of 6 days of obstruction. RESULTS: We found at the early time point (∼2 days after releasing the obstruction), the relative oxygenation in C57Bl/6 mice were lower compared with BALB/c. Diffusion measurements were lower at this time point and reached statistical significance in BALB/c CONCLUSION: These methods may prove valuable in better understanding the natural progression of kidney diseases and in evaluating novel interventions to limit progression.

Cell cycle arrest in a model of colistin nephrotoxicity.

Colistin (polymixin E) is an antibiotic prescribed with resurging frequency for multidrug resistant gram negative bacterial infections. It is associated with nephrotoxicity in humans in up to 55% of cases. Little is known regarding genes involved in colistin nephrotoxicity. A murine model of colistin-mediated kidney injury was developed. C57/BL6 mice were administered saline or colistin at a dose of 16 mg/kg/day in 2 divided intraperitoneal doses and killed after either 3 or 15 days of colistin. After 15 days, mice exposed to colistin had elevated blood urea nitrogen (BUN), creatinine, and pathologic evidence of acute tubular necrosis and apoptosis. After 3 days, mice had neither BUN elevation nor substantial pathologic injury; however, urinary neutrophil gelatinase-associated lipocalin was elevated (P = 0.017). An Illumina gene expression array was performed on kidney RNA harvested 72 h after first colistin dose to identify differentially expressed genes early in drug treatment. Array data revealed 21 differentially expressed genes (false discovery rate < 0.1) between control and colistin-exposed mice, including LGALS3 and CCNB1. The gene signature was significantly enriched for genes involved in cell cycle proliferation. RT-PCR, immunoblot, and immunostaining validated the relevance of key genes and proteins. This murine model offers insights into the potential mechanism of colistin-mediated nephrotoxicity. Further studies will determine whether the identified genes play a causative or protective role in colistin-induced nephrotoxicity.

Quantitative Blood Oxygenation Level Dependent Magnetic Resonance Imaging for Estimating Intra-renal Oxygen Availability Demonstrates Kidneys Are Hypoxemic in Human CKD.

Introduction: Kidney blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) has shown great promise in evaluating relative oxygen availability. This method is quite efficacious in evaluating acute responses to physiological and pharmacologic maneuvers. Its outcome parameter, R2∗ is defined as the apparent spin-spin relaxation rate measured in the presence of magnetic susceptibility differences and it is measured using gradient echo MRI. Although associations between R2∗ and renal function decline have been described, it remains uncertain to what extent R2∗ is a true reflection of tissue oxygenation. This is primarily because of not taking into account the confounding factors, especially fractional blood volume (fBV) in tissue. Methods: This case-control study included 7 healthy controls and 6 patients with diabetes and chronic kidney disease (CKD). Using data before and after administration of ferumoxytol, a blood pool MRI contrast media, the fBVs in kidney cortex and medulla were measured. Results: This pilot study independently measured fBV in kidney cortex (0.23 ± 0.03 vs. 0.17 ± 0.03) and medulla (0.36 ± 0.08 vs. 0.25 ± 0.03) in a small number of healthy controls (n = 7) versus CKD (n = 6). These were then combined with BOLD MRI measurements to estimate oxygen saturation of hemoglobin (StO2) (0.87 ± 0.03 vs. 0.72 ± 0.10 in cortex; 0.82 ± 0.05 vs. 0.72 ± 0.06 in medulla) and partial pressure of oxygen in blood (bloodPO2) (55.4 ± 6.5 vs. 38.4 ± 7.6 mm Hg in cortex; 48.4 ± 6.2 vs. 38.1 ± 4.5 mm Hg in medulla) in control versus CKD. The results for the first time demonstrate that cortex is normoxemic in controls and moderately hypoxemic in CKD. In the medulla, it is mildly hypoxemic in controls and moderately hypoxemic in CKD. Whereas fBV, StO2, and bloodPO2 were strongly associated with estimated glomerular filtration rate (eGFR), R2∗ was not. Conclusion: Our results support the feasibility of quantitatively assessing oxygen availability using noninvasive quantitative BOLD MRI that could be translated to the clinic.

Feasibility of Diffusion Tensor Imaging for Decreasing Biopsy Rates in Breast Imaging: Interim Analysis of a Prospective Study.

Because of the limited specificity of diagnostic imaging, many breast lesions referred for biopsy turn out to be benign. The objective of this study was to evaluate whether diffusion tensor MRI (DTI) parametric maps can be used to safely avoid biopsy of breast lesions. Individuals referred for breast biopsy based on mammogram (MG), ultrasound (US), and/or contrast enhanced (CE)-MRI were recruited. Scans consisting of T2-weighted and DTI sequences were performed. Multiple DTI-derived parametric color maps were evaluated semi-quantitatively to characterize lesions as "definitely benign," "not definitely benign," or "suspicious." All patients subsequently underwent biopsy. In this moderately-sized prospective study, 21 out of 47 pathologically proven benign lesions were characterized by both readers as "definitely benign," which would have precluded the need for biopsy. Biopsy was recommended for 11 out of 13 cancers that were characterized as "suspicious." In the remaining two cancers and 26 of 47 benign lesions, the scans were characterized as "not definitely benign" and hence required biopsy. The main causes for "not definitely benign" scans were small lesion sizes and noise. The results suggest that in appropriately selected patients, DTI may be used to safely reduce the number of unnecessary breast biopsies.

Endotoxemia alters tight junction gene and protein expression in the kidney.

Intact tight junctional (TJ) proteins are required for tubular ion transport and waste excretion. Disruption of TJs may contribute to a decreased glomerular filtration rate in acute kidney injury (AKI) via tubular backleak. The effect of LPS-mediated AKI on murine TJs has not been studied extensively. We hypothesized LPS endotoxin administration to mice would disrupt tubular TJ proteins including zonula occludens-1 (ZO-1), occludin, and claudins. ZO-1 and occludin immunofluorescence 24 h post-LPS revealed a marked change in localization from the usual circumferential fencework pattern to one with substantial fragmentation. Renal ZO-1 expression was significantly reduced 24 h after LPS (decrease of 56.1 ± 7.4%, P < 0.001), with subsequent recovery. ZO-1 mRNA expression was increased 24 h post-LPS (4.34 ± 0.87-fold, P = 0.0019), suggesting disruption of ZO-1 protein is not mediated by transcriptional regulation, but rather by degradation or changes in translation. Similarly, claudin-4 protein expression was decreased despite elevated mRNA. LPS administration resulted in dephosphorylation of occludin and fragmented tubular redistribution. Protein expression of claudin-1, and -3 was increased after LPS. ZO-1, occludin, and claudin-1, -3, and -4 gene expression were increased 48 h after LPS, suggesting a renal response to strengthen TJs following injury. Interestingly, reduced mRNA expression was found only for claudin-8. This study provides further support that LPS-induced AKI is associated with structural injury and is not merely due to hemodynamic changes.

A new model of the distal convoluted tubule.

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

Abnormal immune complex processing and spontaneous glomerulonephritis in complement factor H-deficient mice with human complement receptor 1 on erythrocytes.

Complement receptor 1 (CR1) on human erythrocytes (Es) and complement factor H (CFH) on rodent platelets perform immune adherence, which is a function that allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1(hu)Tg/CFH(-/-) mice spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active immunization with heterologous apoferritin. ICs containing Ag, IgG and C3 bound to Es in CR1(hu)Tg/CFH(-/-) mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement-dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regulator.

C5a/CD88 signaling alters blood-brain barrier integrity in lupus through nuclear factor-κB.

Inflammation is a key factor in a number of neurodegenerative diseases including systemic lupus erythematosus. The complement system is an important mechanism in initiating and amplifying inflammation. Our recent studies demonstrate that C5a, a protein fragment generated during complement activation could alter the blood-brain barrier integrity, and thereby disturb the brain microenvironment. To understand the mechanism by which this occurs, we examined the effects of C5a on apoptosis, translocation of nuclear factor-κB (NF-κb) and the expression of Iκbα, MAPK, CREB and TJ protein, zona occludens (ZO-1) in mouse brain endothelial cells. Apoptosis was examined by DNA laddering and caspase 3 activity and the distribution of the ZO-1 and the p65 subunit of NF-κB were determined by immunofluorescence. Inhibition of CD88 reduced translocation of NF-κb into the nucleus, altered ZO-1 at the interfaces of neighboring cells, decreased caspase 3 activity and prevented apoptosis in these cells. Our results indicate that signaling through CD88 regulates the blood-brain barrier in a NF-κb-dependent manner. These studies suggest that the C5a receptor, CD88 is a promising therapeutic target that will reduce NF-κb-signaling cascades in inflammatory settings.

C5a alters blood-brain barrier integrity in experimental lupus.

The blood-brain barrier (BBB) is a crucial anatomic location in the brain. Its dysfunction complicates many neurodegenerative diseases, from acute conditions, such as sepsis, to chronic diseases, such as systemic lupus erythematosus (SLE). Several studies suggest an altered BBB in lupus, but the underlying mechanism remains unknown. In the current study, we observed a definite loss of BBB integrity in MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) lupus mice by IgG infiltration into brain parenchyma. In line with this result, we examined the role of complement activation, a key event in this setting, in maintenance of BBB integrity. Complement activation generates C5a, a molecule with multiple functions. Because the expression of the C5a receptor (C5aR) is significantly increased in brain endothelial cells treated with lupus serum, the study focused on the role of C5a signaling through its G-protein-coupled receptor C5aR in brain endothelial cells, in a lupus setting. Reactive oxygen species production increased significantly in endothelial cells, in both primary cells and the bEnd3 cell line treated with lupus serum from MRL/lpr mice, compared with those treated with control serum from MRL(+/+) mice. In addition, increased permeability monitored by changes in transendothelial electrical resistance, cytoskeletal remodeling caused by actin fiber rearrangement, and increased iNOS mRNA expression were observed in bEnd3 cells. These disruptive effects were alleviated by pretreating cells with a C5a receptor antagonist (C5aRant) or a C5a antibody. Furthermore, the structural integrity of the vasculature in MRL/lpr brain was maintained by C5aR inhibition. These results demonstrate the regulation of BBB integrity by the complement system in a neuroinflammatory setting. For the first time, a novel role of C5a in the maintenance of BBB integrity is identified and the potential of C5a/C5aR blockade highlighted as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

MRI Mapping of the Blood Oxygenation Sensitive Parameter T2* in the Kidney: Basic Concept.

The role of hypoxia in renal disease and injury has long been suggested but much work still remains, especially as it relates to human translation. Invasive pO2 probes are feasible in animal models but not for human use. In addition, they only provide localized measurements. Histological methods can identify hypoxic tissue and provide a spatial distribution, but are invasive and allow only one-time point. Blood oxygenation level dependent (BOLD) MRI is a noninvasive method that can monitor relative oxygen availability across the kidney. It is based on the inherent differences in magnetic properties of oxygenated vs. deoxygenated hemoglobin. Presence of deoxyhemoglobin enhances the spin-spin relaxation rate measured using a gradient echo sequence, known as R2* (= 1/T2*). While the key interest of BOLD MRI is in the application to humans, use in preclinical models is necessary primarily to validate the measurement against invasive methods, to better understand physiology and pathophysiology, and to evaluate novel interventions. Application of MRI acquisitions in preclinical settings involves several challenges both in terms of logistics and data acquisition. This section will introduce the concept of BOLD MRI and provide some illustrative applications. The following sections will discuss the technical issues associated with data acquisition and analysis.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction chapter is complemented by two separate chapters describing the experimental procedure and data analysis.

Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background.

Chronic kidney disease (CKD) begins with renal injury; the progression thereafter depends upon a number of factors, including genetic background. Unilateral ureteral obstruction (UUO) is a well-described model of renal fibrosis and as such is considered a model of CKD. We used an improved reversible unilateral ureteral obstruction (rUUO) model in mice to study the strain dependence of development of CKD after obstruction-mediated injury. C57BL/6 mice developed CKD after reversal of three or more days of ureteral obstruction as assessed by blood urea nitrogen (BUN) measurements (>40 mg/dl). In contrast, BALB/c mice were resistant to CKD with up to 10 days ureteral obstruction. During rUUO, C57BL/6 mice exhibited pronounced inflammatory and intrinsic proliferative cellular responses, disruption of renal architecture, and ultimately fibrosis. By comparison, BALB/c mice had more controlled and measured extrinsic and intrinsic responses to injury with a return to normal within several weeks after release of ureteral obstruction. Our findings provide a model that allows investigation of the genetic basis of events during recovery from injury that contribute to the development of CKD.

Renal FcRn reclaims albumin but facilitates elimination of IgG.

The widely distributed neonatal Fc receptor (FcRn) contributes to maintaining serum levels of albumin and IgG in adults. In the kidney, FcRn is expressed on the podocytes and the brush border of the proximal tubular epithelium. Here, we evaluated the role of renal FcRn in albumin and IgG metabolism. Compared with wild-type controls, FcRn(-/-) mice had a lower t((1/2)) for albumin (28.7 versus 39.9 h) and IgG (29.5 versus 66.1 h). Renal loss of albumin could account for the former, suggested by the progressive development of hypoalbuminemia in wild-type mice transplanted with FcRn-deficient kidneys. Furthermore, serum albumin levels returned to normal in FcRn(-/-) recipients of wild-type kidneys after removing the native FcRn-deficient kidneys. In contrast, renal loss could not account for the enhanced elimination of IgG in FcRn(-/-) mice. These mice had minimal urinary excretion of native and labeled IgG, which increased to wild-type levels in FcRn(-/-) recipients of a single FcRn-sufficient kidney (t((1/2)) of IgG was 21.7 h). Taken together, these data suggest that renal FcRn reclaims albumin, thereby maintaining the serum concentration of albumin, but facilitates the loss of IgG from plasma protein pools.

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.

TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway.

Curcumin aggravates CNS pathology in experimental systemic lupus erythematosus.

Complement activation and inflammation are key disease features of systemic lupus erythematosus. Curcumin is an anti-inflammatory agent that inhibits the complement cascade. Therefore, we hypothesized that curcumin will be protective in CNS lupus. To assess the effect of curcumin on CNS-lupus, MRL/lpr mice were used. Brain MRI showed that curcumin (30mg/kg body wt. i.p. from 12-20 weeks) worsened regional brain atrophy. The volumes of the lateral and third ventricles are significantly increased (150%-213% and 107%-140%, without and with treatment respectively compared to MRL+/+ controls). The hippocampus was reduced further (83%-81%) by curcumin treatment. In line with increased brain atrophy, there were edematous cells (41% increase in cell size in MRL/lpr compared to MRL+/+ mice. The cell size was further increased by 28% when treated with curcumin; p<0.02) in the cortex. In line with increased atrophy and edema, there was a significant increase (p<0.02) in the mRNA and protein expression of the water channel protein, aquaporin 4 in these mice. The increase in the matrix proteins, glial fibrillary acidic protein and vimentin in lupus mice in the hippocampus was prevented by curcumin. Curcumin increased IgG deposits and decreased C3 deposits in brain with a corresponding increase in immune complexes and decrease in C3 concentration (by 60% in MRL/lpr mice Vs. MRL+/+ mice and a further 26% decrease when treated with curcumin) in circulation. Decrease in C3 could alter the transport of immune complexes leading to an increase in IgG deposits which could induce inflammatory pathways thereby leading to worsening of the disease. The neurological outcome as measured by maze performance indicates that the curcumin treated mice performed poorly compared to the untreated counterparts. Our results for the first time provide evidence that at the dose used in this study, curcumin aggravates some CNS disease manifestations in experimental lupus brain. Therefore, until a safe dose range is established by additional studies, and the validity of the findings is determined in human patients, caution may be warranted in the use of curcumin, even as adjuvant therapy for CNS lupus.

Inhibition of C5a receptor alleviates experimental CNS lupus.

To investigate the role of C5a generated on complement activation in brain, the lupus model, MRL/lpr mice were treated with C5a receptor(R) antagonist (ant). Neutrophil infiltration, ICAM, TNF-alpha and iNOS mRNA expression, neuronal apoptosis and the expression of p-JNK, pSTAT1 and p-Erk were reduced and p-Akt increased on C5aR inhibition in MRL/lpr brains. MRL/lpr serum caused increased apoptosis in neurons showing that lupus had a direct effect on these cells. C5aRant pretreatment prevented the lupus serum induced loss of neuronal cells. Our findings demonstrate for the first time that C5a/C5aR signaling plays an important role in the pathogenesis of CNS lupus.