Bradley K. Hack

Administration of a Soluble Recombinant Complement C3 Inhibitor Protects Against Renal Disease in MRL/lpr Mice

Complement receptor 1-related gene/protein y (Crry) in rodents is a potent membrane complement regulator that inhibits complement C3 activation by both classical and alternative pathways. To clarify the role of complement in lupus nephritis, MRL/lpr mice were given Crry as a recombinant protein (Crry-Ig) from 12 to 24 wk of age. Control groups were given saline or normal mouse IgG. Sera and urine were collected biweekly. Only 1 of 20 (5%) Crry-Ig-treated mice developed renal failure (BUN > 50 mg/dl) compared with 18 of 38 (47.4%) mice in control groups (P = 0.001). BUN levels at 24 wk were reduced from 68.8 +/- 9.7 mg/dl in control groups to 38.5 +/- 3.9 mg/dl in the Crry-Ig-treated group (P < 0.01). Urinary albumin excretion at 24 wk was also significantly reduced from 5.3 +/- 1.4 mg/mg creatinine in the control groups to 0.5 +/- 0.2 mg/mg creatinine in the Crry-Ig-treated group (P < 0.05). Of the histologic data at 24 wk, there was a significant reduction in scores for glomerulosclerosis and C3d, IgG, IgG3, and IgA staining intensity in glomeruli in complement-inhibited animals. Crry-Ig-treated animals were also protected from vasculitic lesions. Although there was no effect on relevant autoimmune manifestations such as anti-double stranded DNA titers or cryoglobulin IgG3 levels, circulating immune complex levels were markedly higher in complement-inhibited animals. Thus, inhibition of complement activation with Crry-Ig significantly reduces renal disease in MRL/lpr lupus mice. The data support the strategy of using recombinant complement C3 inhibitors to treat human lupus nephritis.

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 perm-selectivity. 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.

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.

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‐Tnfrsf6lpr (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.—Jacob, A., Hack, B., Chiang, E., Garcia, J. G. N., Quigg, R. J., Alexander, J. J. C5a alters blood‐brain barrier integrity in experimental lupus. FASEB J. 24, 1682–1688 (2010). www.fasebj.org

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.

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.

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.

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.

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

J. Neurochem. (2011) 119, 1041–1051.

Distinct and separable roles of the complement system in factor H-deficient bone marrow chimeric mice with immune complex disease

Plasma complement factor H (Cfh) is a potent complement regulator, whereas Cfh on the surface of rodent platelets is responsible for immune complex processing. For dissection between the two, bone marrow chimeras between Cfh-deficient (Cfh(-/-)) and wild-type C57BL/6 mice were created. Platelet Cfh protein was tracked with the Cfh status of the bone marrow donor, indicating that platelet Cfh is of intrinsic origin. In an active model of immune complex disease, Cfh(-/-) mice that were reconstituted with wild-type bone marrow had levels of platelet-associated immune complexes comparable to those of wild-type mice and were protected against the excessive glomerular deposition of immune complexes seen in Cfh(-/-) mice, yet these mice still developed glomerular inflammation. In contrast, wild-type mice with Cfh(-/-) bone marrow had reduced platelet-associated immune complexes and extensive glomerular deposition of complement-activating immune complexes, but they did not develop glomerular pathology. The large quantities of glomerular C3 in wild-type mice with Cfh(-/-) bone marrow were in the form of iC3b and C3dg, whereas active C3b remained in Cfh(-/-) recipients of wild-type bone marrow. These data show that plasma Cfh limits complement activation in the circulation and other accessible sites such as the glomerulus, whereas platelet Cfh is responsible for immune complex processing.