Michael G. Robson

Thrombotic micro-angiopathy with sirolimus-based immunosuppression: potentiation of calcineurin-inhibitor-induced endothelial damage?

Thrombotic microangiopathy is a rare but important finding in the context of organ transplantation. Acute renal insufficiency in the setting of hemolysis and thrombocytopenia, a triad that constitutes ‘hemolytic uremic syndrome’, can be associated with, or triggered by, conditions such as verocytotoxin‐producing Escherichia coli, viral infections, malignant hypertension, scleroderma, allograft rejection, lupus erythematosus, pregnancy, and medications including mitomycin C, calcineurin inhibitors, and oral contraceptives. After renal transplantation, it can occur, as either a de novo episode, or recurrent disease. Calcineurin inhibitors have long been associated with post‐transplantation thrombotic microangiopathy. Sirolimus has been used as a primary immunosuppressant in patients transplanted with a history of earlier hemolytic–uremic syndrome, and also as rescue therapy in patients with calcineurin‐inhibitor‐associated thrombotic microangiopathy. We describe four cases where there was significant thrombotic microangiopathy in the context of contemporaneous or contiguous calcineurin inhibitor and sirolimus usage. As the intrarenal cyclosporin concentration is thought to be significantly elevated when cyclosporin and sirolimus are used together, this may explain these findings, and mandates caution in their co‐administration.

Accelerated Nephrotoxic Nephritis Is Exacerbated in C1q-Deficient Mice

C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.

Toll-Like Receptor 4 Ligation on Intrinsic Renal Cells Contributes to the Induction of Antibody-Mediated Glomerulonephritis via CXCL1 and CXCL2

Autoimmune diseases such as glomerulonephritis are exacerbated by infection. This study examined the effect of the Toll-like receptor 4 (TLR4) ligand lipid A on the development of heterologous nephrotoxic nephritis. Administration of nephrotoxic antibody resulted in significant glomerular neutrophil infiltration and albuminuria only when a TLR4 ligand was administered simultaneously. The contribution of TLR4 on renal cells and circulating leukocytes was assessed. Bone marrow chimeras were constructed with TLR4 only on renal cells or bone marrow-derived cells. The administration of nephrotoxic serum and lipid A caused a neutrophil influx in both chimeric groups greater than in sham chimeras that were totally TLR4 deficient but significantly less than in sham chimeras that were totally TLR4 sufficient. Both chimeric groups had greater albuminuria than totally TLR4-deficient sham chimeras; however, the chimeras with TLR4 only on intrinsic renal cells had significantly less than the sham positive group. In situ hybridization showed expression of TLR4 mRNA in mesangial cells and glomerular epithelial cells. For investigation of the potential mechanism by which renal cells could contribute to disease exacerbation, mesangial cells were cultured and found to express mRNA for TLR4, and stimulation of wild-type and TLR4-deficient mesangial cells with LPS caused production of CXC chemokines by wild-type cells only. Treatment of chimeras with TLR4 present only on intrinsic renal cells with anti-CXCL1 and anti-CXCL2 antibody before disease induction significantly reduced renal neutrophil infiltration. These results show that TLR4 on both circulating leukocytes and intrinsic renal cells contributes to the inflammatory effects of antibody deposition within the glomerulus, which depends at least in part on the production of CXC chemokines by intrinsic renal cells.

Toll-like receptors and renal disease.

Toll-like receptors (TLRs) play a central role in the response of both the innate and the adaptive immune system to microbial ligands. There is also evidence that they are stimulated by endogenous ligands. In this review, I discuss evidence that they are important in renal disease. This discussion considers the role of both endogenous and microbial ligands, and also the contribution of TLRs present on leucocytes and on intrinsic renal cells. There is strong evidence of a role for TLR2 and TLR4 in renal ischaemia-reperfusion injury, with the effects probably mediated by endogenous ligands. In systemic lupus erythematosus, stimulation of TLR7 and TLR9 by host-derived nucleic acids is important. TLR7 stimulation exacerbates disease, but the role of TLR9 is complex. I also discuss evidence that they are important in other forms of glomerulonephritis, with evidence derived mainly from experimental models in which exogenous ligands have been administered.

TLR2 Stimulation of Intrinsic Renal Cells in the Induction of Immune-Mediated Glomerulonephritis

Infection may exacerbate organ-specific autoimmune disease such as glomerulonephritis. This may occur in the absence of a measurable effect on the adaptive immune response, and the mechanisms responsible are not fully understood. To investigate this, we have studied the effect of TLR2 ligation by the synthetic ligand Pam3CysSK4 on the development of glomerulonephritis in mice. We demonstrated that glomerular inflammation induced by passive administration of nephrotoxic Ab does not occur in the absence of TLR2 stimulation, with a strong synergy when Ab deposition and TLR2 stimulation occur together. Parameters of glomerular inflammation were neutrophil influx, thrombosis, and albuminuria. To investigate the relative contribution of TLR2 on bone marrow-derived cells and intrinsic renal cells, we constructed bone marrow chimeras. Nephrotoxic Ab and TLR2 ligation caused a neutrophil influx in both types of chimera at a similar level to that seen in sham chimeras totally TLR2 sufficient. Albuminuria was seen in both types of chimera above that seen in sham chimeras that were totally TLR2 deficient. This was greater in chimeras with TLR2 present on bone marrow-derived cells. To find a potential mechanism by which intrinsic renal cells may contribute toward disease exacerbation, mesangial cells were studied and shown to express TLR2 and MyD88. Wild-type but not TLR2-deficient mesangial cells produced CXC chemokines in response to stimulation with Pam3CysSK4. These results demonstrate that TLR2 stimulation on both bone marrow-derived and resident tissue cells plays a role in amplifying the inflammatory effects of Ab deposition in the glomerulus.

Toll-Like Receptor 2 Agonists Exacerbate Accelerated Nephrotoxic Nephritis

Glomerulonephritis can be exacerbated by infection. This study investigated the effect of N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3-lysine (Pam3CysSK4), a synthetic Toll-like receptor 2 (TLR2) ligand, that was given at immunization on disease severity in accelerated nephrotoxic nephritis. Stimulation of TLR by microbial constituents is known to influence the development of the adaptive immunity. It was hypothesized that the TLR2 ligand Pam3CysSK4 can modulate the development of disease in accelerated nephrotoxic nephritis by influencing the development of adaptive immunity. It is shown that Pam3CysSK4, when given at immunization, can increase profoundly the severity of disease, and with the use of TLR2-deficient mice, it is shown that this disease exacerbation depends on the presence of TLR2. Wild-type mice that were given Pam3CysSK4 at immunization and had more severe disease also had a greater amount of antigen-specific IgG1, IgG2b, and IgG3 in the serum and more IgG2b and IgG3 deposited within the glomerulus. They also had increased numbers of glomerular CD4-positive T cells. Therefore, the more severe disease that was seen in the group that was immunized with lipopeptide can be attributed to an influence on the adaptive immune response.

FcγRIII and FcγRIV are indispensable for acute glomerular inflammation induced by switch variant monoclonal antibodies

The relative ability of IgG subclasses to cause acute inflammation and the roles of specific effector mechanisms in this process are not clear. We explored this in an in vivo model of glomerular inflammation in the mouse. Trinitrophenol was planted on the glomerular basement membrane after conjugation to nephrotoxic Ab. The relative nephritogenicity of anti-trinitrophenol switch variant mAbs was then explored and shown to be IgG2a > IgG2b, with no disease caused by IgG1. Using knockout mice, we showed that FcγRIII was necessary for both neutrophil influx and glomerular damage induced by IgG2a and IgG2b. Surprisingly, IgG1 did not cause disease although it binds to FcγRIII. Using blocking Abs, we showed that this was explained by an additional requirement for FcγRIV, which does not bind to IgG1. IgG2a- or IgG2b-induced neutrophil influx was not affected by deficiency of either FcγRI or C3. Bone marrow chimeras were constructed to test the effect of combined deficiency of FcγRI and C3, and there was no effect on IgG2a- or IgG2b-mediated neutrophil influx. However, IgG2b-induced albuminuria and thrombosis were reduced in C3-deficient mice, showing an additional role for complement in IgG2b-mediated glomerular damage. The results show that IgG2a and IgG2b are the pathogenic subclasses in acute neutrophil-mediated glomerular inflammation, with an indispensable role for both FcγRIII and FcγRIV. Additionally, complement contributes to IgG2b-induced glomerular injury.

Animal models of anti‐neutrophil cytoplasmic antibody‐associated vasculitis

OTHER ARTICLES PUBLISHED ON ANCA IN THIS ISSUE

Transferred Antigen-Specific TH17 but not TH1 Cells Induce Crescentic Glomerulonephritis in Mice

To explore the role of antigen-specific CD4(+) T cells in glomerulonephritis, we administered ovalbumin 323-339 peptide conjugated to glomerular-binding polyclonal antibody and induced disease in RAG1(-/-) mice with CD4(+) T cells from OT2 × RAG1(-/-) mice. These OT2 × RAG1(-/-) mice have a transgenic T-cell receptor specific for this peptide. When CD4(+) T cells were primed in vivo, crescentic glomerulonephritis developed after 21 days in mice given peptide-conjugated glomerular-binding antibody but not unconjugated antibody control. We then investigated the relative roles of T(H)1 and T(H)17 cells, using Fab(2) fragments of glomerular-binding antibody to exclude a role for antibody in this model. T cells from OT2 × RAG1(-/-) mice were polarized in vitro, and T(H)1 or T(H)17 cell lines were injected into mice that were also given peptide-conjugated Fab(2) or unconjugated Fab(2) control, giving four experimental groups. After 21 days crescentic glomerulonephritis was seen in mice receiving T(H)17 cells and peptide-conjugated Fab(2) but in none of the other three groups. These results suggest that T(H)17 but not T(H)1 cells can induce crescentic glomerulonephritis.

Toll-Like Receptor 4 Stimulation Triggers Crescentic Glomerulonephritis by Multiple Mechanisms Including a Direct Effect on Renal Cells

A role for toll-like receptor 4 (TLR4) has been suggested in previous studies of glomerulonephritis, but the complex integration of these effects has not been explored. To separate effects on the innate and adaptive immune responses, we use the autologous nephrotoxic nephritis model with two disease induction protocols. First, we give a TLR4 ligand at the time of immunization and show the effects are mediated via TLR4 by comparing wild-type and TLR4-deficient mice. In wild-type mice histological measures of disease and serum creatinine are all at least twice as high as TLR4-deficient mice, due to an enhanced immune response to the nephritogenic sheep IgG. Second, we stimulate TLR4 later in the course of disease development and construct four groups of bone marrow chimeric or sham chimeric mice to study the role of TLR4 on bone marrow or renal cells. The most striking finding is that renal cell TLR4 stimulation increases glomerular crescent formation, with a mean of 21% and 25% in the two groups of mice with renal cell TLR4 compared with 0.1% and 0.6% in the two groups without, with differences mirrored by changes in serum creatinine. These findings, in a single disease model, illustrate that TLR4 stimulation triggers crescentic glomerulonephritis by effects on both the adaptive and innate immune response, with a crucial direct effect on renal cells.