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.     

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.     

Modulation of renal disease in MRL/lpr mice by pharmacologic inhibition of inducible nitric oxide synthase

BACKGROUND: MRL-MPJFaslpr (MRL/lpr) mice spontaneously develop lupus-like disease characterized by immune complex glomerulonephritis and overproduction of nitric oxide (NO). Blocking NO production pharmacologically by a non-specific nitric oxide synthase (NOS) inhibitor ameliorated renal disease in MRL/lpr mice while genetically deficient inducible NOS (iNOS) mice developed proliferative glomerulonephritis similar to wild-type controls.METHODS: To clarify the role of iNOS in the pathogenesis of nephritis in MRL/lpr mice, we treated mice with two different NOS inhibitors. Either NG-monomethyl-l-arginine (L-NMMA), a nonspecific NOS inhibitor, or l-N6-(1-iminoethyl)lysine (L-NIL), an iNOS specific inhibitor, was administered in the drinking water from 10 through 22 weeks of age with disease progression monitored over time. Control mice received water alone.RESULTS: Both L-NMMA and L-NIL blocked NO production effectively in MRL/lpr mice. As expected, neither L-NNMA nor L-NIL had an effect on antibody production, immune complex deposition or complement activation. Although both NOS inhibitors decreased protein excretion, L-NMMA was more effective than L-NIL. Pathologic renal disease was significantly decreased at 19 weeks in both treatment groups. At 22 weeks the L-NIL treated mice, but not the L-NMMA mice, had significantly reduced renal disease scores compared to controls.CONCLUSION: These results indicate that specific inhibition of iNOS blocks the development of pathologic renal disease in MRL/lpr mice.     

Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease

BACKGROUND: Retinoic acid (tRA) is an active metabolite of vitamin A with potent anti-inflammatory properties. We analyzed the effects of tRA on the development of lupus nephritis in MRL/lpr mice. METHODS: MRL/lpr mice received chow supplemented with vehicle or tRA (daily 10 mg/kg) from 8 to 14 weeks until their sacrifice. MRL/wt mice served as an additional control. RESULTS: tRA-treated MRL/lpr mice showed reduced lymphoadenopathy and splenomegaly as compared to vehicle-treated controls. Treatment reduced proteinuria to almost basal levels. Plasma IgG and anti-DNA antibodies increased comparably in both vehicle and tRA-treated mice. Vehicle-treated mice showed characteristic renal lesions. In contrast tRA-treated mice showed almost normal glomerular histology with a pronounced reduction in endocapillary cell proliferation. T-cell and macrophage infiltrates were reduced after tRA treatment within glomeruli and interstitium as compared to vehicle-treated animals. In spite of this, immune complex and complement deposition were comparable in both groups. Adoptively transferred T cells from vehicle-treated to tRA-treated MRL/lpr mice did not induce renal lesions or proteinuria. These beneficial effects of tRA treatment were associated with reduced renal expression of chemokines and inflammatory cytokines. Surprisingly, renal transforming growth factor-beta (TGF-beta) mRNA levels of tRA-treated mice were elevated, possibly indicating that TGF-beta acts as an anti-inflammatory signal in this lupus model. CONCLUSION: tRA treatment reduces lymphoproliferation and glomerulonephritis in MRL/lpr mice. This occurs in spite of unaltered anti-DNA titers and glomerular immune complex deposition, and cannot be overcome by T-cell transfer from nephritic MRL/lpr mice.     

Increased survival and reduced renal injury in MRL/lpr mice treated with a novel sphingosine-1-phosphate receptor agonist

Agonists of the type 1 sphingosine-1-phosphate (S1P) receptor inhibit lymphocyte migration, causing their sequestration in lymphoid tissue. The S1P agonist FTY720 prolongs the survival of organ allografts and blocks T-cell mediated autoimmune diseases in experimental models; however, it is a non-selective agonist of four of the five S1P receptors. In this study female MRL/lpr mice, which develop an aggressive form of spontaneous autoimmune kidney disease, were treated with a more selective agonist of the type 1 receptor (KRP-203) or vehicle at 12 or 16 weeks of age. Eighty percent of the mice treated at 12 weeks, before the onset of visible disease, survived to the 24 weeks end point with decreased tubulointerstitial disease and significantly fewer infiltrating CD4(+) and CD8(+) T-cells. Only half of the control vehicle-treated mice survived. All of the mice treated at 16 weeks survived with reduced proteinuria. Mice in both groups had significant reductions in circulating lymphocytes. Mice receiving KRP-203 for 8-12 weeks had significant reductions in T-cells and consequently less adenopathy. Ex vivo treatment of lymphocytes from MRL/lpr mice with KRP-203 enhanced their apoptosis. Our study indicates that KRP-203 attenuates kidney injury in MRL/lpr mice, in part, by reducing T-cell infiltrates.     

Metabolism of complement factor D in renal failure

Factor D is an essential enzyme of the alternative pathway of complement. Its plasma concentration increases approximately tenfold in end-stage renal failure (ESRF). To analyze its metabolism in humans, we injected purified radiolabelled factor D into 5 healthy individuals and 12 patients with various renal diseases or renal failure. Fractional metabolic rates (FMR) and extravascular/intravascular distributions (EV/IV) were calculated using a compartmental model. The FMR was very rapid in normal individuals (mean 59.6%/hr; range 74.1 to 50.5), significantly diminished in the five patients with ESRF (5.7%/hr; 7.0 to 2.8; P less than 0.004), and correlated well with the creatinine clearance (r = 0.89; P less than 0.001). The extrarenal catabolic rate was not modified in renal failure. Despite a significant inverse correlation between plasma levels of factor D and creatinine clearance [r = 0.68; P less than 0.002], factor D levels were not a sensitive indicator of renal function because the synthesis rate (SR) varied widely from one individual to another (mean SR: 62.9 micrograms/kg/hr; 14.9 to 136.5). Factor D synthesis was not significantly altered by renal function, and did not correlate with C-reactive protein, suggesting that factor D is not an acute phase protein. The proportion of intact factor D elimination in the urine was increased in patients with tubular dysfunction (up to 15% compared to less than 0.2% in normal individuals) confirming that under normal circumstances factor D is filtered through the glomerulus and catabolized by tubular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary complement factor H in renal disease

BACKGROUND: Complement factor H (hCFH) plays a key inhibitory role in the control of the alternative complement pathway. We examined whether urinary hCFH (U-hCFH) levels is useful as an indirect indicator of renal damage. METHODS: Urine samples were obtained from 104 patients with renal disease. Urine was collected with 10 mM EDTA and U-hCFH levels were measured using the BTA TRAK Assay Kit. RESULTS: In the 62 patients with nephritis, the levels of U-hCFH were elevated (range 15-52,198 U/ml) over the normal range (0-14 U/ml). U-hCFH levels of patients with chronic renal failure, lupus nephritis, membranoproliferative glomerulonephritis, focal glomerulosclerosis were higher than that of IgA nephropathy patients (p < 0.05). In the patients with minimal change disease, showed high levels of U-hCFH during the nephrotic syndrome. U-hCFH was correlated significantly with urinary protein and urinary N-acetyl-beta-D-glucosaminidase. CONCLUSIONS: We demonstrated that U-hCFH was detected in the urine of nephritis patients.     

Mesangial immune complex glomerulonephritis due to complement factor D deficiency

Complement factor D is a serine protease essential for the activation of the alternative pathway and is expressed in the kidney, adipocytes, and macrophages. Factor D is found at relatively high levels in glomeruli suggesting that this component of the complement cascade could influence renal pathophysiology. In this study, we utilize mice with a targeted deletion of the activating complement factor D gene and compare these results to mice with targeted deletion of the inhibitory complement factor H gene. Eight-month-old mice with a deleted factor D gene spontaneously develop albuminuria and have reduced creatinine clearance due to mesangial immune complex glomerulonephritis. These mesangial deposits contain C3 and IgM. In contrast to the mesangial location of the immune deposits in the factor D-deficient mice, age-matched factor H-deficient mice develop immune deposits along the glomerular capillary wall. Our observations suggest that complement factor D or alternative pathway activation is needed to prevent spontaneous accumulation of C3 and IgM deposits within the mesangium. Our studies show that the complement factor D gene knockout mice are a novel model of spontaneous mesangial immune complex glomerulonephritis.     

Steroid-responsive cochlear dysfunction in the MRL/lpr autoimmune mouse.

Corticosteroids historically have been used to treat autoimmune sensorineural hearing loss, although little is known of how steroids restore normal inner ear function. Therefore, to identify a potential model for this field of research, this study examined the effects of prednisolone on auditory brain stem response thresholds in the MRL/lpr mouse model of autoimmune sensorineural hearing loss. Mice treated with prednisolone after auditory threshold elevations demonstrated significant improvement and stabilization of thresholds compared with untreated controls. MRL/lpr mice treated with steroids before the onset of autoimmune disease and cochlear dysfunction demonstrated decreased serum immune complexes, higher survival rates, and lower auditory thresholds compared with untreated controls. These positive results suggest the autoimmune mouse may be useful for studies of steroid-responsive mechanisms of the cochlea in autoimmune sensorineural hearing loss, as well as any hearing disorder in which steroid therapy is currently used.     

Effects of excess factor D on early- and late-phase activation of the complement cascade.

The present study was undertaken to examine the effects of excess factor D build-up in the body of end-stage renal disease (ESRD) patients upon the activation of the alternative pathway and the terminal pathway in the fluid phase. First, to clarify the effect of excess factor D on the alternative pathway, purified factor D from an ESRD patient was added to normal serum and the changes in concentrations of C3a-des-Arg and C5a-des-Arg were investigated. The results showed that once the serum factor D level reached a concentration corresponding to 15 micrograms/ml in the serum of the ESRD patient, the C3a-des-Arg and C5a-des-Arg levels had climbed to about 1.7-fold the concentration in normal serum. Next, in order to clarify the effect of excess factor D on the terminal pathway, purified factor D was added to normal serum, and the changes in C5b6 generation were examined. The results indicated that as the factor D level increased in the serum, the C5b6 level rose gradually also; and when the factor D concentration reached 15 micrograms/ml, the C5b6 generation had risen to approximately 1.5-fold the level in normal serum. The present results therefore suggest that factor D build-up in ESRD patients provides a uremic toxin that can cause abnormal activation of the whole complement cascade.     

Fas on renal parenchymal cells does not promote autoimmune nephritis in MRL mice

BACKGROUND: Although Fas on pancreatic islets promotes autoimmune diabetes in mice, the role of Fas expression on kidney parenchymal cells during autoimmune disease is unknown. METHODS: To determine whether Fas on renal parenchymal cells promotes autoimmune renal destruction, we compared apoptosis and pathology in Fas-intact and Fas-deficient kidneys in an autoimmune milieu. For this purpose, we transplanted single, normal kidneys from MRL-++ (Fas-intact) mice (3 months of age) into age-matched, congenic MRL-Faslpr (Fas-deficient) recipients after removal of nephritic kidneys. These Fas-intact kidneys were compared with Fas-deficient nephritic kidneys. RESULTS: There is a progressive increase of FasL on kidney-infiltrating cells and Fas and FasL on renal parenchymal cells in MRL-++ kidneys during engraftment (0, 2, 4-6, and 8 weeks). By comparison, we detected an increase in FasL in MRL-Faslpr kidneys (3 to 5 months of age), whereas Fas was not detectable. The engagement of T cells bearing FasL with Fas expressing tubular epithelial cells (TECs) induced TEC apoptosis in vitro. However, apoptosis and pathology were similar in kidneys (MRL-++, 8 weeks postengraftment vs. MRL-Faslpr, 5 months) with equivalent amounts of FasL-infiltrating cells or FasL TECs, regardless of Fas on renal parenchymal cells. CONCLUSION: The expression of Fas on renal parenchymal cells does not increase apoptosis or promote renal disease in MRL-++ mice. We speculate that the autoimmune milieu evokes mechanisms that mask, counter, or pre-empt Fas-FasL-initiated apoptosis in MRL kidneys.     

三氧化二砷及5-氮胞苷对狼疮鼠的免疫状态和基因甲基化的影响

目的：探讨三氧化二砷（arsenic trioxide，ATO）及5-氮胞苷（5-Aza）对MRL／lpr狼疮小鼠免疫状况和基因甲基化的影响。方法：12周龄MRL／lpr小鼠36只，随机分为3组分别给ATO、5-Aza与生理盐水（NS），疗程为60d。分别检测治疗前后抗dsDNA抗体，治疗后用高效液相方法检测脾脏、淋巴结、胸腺和血液DNA甲基化水平。结果：（1）治疗后ATO组血清抗dsDNA抗体明显低于NS组与5-Aza组（P〈0．01）；（2）治疗后ATO组脾脏及淋巴结重量较NS组与5-Aza组有明显减轻（P〈0．05）；（3）治疗后ATO组脾脏及淋巴结的甲基化水平较Ns组有明显升高（P〈0．05），而血液及胸腺甲基化水平两组间差异无统计学意义，治疗后5-Aza组脾脏、淋巴结及血液的甲基化水平较NS组明显下降（P〈0．05）。结论：ATO能够降低小鼠外周血抗dsDNA抗体水平并抑制淋巴组织的增生，明显提高脾脏和淋巴结的基因甲基化水平。     

Effects of renal failure on complement C3d levels

Elevated plasma concentrations of complement split product C3d have been reported to represent activation of the complement system. In the present study the effect of renal function on C3d concentrations was investigated in patients with various degrees of renal impairment, in patients with chronic renal failure and in CAPD patients. It appeared that elevated plasma C3d concentrations were present in patients with plasma creatinine concentrations in excess of 200 mumol/l regardless of the type of kidney disease. It is very likely that this can be attributed to renal handling (i.e. glomerular filtration, tubular reabsorption and renal catabolism) of C3d in a similar way as has been demonstrated for other low molecular weight proteins. The peritoneal permeability to C3d was slightly less than could be expected on the basis of its molecular weight without evidence of local production of C3d. Renal function should be taken into account in the interpretation of elevated plasma concentrations of C3d.     

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.     

Effects of vitamin D deficiency in the chicken embryo

Summary Vitamin D-deficient chicken embryos were obtained by feeding laying hens a diet in which 5 μg 1,25(OH)₂D₃/kg feed were substituted for the vitamin D₃ supplement in the control diet. Hatchability, total Ca and inorganic P concentration in blood, and tibial ash/dry weight ratio were determined in the vitamin D-deficient embryos and in embryos obtained from hens fed the control diet supplemented with 1100 IU vitamin D₃/kg feed. After 5 weeks on the substituted diet the hens laid eggs that showed decreased hatchability in spite of excellent shell quality. All determinations in blood and bones were made on embryos of eggs laid after 6–12 weeks on the diets. On the 17th day of incubation the embryos derived from hens fed the substituted diet showed significant hypocalcemia and hyperphosphatemia and a low tibial ash/dry weight ratio. Injection of 1,25(OH)₂D₃ 3 days before killing corrected the hypocalcemia of the deficient embryos. Those chicks that managed to hatch had normal levels of calcium and inorganic phosphate 1 day after hatching. These findings support previous suggestions by us and other authors that vitamin D metabolites are required by the embryo in order to mobilize calcium from the shell, and decreased hatchability in vitamin D-deficient embryos is related to a defect in calcium mobilization from the shell. While in previous studies a decrease in hatchability was the only parameter used to judge D deficiency of the embryos in our present studies, the deficiency is confirmed by demonstrating a deficit in mineral metabolism which is a more specific sign of D deficiency.     

Mineral metabolism and arterial functions in end-stage renal disease: potential role of 25-hydroxyvitamin D deficiency

In ESRD, arterial function is abnormal, characterized by decreased capacitive function (arterial stiffening) and reduced conduit function, shown by diminished flow-mediated dilation (FMD). The pathophysiology of these abnormalities is not clear, and this cross-sectional study analyzed possible relationships among arterial alterations and cardiovascular risk factors, including mineral metabolism parameters, such as serum parathormone, and vitamin D "nutritional" and "hormonal" status by measuring serum 25-hydroxyvitamin D [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] levels. Aortic stiffness (pulse wave velocity), brachial artery (BA) distensibility (echotracking; n = 42), BA FMD (hand-warming; n = 37), and arterial calcification scores (echography and plain x-rays) were measured in 52 stable and uncomplicated patients who were on hemodialysis. 25(OH)D(3) and 1,25(OH)(2)D(3) serum levels were low and weakly correlated (r = 0.365, P < 0.05). After adjustment for BP and age, multivariate analyses indicated that 25(OH)D(3) and 1,25(OH)(2)D(3) were negatively correlated with aortic pulse wave velocity (P < 0.001) and positively correlated with BA distensibility (P < 0.01) and FMD (P < 0.001). Arterial calcification scores were not independently associated with 25(OH)D(3) and 1,25(OH)(2)D(3) serum concentrations. These results suggest that nutritional vitamin D deficiency and low 1,25(OH)(2)D(3) could be associated with arteriosclerosis and endothelial dysfunction in patients who have ESRD and are on hemodialysis.