Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Effect of Short-term High-Dose Creatine Supplementation on Measured GFR in a Young Man With a Single Kidney

It currently is unknown whether creatine supplementation is safe for people with or at risk of kidney disease. We report on the short-term effects of creatine supplementation on kidney function in a young man with a single kidney and mildly decreased glomerular filtration rate (GFR). A 20-year-old man who had undergone unilateral nephrectomy and presented with mildly decreased GFR without kidney damage underwent a trial with 35 days of creatine supplementation (20 g/d for 5 days followed by 5 g/d for the next 30 days) and had his kidney function monitored. After the intervention, (51)Cr-EDTA clearance (pre, 81.6 mL/min/1.73 m(2); post, 82.0 mL/min/1.73 m(2)), proteinuria (protein excretion: pre, 130 mg/d; post, 120 mg/d), and electrolyte levels were unchanged. Albuminuria, serum urea level, and estimated creatinine clearance were decreased (pre, 4.6 mg/d; post, 2.9 mg/d; pre, 37 mg/d; post, 28 mg/dL; and pre, 88 mL/min/1.73 m(2); post, 71 mL/min/1.73 m(2), respectively), whereas serum creatinine level was slightly increased (pre, 1.03 mg/dL; post, 1.27 mg/dL), falsely suggesting kidney function impairment. This prospective report suggests that short-term creatine supplementation may not affect kidney function in an individual with a single kidney, mild decreased GFR, and ingesting a high-protein diet (ie, 2.8 g/kg/d). This finding has great relevance considering that creatine-induced kidney disease has been a growing concern, even for healthy people. Am J Kidney Dis 55: e7-e9. (C) 2010 by the National Kidney Foundation, Inc.

Avaliação da técnica de arteriografia renal guiada por fluoroscopia em suínos

The fluoroscopy-guided renal arteriography was evaluated in order to be used as an auxiliary method in investigations and as a way to experimentally induce kidney diseases in swine. The technique was effective to obtain sharp images as well as to determine the area of renal irrigation. Despite its easiness, trained professionals are required to perform it.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Association Between Increased Platelet P-Selectin Expression and Obesity in Patients With Type 2 Diabetes A BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) substudy

OBJECTIVE- To determine whether obesity increases platelet reactivity and thrombin activity in patients with type 2 diabetes plus stable coronary artery disease. RESEARCH DESIGN AND METHODS- We assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.2 (PT1.2, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.2, and 1 mu mol/l adenosine diphosphate (ADP). RESULTS- Concentrations of FPA, PT1.2, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 mu m ADP as assessed by surface expression of P-selectin (r = 0.29, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.2. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration Of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide. CONCLUSIONS- Among patients enrolled in this substudy of BARI 2D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type 2 diabetes.

Prevalence of diabetic peripheral neuropathy and relation to glycemic control therapies at baseline in the BARI 2D cohort

We evaluated the associations between glycemic therapies and prevalence of diabetic peripheral neuropathy (DPN) at baseline among participants in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial on medical and revascularization therapies for coronary artery disease (CAD) and on insulin-sensitizing vs. insulin-providing treatments for diabetes. A total of 2,368 patients with type 2 diabetes and CAD was evaluated. DPN was defined as clinical examination score > 2 using the Michigan Neuropathy Screening Instrument (MNSI). DPN odds ratios across different groups of glycemic therapy were evaluated by multiple logistic regression adjusted for multiple covariates including age, sex, hemoglobin A1c (HbA1c), and diabetes duration. Fifty-one percent of BARI 2D subjects with valid baseline characteristics and MNSI scores had DPN. After adjusting for all variables, use of insulin was significantly associated with DPN (OR = 1.57, 95% CI: 1.15-2.13). Patients on sulfonylurea (SU) or combination of SU/metformin (Met)/thiazolidinediones (TZD) had marginally higher rates of DPN than the Met/TZD group. This cross-sectional study in a cohort of patients with type 2 diabetes and CAD showed association of insulin use with higher DPN prevalence, independent of disease duration, glycemic control, and other characteristics. The causality between a glycemic control strategy and DPN cannot be evaluated in this cross-sectional study, but continued assessment of DPN and randomized therapies in BARI 2D trial may provide further explanations on the development of DPN.

Association of Changes in Bone Remodeling and Coronary Calcification in Hemodialysis Patients: A Prospective Study

Background: Vascular calcification is common and constitutes a prognostic marker of mortality in the hemodialysis population. Derangements of mineral metabolism may influence its development. The aim of this study is to prospectively evaluate the association between bone remodeling disorders and progression of coronary artery calcification (CAC) in hemodialysis patients. Study Design: Cohort study nested within a randomized controlled trial. Setting & Participants: 64 stable hemodialysis patients. Predictor: Bone-related laboratory parameters and bone histomorphometric characteristics at baseline and after 1 year of follow-up. Outcomes: Progression of CAC assessed by means of coronary multislice tomography at baseline and after 1 year of follow-up. Baseline calcification score of 30 Agatston units or greater was defined as calcification. Change in calcification score of 15% or greater was defined as progression. Results: Of 64 patients, 26 (40%) had CAC at baseline and 38 (60%) did not. Participants without CAC at baseline were younger (P < 0.001), mainly men (P = 0.03) and nonwhite (P = 0.003), and had lower serum osteoprotegerin levels (P = 0.003) and higher trabecular bone volume (P = 0.001). Age (P 0.003; beta coefficient = 1.107; 95% confidence interval [Cl], 1.036 to 1.183) and trabecular bone volume (P = 0.006; beta coefficient = 0.828; 95% Cl, 0.723 to 0.948) were predictors for CAC development. Of 38 participants who had calcification at baseline, 26 (68%) had CAC progression in 1 year. Progressors had lower bone-specific alkaline phosphatase (P = 0.03) and deoxypyridinoline levels (P = 0.02) on follow-up, and low turnover was mainly diagnosed at the 12-month bone biopsy (P = 0.04). Low-turnover bone status at the 12-month bone biopsy was the only independent predictor for CAC progression (P = 0.04; beta coefficient = 4.5; 95% Cl, 1.04 to 19.39). According to bone histological examination, nonprogressors with initially high turnover (n = 5) subsequently had decreased bone formation rate (P = 0.03), and those initially with low turnover (n = 7) subsequently had increased bone formation rate (P = 0.003) and osteoid volume (P = 0.001). Limitations: Relatively small population, absence of patients with severe hyperparathyroidism, short observational period. Conclusions: Lower trabecular bone volume was associated with CAC development, whereas improvement in bone turnover was associated with lower CAC progression in patients with high- and low-turnover bone disorders. Because CAC is implicated in cardiovascular mortality, bone derangements may constitute a modifiable mortality risk factor in hemodialysis patients.

Sex, Clinical Symptoms, and Angiographic Findings in Patients With Diabetes Mellitus and Coronary Artery Disease (from the Bypass Angioplasty Revascularization Investigation [BARI] 2 Diabetes Trial)

Previous studies have reported differences in presenting symptoms and angiographic characteristics between women and men undergoing evaluation for suspected coronary artery disease (CAD). We examined the relation between symptoms and extent of CAD in patients with type 2 diabetes mellitus and known CAD enrolled in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial. Of 1,775 patients (533 women, 30%, and 1,242 men, 70%), women were more likely than men to have angina (65% vs 56%, p < 0.001) or an atypical angina/anginal equivalent (71% vs 58%, p < 0.001). More women reported unstable angina (17% vs 13%, p = 0.047) or were in a higher Canadian Cardiology Society class compared to men (Canadian Cardiology Society classes II to IV 78% vs 68%, p = 0.002). Fewer women than men had no symptoms (14% vs 22%, p < 0.001). Women had a lower mean myocardial jeopardy index (42.5 +/- 24.3 vs 47.9 +/- 24.3, p < 0.001), smaller number of total significant lesions (2.3 +/- 17 1.7 vs 2.7 +/- 1.8, p < 0.001), and fewer jeopardized left ventricular regions (p < 0.001 for distribution) or long-term occlusions (29% vs 42%, p < 0.001). After adjustment for relevant covariates, the odds of having CAD symptoms were still higher in women than men (odds ratio for angina 1.31, 95% confidence interval 1.02 to 1.69; odds ratio for atypical angina 1.52, 95% confidence interval 1.17 to 1.96). In conclusion, in a high-risk group of patients with known CAD and diabetes mellitus, women were more symptomatic than men but had less obstructive CAD. These data suggest that factors other than epicardial CAD severity influence symptom presentation in women in this population. (C) 2011 Elsevier Inc. All rights reserved. (Am J Cardiol 2011;107:980-985)

Effects of Optimal Medical Treatment With or Without Coronary Revascularization on Angina and Subsequent Revascularizations in Patients With Type 2 Diabetes Mellitus and Stable Ischemic Heart Disease

Background-In the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial, an initial strategy of coronary revascularization and optimal medical treatment (REV) compared with an initial optimal medical treatment with the option of subsequent revascularization (MED) did not reduce all-cause mortality or the composite of cardiovascular death, myocardial infarction, and stroke in patients with type 2 diabetes mellitus and stable ischemic heart disease. In the same population, we tested whether the REV strategy was superior to the MED strategy in preventing worsening and new angina and subsequent coronary revascularizations. Methods and Results-Among the 2364 men and women (mean age, 62.4 years) with type 2 diabetes mellitus, documented coronary artery disease, and myocardial ischemia, 1191 were randomized to the MED and 1173 to the REV strategy preselected in the percutaneous coronary intervention (796) and coronary artery bypass graft (377) strata. Compared with the MED strategy, the REV strategy at the 3-year follow-up had a lower rate of worsening angina (8% versus 13%; P < 0.001), new angina (37% versus 51%; P = 0.001), and subsequent coronary revascularizations (18% versus 33%; P < 0.001) and a higher rate of angina-free status (66% versus 58%; P = 0.003). The coronary artery bypass graft stratum patients were at higher risk than those in the percutaneous coronary intervention stratum, and had the greatest benefits from REV. Conclusions-In these patients, the REV strategy reduced the occurrence of worsening angina, new angina, and subsequent coronary revascularizations more than the MED strategy. The symptomatic benefits were observed particularly for high-risk patients.

Obstetric Outcome in Pregnant Women on Long-term Dialysis: A Case Series

Background: Although still uncommon, pregnancy frequency in women on maintenance hemodialysis therapy has increased in the past 20 years. Most published reports suggest that intensified hemodialysis regimens result in better pregnancy outcomes. The small number of patients investigated in all reported series is the main limitation of the available studies. Study Design: Retrospective case series. Setting & Participants: Data for all pregnancies that occurred in 1988-2008 in women undergoing maintenance hemodialysis (52 pregnancies) at the Sao Paulo University Medical School (Sao Paulo, Brazil). Outcomes & Measurements: We analyzed maternal and fetal outcomes of 52 pregnancies, as well as their relationship with various clinical, laboratory, and hemodialysis parameters, such as pre-eclampsia, pregnancy before or after dialysis therapy, hemodialysis dose, polyhydramnios, anemia, and predialysis serum urea level. In addition, logistic regression models for a composite adverse fetal outcome (perinatal death or extremely premature delivery) and linear regression models for birth weight were built. Results: 87% overall rate of successful delivery, with a mean gestational age of 32.7 +/- 3.1 weeks. Pre-eclampsia was associated with a poor prognosis compared with pregnancies without pre-eclampsia: a successful delivery rate of 60% versus 92.9% (P = 0.02), extremely premature delivery rate of 77.8% versus 3.3% (P = 0.001), lower gestational age (P = 0.001), and birth weight (P = 0.001). Patients with an adverse composite fetal outcome had a higher frequency of pre-eclampsia (P = 0.001), lower frequency of polyhydramnios (P = 0.03), lower third-trimester hematocrit (P = 0.03), and higher predialysis serum urea level (P = 0.03). The same results were seen for birth weight. Limitations: Retrospective data analysis. The absence of creatinine clearance measurements did not allow evaluation of the impact of residual renal function on fetal outcome. Conclusions: Outcomes of pregnancy in women undergoing hemodialysis often are good. Preeclampsia, third-trimester hematocrit, polyhydramnios, and predialysis serum urea level are important variables associated with fetal outcome and birth weight. Am J Kidney Dis 56:77-85. (C) 2010 by the National Kidney Foundation, Inc.Inc

Factors Related to the Selection of Surgical Versus Percutaneous Revascularization in Diabetic Patients With Multivessel Coronary Artery Disease in the BARI 2D (Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes) Trial

Objectives We evaluated demographic, clinical, and angiographic factors influencing the selection of coronary artery bypass graft (CABG) surgery versus percutaneous coronary intervention (PCI) in diabetic patients with multivessel coronary artery disease (CAD) in the BARI 2D (Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes) trial. Background Factors guiding selection of mode of revascularization for patients with diabetes mellitus and multivessel CAD are not clearly defined. Methods In the BARI 2D trial, the selected revascularization strategy, CABG or PCI, was based on physician discretion, declared independent of randomization to either immediate or deferred revascularization if clinically warranted. We analyzed factors favoring selection of CABG versus PCI in 1,593 diabetic patients with multivessel CAD enrolled between 2001 and 2005. Results Selection of CABG over PCI was declared in 44% of patients and was driven by angiographic factors including triple vessel disease (odds ratio [OR]: 4.43), left anterior descending stenosis >= 70% (OR: 2.86), proximal left anterior descending stenosis >= 50% (OR: 1.78), total occlusion (OR: 2.35), and multiple class C lesions (OR: 2.06) (all p < 0.005). Nonangiographic predictors of CABG included age >= 65 years (OR: 1.43, p = 0.011) and non-U.S. region (OR: 2.89, p = 0.017). Absence of prior PCI (OR: 0.45, p < 0.001) and the availability of drug-eluting stents conferred a lower probability of choosing CABG (OR: 0.60, p = 0.003). Conclusions The majority of diabetic patients with multivessel disease were selected for PCI rather than CABG. Preference for CABG over PCI was largely based on angiographic features related to the extent, location, and nature of CAD, as well as geographic, demographic, and clinical factors. (Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes [BARI 2D]; NCT00006305) (J Am Coll Cardiol Intv 2009;2:384-92) (C) 2009 by the American College of Cardiology Foundation

Novel inactivating mutations in the GH secretagogue receptor gene in patients with constitutional delay of growth and puberty

Background: A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature. Objective: To analyze GHSR in idiopathic short stature (ISS) children including a subgroup of constitutional delay of growth and puberty (CDGP) patients. Subjects and methods: The GHSR coding region was directly sequenced in 96 independent patients with ISS, 31 of them with CDGP, in 150 adults, and in 197 children with normal stature. The pharmacological consequences of GHSR non-synonymous variations were established using in vitro cell-based assays. Results: Five different heterozygous point variations in GHSR were identified (c.-6 G>C, c.251G>T (p.Ser84Ile), c.505G>A (p.Ala169Thr), c.545 T>C (p.Val182Ala), and c.1072G>A (p.Ala358Thr)), all in patients with CDGP. Neither these allelic variants nor any other mutations were found in 694 alleles from controls. Functional studies revealed that two of these variations (p.Ser84Ile and p. Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. These mutations were identified in two female patients with CDGP (at the age of 13 years, their height SDS were -2.4 and -2.3). Both patients had normal progression of puberty and reached normal adult height (height SDS of -0.7 and -1.4) without treatment. Conclusion: This is the first report of GHSR mutations in patients with CDGP. Our data raise the intriguing possibility that abnormalities in ghrelin receptor function may influence the phenotype of individuals with CDGP.

Evaluation of the vaccination status in pediatric renal transplant recipients

To assess the immunization status of pediatric renal transplant patients followed at a single center in Brazil, vaccination charts of all patients aged between one and 18 yr were analyzed both pre- and post-transplantation. Appropriate immunization was defined according to the National Immunization Program (routine vaccines) - for all Brazilian children - and the Special Immunobiological Agents Program that also includes special vaccines for immunodeficient or other high-risk children. A total of 46 patients was evaluated (mean age 13.7 yr; range 4-17 yr). Vaccination charts were found to be up to date in only two patients (4.3%) pretransplant and in two (4.3%) post-transplant. Although 36 patients (62.2%) in the pretransplant phase and 24 (52.1%) in the post-transplant phase had been vaccinated according to the National Immunization Program, they had not received the special vaccines indicated for their immunocompromised condition. Therefore, despite being followed at a referral center, almost all patients presented an incomplete immunization status pre- and post-transplant. This probably reflects missed opportunities and medical/parental apprehension related to vaccination of patients with chronic renal insufficiency, dialysis or kidney transplantation. Efforts should be made to ensure adequate vaccination in children with kidney diseases, especially before kidney transplantation.

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Loxosceles gaucho Venom-Induced Acute Kidney Injury - In Vivo and In Vitro Studies

Background: Accidents caused by Loxosceles spider may cause severe systemic reactions, including acute kidney injury (AKI). There are few experimental studies assessing Loxosceles venom effects on kidney function in vivo. Methodology/Principal Findings: In order to test Loxosceles gaucho venom (LV) nephrotoxicity and to assess some of the possible mechanisms of renal injury, rats were studied up to 60 minutes after LV 0.24 mg/kg or saline IV injection (control). LV caused a sharp and significant drop in glomerular filtration rate, renal blood flow and urinary output and increased renal vascular resistance, without changing blood pressure. Venom infusion increased significantly serum creatine kinase and aspartate aminotransferase. In the LV group renal histology analysis found acute epithelial tubular cells degenerative changes, presence of cell debris and detached epithelial cells in tubular lumen without glomerular or vascular changes. Immunohistochemistry disclosed renal deposition of myoglobin and hemoglobin. LV did not cause injury to a suspension of fresh proximal tubules isolated from rats. Conclusions/Significance: Loxosceles gaucho venom injection caused early AKI, which occurred without blood pressure variation. Changes in glomerular function occurred likely due to renal vasoconstriction and rhabdomyolysis. Direct nephrotoxicity could not be demonstrated in vitro. The development of a consistent model of Loxosceles venom-induced AKI and a better understanding of the mechanisms involved in the renal injury may allow more efficient ways to prevent or attenuate the systemic injury after Loxosceles bite.