Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs-also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/ or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-) reprogram macrophages by releasing prostaglandin E 2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups. © 2008 Nature Publishing GroupCorrespondence should be addressed to E.M. (E-mail: mezeye@mail.nih.gov).. 6 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS K.N., A.L., P.S.T.Y., R.A.S. and E.M. formulated the basic hypotheses and experimental design; K.N., A.L., E.M., P.S.T.Y. and R.A.S. collected and evaluated data on survival and organ injury; K.N. and A.L. performed the in vivo experiments; A.L., P.S.T.Y., A.P., K.D., K.L. and X.H. assisted in the in vivo experiments and histology; P.G.R. consulted on BMSC biology; K.N. formulated the molecular mechanism hypothesis and designed and performed in vitro and ex vivo assays; B.H.K. helped to test the involvement of the prostaglandin receptors; J.M.B. and B.M. contributed to testing the involvement of COX2; B.M. performed the measurements for tissue peroxidase; I.J. performed FACS experiments; E.M. wrote the initial manuscript and prepared the figures; all of the authors edited the manuscript.Note: Supplementary information is available on the Nature Medicine website. In the last few years, it has been discovered that BMSCs are potent modulators of immune responses 2-5 . We wondered whether such cells could bring the immune response back into balance, thus attenuating the underlying pathophysiology that eventually leads to severe sepsis, septic shock and death 6,7 . NIH Public AccessAs a model of sepsis, we chose cecal ligation and puncture (CLP), a procedure that has been used for more than two decades 8 . This mouse model closely resembles the human disease: it has a focal origin (cecum), is caused by multiple intestinal organisms, and results in septicemia with release of bacterial toxins into the circulation. With no treatment, the ma...

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.

CLP in aged mice causes functional and histologic changes consistent with human ARF. A single dose of ethyl pyruvate inhibits renal and multiple organ damage, and is still effective when given 12 hours after surgery.

Serum creatinine is used clinically to detect and evaluate acute kidney injury (AKI) and chronic kidney disease (CKD), 1,2 although the limitations of serum creatinine for the early detection and accurate estimation of renal injury are widely known. In AKI, serum creatinine does not accurately reflect the GFR because the patient is not in steady state. 3 Furthermore, serum creatinine is also influenced by tubular creatinine secretion and by nonrenal factors such as muscle mass, liver function, and nonrenal (gastrointestinal) elimination.Sepsis remains a serious problem in critically ill patients, and mortality from sepsis is increased dramatically when complicated by AKI; therefore, early detection and accurate evaluation of AKI is important in patients with sepsis. We modified the cecal ligation and puncture (CLP) mouse sepsis model to make it more clinically relevant, and our model developed sepsis-induced AKI. 4 -6 We found that serum creatinine increased to a lesser extent in CLP (approximately 0.5 mg/dl) than ischemia/reperfusion or cisplatin administration (Ͼ1 mg/dl) within 24 h of injury. To explore nonrenal factors that might account for this disparity, we used bilateral nephrectomy (BNx), a technique used recently by others to study the contribution of the kidney to cytokine metabolism and acute lung injury. 7 First, we evaluated sepsis in bilaterally nephrectomized mice. We induced sepsis by CLP surgery with 8-mm cecal ligation, which causes modest sublethal sepsis in normal outbred CD-1 mice. 8 All animals survived until they were killed at 18 h after surgery. CLP surgery in non-nephrectomized mice caused a numerically small, but not significant increase of serum creatinine (sham BNx ϩCLP group). As expected, we found large increases of serum creatinine in the BNxϩsham CLP group; however, induction of sepsis at the time of BNx significantly decreased serum creatinine (BNxϩCLP group) compared with nonseptic BNx alone (BNxϩsham CLP group; Figure 1A), raising doubt about whether serum creatinine accurately reflects impaired kidney function during sepsis. In contrast, nonrenal organ injury markers (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and lactate dehydrogenase [LDH]) and serum TNF-␣ were higher in the BNxϩCLP group than in the BNxϩsham CLP group, confirming the presence of severe sepsis (Figure 1, ABSTRACTAlthough diagnosis and staging of acute kidney injury uses serum creatinine, acute changes in creatinine lag behind both renal injury and recovery. The risk for mortality increases when acute kidney injury accompanies sepsis; therefore, we sought to explore the limitations of serum creatinine in this setting. In mice, induction of sepsis by cecal ligation and puncture in bilaterally nephrectomized mice increased markers of nonrenal organ injury and serum TNF-␣. Serum creatinine, however, was significantly lower in septic animals than in animals subjected to bilateral nephrectomy and sham cecal ligation and puncture. Under these conditions treatment with chloroquine decrease...

Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracellular kidney injury biomarkers. To study whether they contain transcription factors, we collected urine from two acute kidney injury models (cisplatin or ischemia-reperfusion), two podocyte injury models (puromycin-treated rats or podocin-Vpr transgenic mice) and from patients with focal segmental glomerulosclerosis, acute kidney injury and matched controls. Exosomes were isolated by differential centrifugation and found to contain activating transcription factor 3 (ATF3) and Wilms Tumor 1 (WT-1) proteins detected by Western blot. These factors were found in the concentrated exosomal fraction, but not in whole urine. ATF3 was continuously present in urine exosomes of the rat models following acute injury at times earlier than the increase in serum creatinine. ATF3 was found in exosomes isolated from patients with acute kidney injury but not from patients with chronic kidney disease or controls. Urinary WT-1 was present in animal models before significant glomerular sclerosis and in 9/10 patients with focal segmental glomerulosclerosis but not in 8 controls. Our findings suggest that transcription factor ATF3 may provide a novel renal tubular cell biomarker for acute kidney injury while WT-1 may detect early podocyte injury. Measurement of urinary exosomal transcription factors may offer insight into cellular regulatory pathways.

Star RA. Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury.

The remnant kidney model in C57BL6 mice does not develop progressive chronic kidney disease (CKD). In this study we modified the model to mimic features of human CKD and to define accelerants of disease progression using three strains of mice. Following the procedure there was a progressive increase in albuminuria, progressive loss in renal function, severe glomerulosclerosis and interstitial fibrosis, hypertension, cardiac fibrosis, and anemia by 4 weeks in CD-1 mice and by 12 weeks in 129S3 mice. In contrast, even after 16 weeks, the C57BL/6 mice with a remnant kidney had modestly increased albuminuria without increased blood pressure and without developing CKD or cardiac fibrosis. The baseline blood pressure, determined by radiotelemetry in conscious animals, correlated with CKD progression rates in each strain. Administering angiotensin II overcame the resistance of C57BL/6 mice to CKD following renal mass reduction displaying high blood pressure and albuminuria, severe glomerulosclerosis, and loss of renal function by 4 weeks. Decreasing blood pressure with olmesartan but not hydralazine in CD-1 mice with a remnant kidney reduced CKD progression and cardiac fibrosis. C57BL/6 mice with a remnant kidney and DOCA-salt hypertension developed modest CKD. Each strain had similar degrees of interstitial fibrosis in three different normotensive models of renal fibrosis. Thus reducing renal mass in CD-1 or 129S3 mice mimics many features of human CKD. Angiotensin II can convert the C57BL/6 strain from CKD-resistant to susceptible in this disease model.

Acute kidney injury (AKI) occurs in about half of patients in septic shock and the mortality of AKI with sepsis is extremely high. An effective therapeutic intervention is urgently required. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that also have pleiotropic actions. They have been reported to increase the survival of septic or infectious patients. But the effect of simvastatin, a widely used statin, on sepsis-induced AKI is unknown. The effects of simvastatin and tumor necrosis factor (TNF)-alpha neutralizing antibody were studied in a clinically relevant model of sepsis-induced AKI using cecal ligation and puncture (CLP) in elderly mice. Simvastatin significantly improved CLP-induced mortality and AKI. Simvastatin attenuated CLP-induced tubular damage and reversed CLP-induced reduction of intrarenal microvascular perfusion and renal tubular hypoxia at 24 h. Simvastatin also restored towards normal CLP-induced renal vascular protein leak and serum TNF-alpha. Neither delayed simvastatin therapy nor TNF-alpha neutralizing antibody improved CLP-induced AKI. Simvastatin improved sepsis-induced AKI by direct effects on the renal vasculature, reversal of tubular hypoxia, and had a systemic anti-inflammatory effect.