Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

Urinary MicroRNA biomarkers of nephrotoxicity in Macaca fascicularis.

Drug-induced kidney injury (DIKI) refers to kidney damage resulting from the administration of medications. The aim of this project was to identify reliable urinary microRNA (miRNAs) biomarkers that can be used as potential predictors of DIKI before disease diagnosis. This study quantified a panel of six miRNAs (miRs-210-3p, 423-5p, 143-3p, 130b-3p, 486-5p, 193a-3p) across multiple time points using urinary samples from a previous investigation evaluating effects of a nephrotoxicant in cynomolgus monkeys. Exosome-associated miRNA exhibited distinctive trends when compared to miRNAs quantified in whole urine, which may reflect a different urinary excretion mechanism of miRNAs than those released passively into the urine. Although further research and mechanistic studies are required to elucidate how these miRNAs regulate signaling in disease pathways, we present, for the first time, data that several miRNAs displayed strong correlations with histopathology scores, thus indicating their potential use as biomarkers to predict the development of DIKI in preclinical studies and clinical trials. Also, these findings can potentially be translated into other non-clinical species or human for the detection of DIKI.

Class B Scavenger Receptors BI and BII Protect against LPS-Induced Acute Lung Injury in Mice by Mediating LPS.

Recent studies suggest an anti-inflammatory protective role for class B scavenger receptor BI (SR-BI) in endotoxin-induced inflammation and sepsis. Other data, including ours, provide evidence for an alternative role of SR-BI, facilitating bacterial and endotoxin uptake and contributing to inflammation and bacterial infection. Enhanced endotoxin susceptibility of SR-BI-deficient mice due to their anti-inflammatory glucocorticoid deficiency complicates the understanding of SR-BI's role in endotoxemia/sepsis, calling for the use of alternative models. In this study, using human SR-BI (hSR-BI) and hSR-BII transgenic mice, we found that SR-BI and, to a lesser extent, its splicing variant SR-BII protect against LPS-induced lung damage. At 20 h after intratracheal LPS instillation, the extent of pulmonary inflammation and vascular leakage was significantly lower in hSR-BI and hSR-BII transgenic mice than in wild-type mice. Higher bronchoalveolar lavage fluid (BALF) inflammatory cell count and protein content and lung tissue neutrophil infiltration found in wild-type mice were associated with markedly (2 to 3 times) increased proinflammatory cytokine production compared to these parameters in transgenic mice following LPS administration. The markedly lower endotoxin levels detected in BALF of transgenic versus wild-type mice and the significantly increased BODIPY-LPS uptake observed in lungs of hSR-BI and hSR-BII mice 20 h after the i.t. LPS injection suggest that hSR-BI- and hSR-BII-mediated enhanced LPS clearance in the airways could represent the mechanism of their protective role against LPS-induced acute lung injury.

Methodological considerations for measuring biofluid-based microRNA biomarkers.

MicroRNAs (miRNAs) are small non-coding RNA that regulate the expression of messenger RNA and are implicated in almost all cellular processes. Importantly, miRNAs can be released extracellularly and are stable in these matrices where they may serve as indicators of organ or cell-specific toxicity, disease, and biological status. There has thus been great enthusiasm for developing miRNAs as biomarkers of adverse outcomes for scientific, regulatory, and clinical purposes. Despite advances in measurement capabilities for miRNAs, miRNAs are still not routinely employed as noninvasive biomarkers. This is in part due to the lack of standard approaches for sample preparation and miRNA measurement and uncertainty in their biological interpretation. Members of the microRNA Biomarkers Workgroup within the Health and Environmental Sciences Institute's (HESI) Committee on Emerging Systems Toxicology for the Assessment of Risk (eSTAR) are a consortium of private- and public-sector scientists dedicated to developing miRNAs as applied biomarkers. Here, we explore major impediments to routine acceptance and use of miRNA biomarkers and case examples of successes and deficiencies in development. Finally, we provide insight on miRNA measurement, collection, and analysis tools to provide solid footing for addressing knowledge gaps toward routine biomarker use.

Circadian variation in the release of small extracellular vesicles can be normalized by vesicle number or TSG101.

Numerous candidate biomarkers in urine extracellular vesicles (EVs) have been described for kidney diseases, but none are yet in clinical use, possibly due to a lack of proper normalization. Proper normalization corrects for normal biological variation in urine flow rate or concentration, which can vary by over one order of magnitude. Here, we observed inter- and intra-animal variation in urine excretion rates of small EVs (<200 nm in diameter) in healthy rats as a series of six 4-h fractions. To visualize intra-animal variation, we normalized a small EV excretion rate to a peak excretion rate, revealing a circadian pattern for each rat. This circadian pattern was distinct from urine volume, urine albumin, urine creatinine, and urine albumin-to-creatinine ratio. Furthermore, urine small EV excretion was not significantly altered by sex, food/water deprivation, or ischemic acute kidney injury. Urine excretion of the exosomal/small EV marker protein tumor susceptibility gene 101 (TSG101) displayed a similar circadian pattern to urine small EV excretion; both measurements were highly correlated (R2 = 0.85), with an average stoichiometry of 10.0 molecules of TSG101/vesicle in healthy rats. The observed stoichiometry of TSG101/vesicle in rat urine translated to human spot urine samples (10.2 molecules/vesicle) and cultured kidney-derived cell lines (human embryonic kidney-293 and normal rat kidney 52E cells). Small EV number and its surrogate, TSG101 protein, can normalize for circadian variation when testing candidate biomarkers in small EVs. Just as creatinine has emerged as the customary normalization factor for liquid-phase urine biomarkers, vesicle number and its surrogate, molecules of exosome/small EV-associated TSG101, should be considered as viable, normalizing factors for small EV biomarkers.

Gut Leakage of Fungal-Derived Inflammatory Mediators: Part of a Gut-Liver-Kidney Axis in Bacterial Sepsis.

Sepsis is a life-threatening response to systemic infection. In addition to frank gastrointestinal (GI) rupture/puncture, sepsis can also be exacerbated by translocation of pathogen-associated molecular patterns (PAMPs) from the GI tract to the systemic circulation (gut origin of sepsis). In the human gut, Gram-negative bacteria and Candida albicans are abundant, along with their major PAMP components, endotoxin (LPS) and (1 → 3)-ß-D-glucan (BG). Whereas the influence of LPS in bacterial sepsis has been studied extensively, exploration of the role of BG in bacterial sepsis is limited. Post-translocation, PAMPs enter the circulation through lymphatics and the portal vein, and are detoxified and then excreted via the liver and the kidney. Sepsis-induced liver and kidney injury might therefore affect the kinetics and increase circulating PAMPs. In this article, we discuss the current knowledge of the impact of PAMPs from both gut mycobiota and microbiota, including epithelial barrier function and the "gut-liver-kidney axis," on bacterial sepsis severity.

The role of adenosine 1a receptor signaling on GFR early after the induction of sepsis.

Sepsis and acute kidney injury (AKI) synergistically increase morbidity and mortality in the ICU. How sepsis reduces glomerular filtration rate (GFR) and causes AKI is poorly understood; one proposed mechanism includes tubuloglomerular feedback (TGF). When sodium reabsorption by the proximal tubules is reduced in normal animals, the macula densa senses increased luminal sodium chloride, and then adenosine-1a receptor (A1aR) signaling triggers tubuloglomerular feedback, reducing GFR through afferent arteriole vasoconstriction. We measured GFR and systemic hemodynamics early during cecal ligation and puncture-induced sepsis in wild-type and A1aR-knockout mice. A miniaturized fluorometer was attached to the back of each mouse and recorded the clearance of FITC-sinistrin via transcutaneous fluorescence to monitor GFR. Clinical organ injury markers and cytokines were measured and hemodynamics monitored using implantable transducer telemetry devices. In wild-type mice, GFR was stable within 1 h after surgery, declined by 43% in the next hour, and then fell to less than 10% of baseline after 2 h and 45 min. In contrast, in A1aR-knockout mice GFR was 37% below baseline immediately after surgery and then gradually declined over 4 h. A1aR-knockout mice had similar organ injury and inflammatory responses, albeit with lower heart rate. We conclude that transcutaneous fluorescence can accurately monitor GFR and detect changes rapidly during sepsis. Tubuloglomerular feedback plays a complex role in sepsis; initially, TGF helps maintain GFR in the 1st hour, and over the subsequent 3 h, TGF causes GFR to plummet. By 18 h, TGF has no cumulative effect on renal or extrarenal organ damage.

Two approaches for estimating the lower limit of quantitation (LLOQ) of microRNA levels assayed as exploratory biomarkers by RT-qPCR.

BACKGROUND: Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation. RESULTS: In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates. CONCLUSIONS: Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice.

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11-directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal-regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.

Quantification of Exosomes.

Exosomes are released by cells as self-contained vesicles with an intact lipid bilayer that encapsulates a small portion of the parent cell. Exosomes have been studied widely as information-rich sources of potential biomarkers that can reveal cellular physiology. We suggest that quantification is essential to understand basic biological relationships between exosomes and their parent cells and hence the underlying interpretation of exosome signals. The number of methods for quantifying exosomes has expanded as interest in exosomes has increased. However, a consensus on proper quantification has not developed, making each study difficult to compare to another. Overcoming this ad hoc approach will require widely available standards that have been adequately characterized, and multiple comparative studies across platforms. We outline the current status of these technical approaches and our view of how they can become more coherent. J. Cell. Physiol. 232: 1587-1590, 2017. © 2016 Wiley Periodicals, Inc.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation.

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein A-I-mimetic peptide 5A antagonizes CD36 in vitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild-type to CD36 knockout mice and wild-type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild-type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36 knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild-type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreased renal expression of inflammasome genes. Thus, CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression.

Pulsed focused ultrasound pretreatment improves mesenchymal stromal cell efficacy in preventing and rescuing established acute kidney injury in mice.

Animal studies have shown that mesenchymal stromal cell (MSC) infusions improve acute kidney injury (AKI) outcomes when administered early after ischemic/reperfusion injury or within 24 hours after cisplatin administration. These findings have spurred several human clinical trials to prevent AKI. However, no specific therapy effectively treats clinically obvious AKI or rescues renal function once advanced injury is established. We investigated if noninvasive image-guided pulsed focused ultrasound (pFUS) could alter the kidney microenvironment to enhance homing of subsequently infused MSC. To examine the efficacy of pFUS-enhanced cell homing in disease, we targeted pFUS to kidneys to enhance MSC homing after cisplatin-induced AKI. We found that pFUS enhanced MSC homing at 1 day post-cisplatin, prior to renal functional deficits, and that enhanced homing improved outcomes of renal function, tubular cell death, and regeneration at 5 days post-cisplatin compared to MSC alone. We then investigated whether pFUS+MSC therapy could rescue established AKI. MSC alone at 3 days post-cisplatin, after renal functional deficits were obvious, significantly improved 7-day survival of animals. Survival was further improved by pFUS and MSC. pFUS prior to MSC injections increased IL-10 production by MSC that homed to kidneys and generated an anti-inflammatory immune cell profile in treated kidneys. This study shows pFUS is a neoadjuvant approach to improve MSC homing to diseased organs. pFUS with MSC better prevents AKI than MSC alone and allows rescue therapy in established AKI, which currently has no meaningful therapeutic options.

Microparticles: markers and mediators of sepsis-induced microvascular dysfunction, immunosuppression, and AKI.

Sepsis is a severe and complex syndrome that lacks effective prevention or therapeutics. The effects of sepsis on the microvasculature have become an attractive area for possible new targets and therapeutics. Microparticles (MPs) are cell membrane-derived particles that can promote coagulation, inflammation, and angiogenesis, and they can participate in cell-to-cell communication. MPs retain cell membrane and cytoplasmic constituents of their parental cells, including two procoagulants: phosphatidylserine and tissue factor. We highlight the role of microparticles released by endothelial and circulating cells after sepsis-induced microvascular injury, and we discuss possible mechanisms by which microparticles can contribute to endothelial dysfunction, immunosuppression, and multiorgan dysfunction--including sepsis-AKI. Once viewed as cellular byproducts, microparticles are emerging as a new class of markers and mediators in the pathogenesis of sepsis.

Comparison of serum creatinine and serum cystatin C as biomarkers to detect sepsis-induced acute kidney injury and to predict mortality in CD-1 mice.

Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed when based on serum creatinine (SCr) changes, due in part, to decreased creatinine production. During experimental sepsis, we compared serum cystatin C (sCysC), SCr, and blood urea nitrogen (BUN) to inulin glomerular filtration rate (iGFR) before or 3-18 h after cecal ligation and puncture (CLP)-induced sepsis in CD-1 mice. sCysC had a faster increase and reached peak levels more rapidly than SCr in both sepsis and bilateral nephrectomy (BiNx) models. sCysC was a better surrogate of iGFR than SCr during sepsis. Combining sCysC with SCr values into a composite biomarker improved correlation with iGFR better than any biomarker alone or any other combination. We determined the renal contribution to sCysC handling with BiNx. sCysC and SCr were lower post-BiNx/CLP than post-BiNx alone, despite increased inflammatory and nonrenal organ damage biomarkers. Sepsis decreased CysC production in nephrectomized mice without changing body weight or CysC space. Sepsis decreased sCysC production and increased nonrenal clearance, similar to effects of sepsis on SCr. sCysC, SCr, and BUN were measured 6 h postsepsis to link AKI with mortality. Mice with above-median sCysC, BUN, or SCr values 6 h postsepsis died earlier than mice with below-median values, corresponding to a substantial AKI association with sepsis mortality in this model. sCysC performs similarly to SCr in classifying mice at risk for early mortality. We conclude that sCysC detects AKI early and better reflects iGFR in CLP-induced sepsis. This study shows that renal biomarkers need to be evaluated in specific contexts.

Class B scavenger receptor types I and II and CD36 mediate bacterial recognition and proinflammatory signaling induced by Escherichia coli, lipopolysaccharide, and cytosolic chaperonin 60.

Class B scavenger receptors (SR-B) are lipoprotein receptors that also mediate pathogen recognition, phagocytosis, and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family, namely, CLA-1, CLA-2, and CD36, mediate recognition of bacteria not only through interaction with cell wall LPS but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3- to 5-fold) increased binding and endocytosis of Escherichia coli, LPS, and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS, and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36 > CLA-2 > CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II-null mice by 40-50 and 30-40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40-60% lower in CD36(-/-) relative to wild-type mice, whereas increased cytokine responses were found in SR-BI/II(-/-) mice. While investigating the discrepancy of in vitro versus in vivo data in SR-BI/II deficiency, SR-BI/II(-/-) mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in wild-type mice. SR-BI/II(-/-) mice with mineralocorticoid replacement demonstrated an ∼40-50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice.

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.

Macrophage depletion protects against cisplatin-induced ototoxicity and nephrotoxicity.

Cisplatin is a widely used anticancer drug with notable side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced toxicities, we used PLX3397, a U.S. Food and Drug Administration-approved inhibitor of the colony-stimulating factor 1 receptor, to eliminate tissue-resident macrophages. Mice treated with cisplatin alone had considerable hearing loss (ototoxicity) and kidney injury (nephrotoxicity). Macrophage ablation resulted in significantly reduced hearing loss and had greater outer hair cell survival. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together, our data indicate that ablation of tissue-resident macrophages represents an important strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

Urinary exosomal Wilms' tumor-1 as a potential biomarker for podocyte injury.

Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-ß1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-ß1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunteers or steroid-sensitive nephrotic syndrome (SSNS) patients. Urinary exosomal WT-1 was also significantly decreased in patients in remission for either FSGS or SSNS or following steroid treatment in six SSNS subjects. We conclude that urinary exosomal WT-1 is a promising noninvasive biomarker with apparent podocyte specificity that can detect early progression and treatment-induced regression of podocyte injury in FSGS or SSNS. These results warrant longitudinal, prospective studies in a large cohort with a range of podocyte diseases.

Calpastatin controls polymicrobial sepsis by limiting procoagulant microparticle release.

RATIONALE: Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release. OBJECTIVES: We investigated the contribution of calpain in eliciting tissue damage during sepsis. METHODS: To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. MEASUREMENTS AND MAIN RESULTS: In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy. CONCLUSIONS: These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.