RNA Sequencing Identifies Novel Translational Biomarkers of Kidney Fibrosis.

CKD is the gradual, asymptomatic loss of kidney function, but current tests only identify CKD when significant loss has already happened. Several potential biomarkers of CKD have been reported, but none have been approved for preclinical or clinical use. Using RNA sequencing in a mouse model of folic acid-induced nephropathy, we identified ten genes that track kidney fibrosis development, the common pathologic finding in patients with CKD. The gene expression of all ten candidates was confirmed to be significantly higher (approximately ten- to 150-fold) in three well established, mechanistically distinct mouse models of kidney fibrosis than in models of nonfibrotic AKI. Protein expression of these genes was also high in the folic acid model and in patients with biopsy-proven kidney fibrosis. mRNA expression of the ten genes increased with increasing severity of kidney fibrosis, decreased in response to therapeutic intervention, and increased only modestly (approximately two- to five-fold) with liver fibrosis in mice and humans, demonstrating specificity for kidney fibrosis. Using targeted selected reaction monitoring mass spectrometry, we detected three of the ten candidates in human urine: cadherin 11 (CDH11), macrophage mannose receptor C1 (MRC1), and phospholipid transfer protein (PLTP). Furthermore, urinary levels of each of these three proteins distinguished patients with CKD (n=53) from healthy individuals (n=53; P<0.05). In summary, we report the identification of urinary CDH11, MRC1, and PLTP as novel noninvasive biomarkers of CKD.

Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis.

Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250mg/kgi.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (>2-fold, p<0.05), we chose to further investigate miR-18a-5p, which is expressed during the acute stage of the injury; miR-132-3p, which is upregulated during transition between acute and fibrotic injury; and miR-146b-5p, which is highly expressed at the peak of fibrosis. Using qRT-PCR, we confirmed the increased expression of these candidate miRNAs in the folic acid model as well as in other established mouse models of acute injury (ischemia/reperfusion injury) and fibrosis (unilateral ureteral obstruction). In situ hybridization confirmed high expression of miR-18a-5p, miR-132-3p and miR-146b-5p throughout the kidney cortex in mice and humans with severe kidney injury or fibrosis. When primary human proximal tubular epithelial cells were treated with model nephrotoxicants such as cadmium chloride (CdCl2), arsenic trioxide, aristolochic acid (AA), potassium dichromate (K2Cr2O7) and cisplatin, miRNA-132-3p was upregulated 4.3-fold after AA treatment and 1.5-fold after K2Cr2O7 and CdCl2 treatment. These results demonstrate the application of temporal small RNA sequencing to identify miR-18a, miR-132 and miR-146b as differentially expressed miRNAs during distinct phases of kidney injury and fibrosis progression.

Pharmacological and genetic depletion of fibrinogen protects from kidney fibrosis.

Fibrinogen (Fg) has been implicated in the pathogenesis of several fibrotic disorders by acting as a profibrotic ligand for a variety of cellular surface receptors and by modulating the provisional fibrin matrix formed after injury. We demonstrated increased renal Fg expression after unilateral ureteral obstruction and folic acid (FA) nephropathy in mice, respectively. Urinary Fg excretion was also increased in FA nephropathy. Using in vitro and in vivo approaches, our results suggested that IL-6 mediates STAT3 activation in kidney fibrosis and that phosphorylated (p)STAT3 binds to Fgα, Fgß, and Fgγ promoters in the kidney to regulate their transcription. Genetically modified Fg heterozygous mice (∼75% of normal plasma Fg levels) exhibited only 3% kidney interstitial fibrosis and tubular atrophy after FA nephropathy compared with 24% for wild-type mice. Fibrinogenolysis through Ancrod administration after FA reduced interstitial fibrosis more than threefold compared with vehicle-treated control mice. Mechanistically, we show that Fg acts synergistically with transforming growth factor (TGF)-ß1 to induce fibroblast proliferation and activates TGF-ß1/pSMAD2 signaling. This study offers increased understanding of Fg expression and molecular interactions with TGF-ß1 in the progression to kidney fibrosis and, importantly, indicates that fibrinogenolytics like Ancrod present a treatment opportunity for a yet intractable disease.

Cecal ligation and puncture-induced murine sepsis does not cause lung injury.

OBJECTIVE: The cause of death in murine models of sepsis remains unclear. The primary purpose of this study was to determine if significant lung injury develops in mice predicted to die after cecal ligation and puncture-induced sepsis compared with those predicted to live. DESIGN: Prospective, laboratory controlled experiments. SETTING: University research laboratory. SUBJECTS: Adult, female, outbred Institute of Cancer Research mice. INTERVENTIONS: Mice underwent cecal ligation and puncture to induce sepsis. Two groups of mice were euthanized at 24 and 48 hrs postcecal ligation and puncture and samples were collected. These mice were further stratified into groups predicted to die (Die-P) and predicted to live (Live-P) based on plasma interleukin-6 levels obtained 24 hrs postcecal ligation and puncture. Multiple measures of lung inflammation and lung injury were quantified in these two groups. Results from a group of mice receiving intratracheal normal saline without surgical intervention were also included as a negative control. As a positive control, bacterial pneumonia was induced with Pseudomonas aeruginosa to cause definitive lung injury. Separate mice were followed for survival until Day 28 postcecal ligation and puncture. These mice were used to verify the interleukin-6 cutoffs for survival prediction. MEASUREMENTS AND MAIN RESULTS: After sepsis, both the Die-P and Live-P mice had significantly suppressed measures of respiratory physiology but maintained normal levels of arterial oxygen saturation. Bronchoalveolar lavage levels of pro- and anti-inflammatory cytokines were not elevated in the Die-P mice compared with the Live-P. In addition, there was no increase in the recruitment of neutrophils to the lung, pulmonary vascular permeability, or histological evidence of damage. In contrast, all of these pulmonary injury and inflammatory parameters were increased in mice with Pseudomonas pneumonia. CONCLUSIONS: These data demonstrate that mice predicted to die during sepsis have no significant lung injury. In murine intra-abdominal sepsis, pulmonary injury cannot be considered the etiology of death in the acute phase.

Acute oral ethanol exposure triggers asthma in cockroach allergen-sensitized mice.

Asthma may be triggered by multiple mediators, including allergen-IgE cross-linking and non-IgE mechanisms. Several clinical studies have shown acute ethanol consumption exacerbates asthma, yet no animal model exists to study this process. We developed a model of ethanol-triggered asthma in allergen-sensitized mice to evaluate the mechanisms of ethanol inducing asthma-like responses. Outbred mice were exposed to cockroach allergens on Days 0 and 14; and on Day 21, mice received ethanol by oral gavage. Tracer studies confirmed alcohol aspiration did not occur. Within 30 minutes, alcohol induced degranulation of over 74% of mast cells, and multiple parameters of asthma-like pulmonary inflammation were triggered. Ethanol-gavaged mice had a fivefold increased production of eotaxin-2 (534 pg/mL) and a sevenfold increase in bronchoalveolar eosinophils (70,080 cells). Ethanol induced a 10-fold increase in IL-13, from 84 pg/mL in sensitized mice to 845 pg/mL in ethanol-gavaged sensitized mice. In cockroach allergen-sensitized mice, ethanol triggered asthma-like changes in respiratory physiology and a significant fivefold increase in airway mucin production. Importantly, none of these asthmatic exacerbations were observed in normal mice gavaged with ethanol. Cromolyn sodium effectively stabilized mast cells, yet increased mucin production and bronchoalveolar eosinophil recruitment. Together, these data show a single oral alcohol exposure will trigger asthma-like pulmonary inflammation in allergen-sensitized mice, providing a novel asthma model.

Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival.

Neutrophils are critical for the rapid eradication of bacterial pathogens, but they also contribute to the development of multiple organ failure in sepsis. We hypothesized that increasing early recruitment of neutrophils to the focus of infection will increase bacterial clearance and improve survival. Sepsis was induced in mice, using cecal ligation and puncture (CLP); blood samples were collected at 6 and 24 h; and survival was followed for 28 d. In separate experiments, peritoneal bacteria and inflammatory cells were measured. Septic mice predicted to die based on IL-6 levels (Die-P) had higher concentrations of CXCL1 and CXCL2 in the peritoneum and plasma compared with those predicted to live (Live-P). At 6 h, Live-P and Die-P had equivalent numbers of peritoneal neutrophils and bacteria. In Die-P mice the number of peritoneal bacteria increased between 6 and 24 h post-CLP, whereas in Live-P it decreased. The i.p. injection of CXCL1 and CXCL2 in naive mice resulted in local neutrophil recruitment. When given immediately after CLP, CXC chemokines increased peritoneal neutrophil recruitment at 6 h after CLP. This early increase in neutrophils induced by exogenous chemokines resulted in significantly fewer peritoneal bacteria by 24 h [CFU (log) = 6.04 versus 4.99 for vehicle versus chemokine treatment; p < 0.05]. Chemokine treatment significantly improved survival at both 5 d (40 versus 72%) and 28 d (27 versus 52%; p < 0.02 vehicle versus chemokines). These data demonstrate that early, local treatment with CXC chemokines enhances neutrophil recruitment and clearance of bacteria as well as improves survival in the CLP model of sepsis.

MicroRNA-155 deficient mice experience heightened kidney toxicity when dosed with cisplatin.

The development of nephrotoxicity limits the maximum achievable dosage and treatment intervals for cisplatin chemotherapy. Therefore, identifying mechanisms that regulate this toxicity could offer novel methods to optimize cisplatin delivery. MicroRNAs are capable of regulating many different genes, and can influence diverse cellular processes, including cell death and apoptosis. We previously observed miR-155 to be highly increased following ischemic or toxic injury to the kidneys and, therefore, sought to determine whether mice deficient in miR-155 would respond differently to kidney injury. We treated C57BL/6 and miR-155(-/-) mice with 20 mg/kg of cisplatin and found a significantly higher level of kidney injury in the miR-155(-/-) mice. Genome-wide expression profiling and bioinformatic analysis indicated the activation of a number of canonical signaling pathways relating to apoptosis and oxidative stress over the course of the injury, and identified potential upstream regulators of these effects. One predicted upstream regulator was c-Fos, which has two confirmed miR-155 binding sites in its 3' UTR and, therefore, can be directly regulated by miR-155. We established that the miR-155(-/-) mice had significantly higher levels of c-Fos mRNA and protein than the C57BL/6 mice at 72 h after cisplatin exposure. These data indicate a role for miR-155 in the cisplatin response and suggest that targeting of c-Fos could be investigated to reduce cisplatin-induced nephrotoxicity.

Early murine polymicrobial sepsis predominantly causes renal injury.

Multiple organ failure in sepsis substantially increases mortality. This study examined if there was greater hepatic, pancreatic, splenic, or renal injury in mice that would die during sepsis induced by cecal ligation and puncture (CLP) compared with that of those that would survive. Mice were stratified into groups predicted to die (Die-P) or predicted to live (Live-P) in the first 5 days after CLP based on plasma interleukin 6 levels. Groups were sacrificed to harvest organs for histology. Separate animals were followed for survival with daily blood sampling to examine renal function. No significant histological evidence of organ injury was observed in either the Live-P or Die-P mice. Minimal hepatic injury occurred as plasma aspartate transaminase demonstrated less than a 2-fold increase over normal in both groups. In addition, pancreatic injury was minimal as there was also less than a 2-fold increase in plasma amylase levels. In contrast, blood urea nitrogen levels were nearly five times higher within 24 h in Die-P mice compared with those of mice predicted to live. Mice with blood urea nitrogen levels higher than 44 mg/dL had a 17.6 higher relative risk of dying (95% confidence interval, 4.5-69.4). Cystatin C, a more specific kidney function biomarker, was also elevated at 24 h after CLP. When the cystatin C levels were analyzed relative to the hours before death, rather than hours after CLP, they were also significantly increased in mice Dead by day 5 compared with those Alive after day 5. We conclude that limited liver, pancreas, and spleen injury develops during murine CLP-induced sepsis while significant kidney injury is present. The renal injury becomes worse closer to death.

Untreated type 1 diabetes increases sepsis-induced mortality without inducing a prelethal cytokine response.

Diabetes mellitus is the leading comorbidity in patients with sepsis, but its impact upon survival and immunoinflammatory signaling in sepsis is undetermined. We investigated the effect of untreated diabetes mellitus upon survival and immunoinflammatory responses in the acute phase (days 1-5) of murine polymicrobial sepsis using the AKITA model of type 1 diabetes. Diabetic female C57BL/6-Ins2 (AKITA) and C57BL/6 wild-type (WT) mice underwent cecal ligation and puncture (CLP), blood (20 µL) was sampled for 5 days, and survival was monitored for 28 days. By day 5, all 8 AKITA mice died compared with 10 of 28 deaths in WT mice. Blood glucose declined post-CLP in all groups (most dramatically in AKITAs by 75%). To compare the evolution of inflammatory profiles, mice were retrospectively divided based on outcome into AKITA, WT-Died, and WT-Survived (within days 1-5). Hypoglycemia developed in all groups, which resolved in WT-Survived (97 mg/dL at 96 h) but intensified in WT-Died and AKITAs (∼30 mg/dL). Dramatic increases in both proinflammatory and anti-inflammatory cytokines were observed in WT-Died (i.e., interleukin 6, 38.2 ± 17.8 ng/mL at 24 h), which contrasted with a lack of prelethal cytokine response in AKITA mice (interleukin 6, 4.3 ± 3.4 ng/mL at 24 h). A prelethal composite cytokine score was calculated on values obtained 24 h before death. This score was 3-fold lower for proinflammatory cytokines and 6-fold lower for anti-inflammatory mediators in the AKITA mice compared with the WT-Died mice but identical to the composite score in WT-Survived. These data demonstrate that untreated type I diabetes mellitus severely exacerbates sepsis mortality without inducing a prelethal release of systemic proinflammatory or anti-inflammatory cytokines.