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Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1 to α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved anti-angiogenic activity in human endothelial cells. Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.

Recent studies show the importance of mitochondrial dysfunction in the initiation and progression of acute kidney injury (AKI). However, no biomarkers exist linking renal injury to mitochondrial function and integrity. To this end, we evaluated urinary mitochondrial DNA (UmtDNA) as a biomarker of renal injury and function in humans with AKI following cardiac surgery. mtDNA was isolated from the urine of patients following cardiac surgery and quantified by quantitative PCR. Patients were stratified into no AKI, stable AKI, and progressive AKI groups based on Acute Kidney Injury Network (AKIN) staging. UmtDNA was elevated in progressive AKI patients and was associated with progression of patients with AKI at collection to higher AKIN stages. To evaluate the relationship of UmtDNA to measures of renal mitochondrial integrity in AKI, mice were subjected to sham surgery or varying degrees of ischemia followed by 24 h of reperfusion. UmtDNA increased in mice after 10-15 min of ischemia and positively correlated with ischemia time. Furthermore, UmtDNA was predictive of AKI in the mouse model. Finally, UmtDNA levels were negatively correlated with renal cortical mtDNA and mitochondrial gene expression. These translational studies demonstrate that UmtDNA is associated with recovery from AKI following cardiac surgery by serving as an indicator of mitochondrial integrity. Thus UmtDNA may serve as valuable biomarker for the development of mitochondrial-targeted therapies in AKI.

Background Hemolytic uremic syndrome (HUS) can occur as a primary process due to mutations in complement genes or secondary to another underlying disease. HUS sometimes occurs in the setting of glomerular diseases, and it has been described in association with Denys-Drash syndrome (DDS), which is characterized by the triad of abnormal genitourinary development; a pathognomonic glomerulopathy, diffuse mesangial sclerosis; and the development of Wilms tumor. Case presentation We report the case of a 46, XX female infant who presented with HUS and biopsy-proven thrombotic microangiopathy. Next generation sequencing of genes with known mutations causative of atypical HUS found that she was homozygous for the Complement Factor H H3 haplotype and heterozygous for a variant of unknown significance in the DGKE gene. Whole exome sequencing identified a de novo heterozygous WT1 c.1384C > T; p.R394W mutation, which is classically associated with Denys-Drash syndrome (DDS). At the time of bilateral nephrectomy five months after her initial biopsy, she had diffuse mesangial sclerosis, typical of Denys-Drash syndrome, without evidence of thrombotic microangiopathy. Conclusion This unique case highlights HUS as a rare but important manifestation of WT1 mutation and provides new insight into the genetics underlying this association.

Summary Background Prognostic biomarkers that predict the severity of AKI at an early time point are needed. Urinary angiotensinogen was recently identified as a prognostic AKI biomarker. The study hypothesis is that urinary renin could also predict AKI severity and that in combination angiotensinogen and renin would be a strong predictor of prognosis at the time of AKI diagnosis. Design, setting, participants, & measurements In this multicenter, retrospective cohort study, urine was obtained from 204 patients who developed AKI after cardiac surgery from August 2008 to June 1, 2012. All patients were classified as having Acute Kidney Injury Network (AKIN) stage 1 disease by serum creatinine criteria at the time of sample collection. Urine output was not used for staging. Urinary angiotensinogen and renin were measured, and the area under the receiver-operating characteristic curve (AUC) was used to test for prediction of progression to AKIN stage 3 or in-hospital 30-day mortality. These biomarkers were added stepwise to a clinical model, and improvement in prognostic predictive performance was evaluated by category free net reclassification improvement (cfNRI) and chi-squared automatic interaction detection (CHAID). Results Both the urinary angiotensinogen-to-creatinine ratio (uAnCR; AUC, 0.75; 95% confidence interval [CI], 0.65 to 0.85) and the urinary renin-to-creatinine ratio (uRenCR; AUC, 0.70; 95% CI, 0.57 to 0.83) predicted AKIN stage 3 or death. Addition of uAnCR to a clinical model substantially improved prediction of the outcome (AUC, 0.85; cfNRI, 0.673), augmenting sensitivity and specificity. Further addition of uRenCR increased the sensitivity of the model (cfNRIevents, 0.44). CHAID produced a highly accurate model (AUC, 0.91) and identified the combination of uAnCR >337.89 ng/mg and uRenCR >893.41 pg/mg as the strongest predictors (positive predictive value, 80.4%; negative predictive value, 90.7%; accuracy, 90.2%). Conclusion The combination of urinary angiotensinogen and renin predicts progression to very severe disease in patients with early AKI after cardiac surgery.

Summary Background Biomarkers of AKI that can predict which patients will develop severe renal disease at the time of diagnosis will facilitate timely intervention in populations at risk of adverse outcomes. Design, setting, participants, & measurements Liquid chromatography/tandem mass spectrometry was used to identify 30 potential prognostic urinary biomarkers of severe AKI in a group of patients that developed AKI after cardiac surgery. Angiotensinogen had the best discriminative characteristics. Urinary angiotensinogen was subsequently measured by ELISA and its prognostic predictive power was verified in 97 patients who underwent cardiac surgery between August 1, 2008 and October 6, 2011. Results The urine angiotensinogen/creatinine ratio (uAnCR) predicted worsening of AKI, Acute Kidney Injury Network (AKIN) stage 3, need for renal replacement therapy, discharge >7 days from sample collection, and composite outcomes of AKIN stage 2 or 3, AKIN stage 3 or death, and renal replacement therapy or death. The prognostic predictive power of uAnCR was improved when only patients classified as AKIN stage 1 at the time of urine sample collection (n=79) were used in the analysis, among whom it predicted development of stage 3 AKI or death with an area under the curve of 0.81. Finally, category free net reclassification improvement showed that the addition of uAnCR to a clinical model to predict worsening of AKI improved the predictive power. Conclusions Elevated uAnCR is associated with adverse outcomes in patients with AKI. These data are the first to demonstrate the utility of angiotensinogen as a prognostic biomarker of AKI after cardiac surgery.

AKI is a common clinical condition associated with a number of adverse outcomes. More timely diagnosis would allow for earlier intervention and could improve patient outcomes. The goal of early identification of AKI has been the primary impetus for AKI biomarker research, and has led to the discovery of numerous novel biomarkers. However, in addition to facilitating more timely intervention, AKI biomarkers can provide valuable insight into the molecular mechanisms of this complex and heterogeneous disease. Furthermore, AKI biomarkers could also function as molecular phenotyping tools that could be used to direct clinical intervention. This review highlights the major studies that have characterized the diagnostic and prognostic predictive power of these biomarkers. The mechanistic relevance of neutrophil gelatinase–associated lipocalin, kidney injury molecule 1, IL-18, liver-type fatty acid–binding protein, angiotensinogen, tissue inhibitor of metalloproteinase-2, and IGF-binding protein 7 to the pathogenesis and pathobiology of AKI is discussed, putting these biomarkers in the context of the progressive phases of AKI. A biomarker-integrated model of AKI is proposed, which summarizes the current state of knowledge regarding the roles of these biomarkers and the molecular and cellular biology of AKI.

Diabetic nephropathy is the leading cause of end-stage renal disease. The urinary albumin to creatinine ratio is used as a predictor for the development of nephropathy but it is neither sensitive nor specific. Here we used liquid chromatography/mass spectrometry on urine of eight normoalbuminuric patients with type 2 diabetes from the VA Diabetes Trial to identify candidate markers for loss of renal function. Initial verification of seven markers (agrin, haptoglobin, mannan-binding lectin serine protease 2, LAMP-2, angiotensinogen, NGAL, and uromodulin) in the urine of an additional 30 patients showed that haptoglobin was the best predictor of early renal functional decline. We then measured this in the urine of 204 patients with type 2 diabetes who did not yet have significant kidney disease (estimated glomerular filtration rate stage 2 or better and an albumin to creatinine ratio300 mg/g). In comparing the highest to lowest tertiles, the odds ratio for having early renal function decline was 2.70 (CI: 1.15, 6.32) using the haptoglobin to creatinine ratio compared with 2.50 (CI 1.14, 5.48) using the albumin to creatinine ratio after adjusting for treatment group and use of ACE inhibitors. Addition of the haptoglobin to creatinine ratio to a model using the albumin to creatinine ratio to predict early renal function decline resulted in improved predictive performance. Thus, the haptoglobin to creatinine ratio may be useful to predict patients with type 2 diabetes at risk of nephropathy before the development of macroalbuminuria or reduced glomerular filtration rate.

Biomarkers for acute kidney injury (AKI) have been used to predict the progression of AKI, but a systematic comparison of the prognostic ability of each biomarker alone or in combination has not been performed. In order to assess this, we measured the concentration of 32 candidate biomarkers in the urine of 95 patients with AKIN stage 1 after cardiac surgery. Urine markers were divided into eight groups based on the putative pathophysiological mechanism they reflect. We then compared the ability of the markers alone or in combination to predict the primary outcome of worsening AKI or death (23 patients) and the secondary outcome of AKIN stage 3 or death (13 patients). IL-18 was the best predictor of both outcomes (AUC of 0.74 and 0.89). L-FABP (AUC of 0.67 and 0.85), NGAL (AUC of 0.72 and 0.83), and KIM-1 (AUC of 0.73 and 0.81) were also good predictors. Correlation between most of the markers was generally related to their predictive ability, but KIM-1 had a relatively weak correlation with other markers. The combination of IL-18 and KIM-1 had a very good predictive value with an AUC of 0.93 to predict AKIN 3 or death. Thus, a combination of IL-18 and KIM-1 would result in improved identification of high-risk patients for enrollment in clinical trials.

Progression of hyperglycemia-induced renal injury is a contributing factor for diabetic nephropathy (DN)-induced end-stage renal disease (ESRD), and development of novel therapeutic strategies that act early to prevent progression of DN and ESRD are important. We examined the efficacy and mechanism(s) of suramin on hyperglycemia-induced renal injury before development of overt histological damage. Two groups of male Sprague-Dawley rats received streptozotocin (STZ) and one group received saline. Three weeks later, one STZ group received suramin (10 mg/kg). All animals were euthanized 1 week later (4 weeks). Although there was a decrease in creatinine clearance between control and STZ ± suramin rats, there was no difference in creatinine clearance between STZ rats ± suramin intervention. Liquid chromatography-tandem mass spectroscopy-based analysis revealed increases in urinary proteins that are early indicators of DN (e.g., cystatin C, clusterin, cathepsin B, retinol binding protein 4, and peroxiredoxin-1) in the STZ group, which were blocked by suramin. Endothelial intracellular adhesion molecule-1 (ICAM-1) activation, leukocyte infiltration, and inflammation; transforming growth factor-β1 (TGF-β1) signaling; TGF-β1/SMAD-3-activated fibrogenic markers fibronectin-1, α-smooth muscle actin, and collagen 1A2; activation of proinflammatory and profibrotic transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription factor-3 (STAT-3), respectively, were all increased in STZ rats and suramin blocked these changes. In conclusion, delayed administration of suramin attenuated 1) urinary markers of DN, 2) inflammation by blocking NF-κB activation and ICAM-1-mediated leukocyte infiltration, and 3) fibrosis by blocking STAT-3 and TGF-β1/SMAD-3 signaling. These results support the potential use of suramin in DN.

Abstract 1278 Background: One of the chief obstacles to improving the outcomes of patients with acute Graft-Versus-Host disease (aGVHD) is accurate and rapid diagnosis. Currently it is diagnosed clinically, once the symptoms are fully manifested and most of the implicated biological processes are established. A biomarker that could predict disease during the asymptomatic period would allow for earlier intensification of immunosuppressant therapy, which could improve patient outcomes. Exosomes, externalized membrane bound nanovesicles that are derived from the multivesicular body, are an attractive target for aGVHD biomarker discovery. They contain a variety of cellular components and are easily obtained from bodily fluids, including plasma. Furthermore, the cell types implicated in the pathophysiology of aGVHD are known to produce exosomes with potent immunomodulatory effects. We utilized a proteomic approach to identify biomarker candidates among exosome-associated proteins utilizing patient samples obtained early after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: We utilized a differential centrifugation protocol to isolate exosomes from cryopreserved plasma and confirmed the presence of exosomes in the final pellet both by Western blotting of known exosome markers and by electron microscopy. We then used this protocol to isolate exosome-enriched pellets from 5ml plasma samples obtained on posttransplantation day 7 from 3 patients (cases) who later developed severe aGVHD (grade 3 or 4) and 4 patients did not develop aGVHD of any grade (controls). We performed proteomic analysis on pellets using liquid chromatography-tandem mass spectrometry and iTRAQ labeling, which allowed us to compare the relative quantities of identified proteins between cases and controls. Results: We made 33 protein identifications. iTRAQ analysis comparing the relative abundances of the identified proteins in cases and controls did not demonstrate any statistically significant differences between the two groups. This is likely due to the small sample size of our dataset. Despite this limitation, our data did show trends toward differences in the relative abundances of some proteins. Specifically, there was a trend toward increased IgG3 constant region in cases (median case:control ratio= 1.36; credible interval of case:control ratio= 0.857, 2.19). Additionally, there were trends toward increased lambda light chain constant region in cases (median case:control ratio= 1.35; credible interval of case:control ratio= 0.844, 2.07) and decreased kappa light chain constant region in cases (median case:control ratio=0.847; credible interval of case:control ratio=.0586, 1.21). We performed further statistical analysis to compare the kappa:lambda ratios in cases compared controls. Briefly, we calculated a ratio of treatment effects by dividing the case kappa:lambda ratio by the control kappa:lambda ratio. A value of 1 would indicate that the ratios do not differ between cases and controls. We observed a median value of 0.63, with a 95% CI of 0.377, 1.027 (see Figure 1 for a histogram of the posterior distribution of kappa:lambda effect ratio). Comparing the median treatment effect of the kappa:lambda ratio to a null distribution which we generated by bootstrap sampling 5,000 pairs of proteins that were identified in our iTraq analysis, we calculated a p-value of .011. Conclusion: These results indicate that there are early differences in B cell activity between patients who go on to develop severe aGVHD and those who do not. Monitoring allo-HSCT patients for markers of B cell activation and expansion could be clinically useful with regard to early diagnosis of aGVHD. Further studies will be conducted to test the hypothesis that aberrations in kappa:lambda ratios early after allo-HSCT predict the occurrence of severe aGVHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 1913 Introduction: Molecular cytogenetic evaluation of chromosome abnormalities has proven to be an important tool to determine prognosis in newly diagnosed multiple myeloma (MM) patients. The majority of MM patients will have one or more cytogenetic abnormality detectable by fluorescence in situ hybridization (FISH). Deletion of 17p13, t(4;14)(p16;q32) and t(14;16)(q23;q32) define high-risk MM, while abnormalities common in standard risk MM are hyperdiploid karyotype and t(11;14)(q13;q32). The prognostic importance of deletion of 13q14 detected by FISH is controversial with worse outcome noted in some but not all series. Even though a strong correlation between age and patter of chromosome abnormalities has been demonstrated in other hematologic malignancies (e.g. acute leukemias), such association has not been clearly defined in MM. We utilized a cohort with high frequency of young patients to further explore the correlation between FISH abnormalities and age in MM. Methods: This is a retrospective analysis of all patients seen at our institution with diagnosis of MM from 2002 to 2010 and for whom comprehensive FISH analysis of MM cells was available. Patients were divided in two groups according to age. The proportions of specific FISH findings were compared between the two groups using chi-square test. Results: One hundred patients were identified with a median age of 59 years (range 20–81). We defined two groups containing younger (age < 60 years, N=51) and older patients (age ≥ 60 years, N=49). FISH abnormalities were detected in 66% (99%C.I. 56.7–75.3%) of patients. Older MM patients had a significantly higher incidence of l t(4:14) abnormalities (16.3% vs 3.9% p=0.039) and IgH rearrangements in general (36.7% vs 15.7% p=0.02). There were no significant differences between the two groups for the frequency of any of the remaining FISH abnormalities (see Figure). Similarly, no pattern of association was seen between FISH panel and initial International Staging System. Conclusions: Even though there does not seem to be a strong association between age and MM FISH abnormalities, our findings suggest a higher frequency of translocations involving IgH, particularly t(4;14), among older patients. It is unlikely however that the differences in outcomes between younger and older MM patients are due to intrinsic biological differences. Disclosures: No relevant conflicts of interest to declare.