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Abstract: While exposure to chemical mixtures is an everyday reality, an understanding of their combined effects, and any potential prediction thereof, is extremely limited. Realistic exposures potentially consist of hundreds to thousands of chemicals per day, but even relatively simple binary mixture interactions can be inherently difficult to predict based upon the lack of temporal and spatial mechanisms for the individual constituents. To this end, we explore the concept of capitalizing on downstream convergence of intracellular signal transduction to experimentally simplify the means of determining xenobiotics that, when combined, could result in increased or unexpected toxicity. In a proof of principle study, we exposed HepG2 cells to deguelin, a natural isoflavonoid, alone and in combination with KCN, and determined the relative post-translational phosphorylation responses to several key proteins related to mitochondrial outer membrane permeabilization. Dose-dependent phosphorylation activity provides a clear identification of threshold response to low-level exposures, and crosstalk amongst selected proteins correctly forecasts mixtures interactions that may lead to increased toxicity. We then used Bliss Independence to determine if the experimentally measured mixture phosphorylation responses could be predicted with individual responses. Independence accurately predicted mixture interactions for deguelin and KCN (87.5%). To more fully exhaust independence as a model for determining potential pharmacodynamic interactions, we exposed HepG2 cells to deguelin and staurosporine, a broad kinase inhibitor; independence accurately predicted these mixture responses (77.5%). In this study, we demonstrate the potential of a new in vitro approach for the prediction of toxic mixtures interactions that is fundamentally driven by the interdependence of signal transduction and apoptosis. [Copyright &y& Elsevier]

Abstract: The pharmaceutical world has greatly benefited from the well-characterized structure–function relationships of toxins with endogenous biomolecules, such as ion-channels, receptors, and signaling molecules. Thus, therapeutics derived from toxins have been aggressively pursued. However, the multifunctional role of various toxins may lead to undesirable off-target effects, hindering their use as therapeutic agents. In this paper, we suggest that previously unsuccessful toxins (due to off-target effects) may be revisited with mixtures by utilizing the pharmacodynamic response to the potential primary therapeutic as a starting point for finding new targets to ameliorate the unintended responses. In this proof of principle study, the pharmacodynamic response of HepG2 cells to a potential primary therapeutic (deguelin, a plant-derived chemopreventive agent) was monitored, and a possible secondary target (p38MAPK) was identified. As a single agent, deguelin decreased cellular viability at higher doses (>10 μM), but inhibited oxygen consumption over a wide dosing range (1.0–100 μM). Our results demonstrate that inhibition of oxygen consumption is related to an increase in p38MAPK phosphorylation, and may only be an undesired side effect of deguelin (i.e., one that does not contribute to the decrease in HepG2 viability). We further show that deguelin's negative effect on oxygen consumption can be diminished while maintaining efficacy when used as a therapeutic mixture with the judiciously selected secondary inhibitor (SB202190, p38MAPK inhibitor). These preliminary findings suggest that an endogenous response-directed mixtures approach, which uses a pharmacodynamic response to a primary therapeutic to determine a secondary target, allows previously unsuccessful toxins to be revisited as therapeutic mixtures. [Copyright &y& Elsevier]

Accurate diagnosis and typing of renal amyloidosis is critical for prognosis, genetic counseling, and treatment. Laser microdissection and mass spectrometry are emerging techniques for the analysis and diagnosis of many renal diseases. Here we present the results of laser microdissection and mass spectrometry performed on 127 cases of renal amyloidosis during 2008-2010. We found the following proteins in the amyloid deposits: immunoglobulin light and heavy chains, secondary reactive serum amyloid A protein, leukocyte cell-derived chemotaxin-2, fibrinogen-α chain, transthyretin, apolipoprotein A-I and A-IV, gelsolin, and β-2 microglobulin. Thus, laser microdissection of affected areas within the kidney followed by mass spectrometry provides a direct test of the composition of the deposit and forms a useful ancillary technique for the accurate diagnosis and typing of renal amyloidosis in a single procedure. [ABSTRACT FROM AUTHOR]

The antiapoptotic BCL2 family member MCL1 is normally up- and down-modulated in response to environmental signals and conditions, but is constitutively expressed in cancer where it promotes cell survival and drug resistance. A post-translational modification identified here, truncation at the N terminus, was found to act along with previously described ERK- and GSK3-induced phosphorylation events to regulate the turnover of the MCL1 protein and thus its availability for antiapoptotic effects. Although both N-terminally truncated and full-length MCL1 contain sequences enriched in proline, glutamic acid, serine, and threonine and were susceptible to proteasomal degradation, the truncated form decayed less rapidly and was maintained for an extended period in the presence of ERK activation. This was associated with extended cell survival because the truncated form of MCL1 (unlike those of BCL2 and BCLX) retained antiapoptotic activity. N-terminal truncation slightly increased the electrophoretic mobility of MCL1 and differed from the phosphorylation/band shift to decreased mobility, which occurs in the G2/M phase and was not found to affect MCL1 turnover. The N-terminally truncated form of MCL1 was expressed to varying extents in normal lymphoid tissues and was the predominant form present in lymphomas from transgenic mice and human tumor lines of B-lymphoid origin. The degradation versus stabilized expression of antiapoptotic MCL1 is thus controlled by N-terminal truncation as well as by ERK- and GSK3 (but not G2/M)-induced phosphorylation. These modifications may contribute to dysregulated MCL1 expression in cancer and represent targets for promoting its degradation to enhance tumor cell death. [ABSTRACT FROM AUTHOR]

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex–mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD.Kidney International (2009) 75, 952–960; doi:10.1038/ki.2008.657; published online 28 January 2009 [ABSTRACT FROM AUTHOR]

Amyloidosis derived from leukocyte chemotactic factor 2 (ALECT2) is a recently described disease. Here, we report the characteristics and outcome of 72 patients with renal ALECT2, which included 19 who had another kidney disease on biopsy. Ninety-two percent of patients were Hispanics and over half were elderly. Three had other organ, but not cardiac, amyloidosis involvement. All patients without concurrent disease, except three, presented with chronic renal insufficiency. Proteinuria was variable and absent in a third, whereas nephrotic syndrome and hematuria were rare. After a median follow-up of 26 months, one-third developed end-stage renal disease (ESRD). The median renal survival was 62 months. Independent predictors of renal survival were serum creatinine at diagnosis, with a value of 2.0 mg/dl being the best cutoff for predicting ESRD, percentage global glomerulosclerosis, and presence of diabetes. Only four patients died and four had received chemotherapy for an erroneous diagnosis of immunoglobulin light chain-derived amyloidosis. Five patients underwent kidney transplantation; none had graft loss but one had disease recurrence. Patient survival is superior to renal immunoglobulin light chain-derived amyloidosis and reactive amyloidosis largely due to the absence of cardiac involvement. Thus, renal ALECT2 mainly affects elderly Hispanics who typically present with chronic renal insufficiency and bland urine sediment, with or without proteinuria. [ABSTRACT FROM AUTHOR]

Abstract: Background: Proper identification of cardiac amyloid type is essential for patient management, and has historically relied upon immunohistochemical- or immunofluorescence-based methods, often correlated with serum and urine protein electrophoresis (SPEP and UPEP) with immunofixation electrophoresis (IFE), and/or free light chain immunoassay (FLC). The recent implementation of mass spectrometry-based proteomic analysis for clinical amyloid typing allows us to determine the validity of these tests to predict amyloid type. Validity of SPEP/UPEP/IFE and FLC assays in cardiac amyloid prediction was examined. Methods: Retrospective analysis of two tertiary care populations (n=143, 2001–2010), of cardiac biopsy-proven amyloidosis, was performed. Results: Amyloid of transthyretin (ATTR) type was found in 81 (57%) of 143 patients and immunoglobulin light chain amyloid was found in the remaining 62 (43%). SPEP/UPEP/IFE detected a monoclonal gammopathy in 76 individuals, 56 with AL and 20 with ATTR amyloid and was overall a poor predictor of AL amyloid in this patient population: specificity (75%; 95% CI, 65-83%) and positive predictive value (PPV 74%; 95% CI, 63–82%). The FLC assay detected an abnormal kappa/lambda ratio in 61 patients, 53 with AL and 8 with ATTR amyloid and was a better predictor of AL amyloid type in this patient population: specificity (90%, 95% CI, 82–95%) and PPV (87%, 95% CI, 76–93%). Conclusions: ATTR was the predominant amyloid type in this large cohort of endomyocardial biopsies characterized by mass spectrometry. Although FLC performs better than SPEP/UPEP/IFE, the performance of blood and urine studies for monoclonal proteins are not adequate to classify amyloid type. Summary: This large-scale retrospective analysis of cardiac amyloidosis shows that blood and urine monoclonal protein studies are not, by themselves, robust predictors of cardiac amyloid type in patients undergoing endomyocardial biopsy. [Copyright &y& Elsevier]

Little is known about the rare entities of heavy- and light-chain amyloidosis (AHL) and heavy-chain amyloidosis (AH). Here, we report the renal and hematological characteristics, pathology, and outcome of 16 patients with renal AH/AHL (5 with AH and 11 with AHL) and compare them with 202 patients with renal light-chain amyloidosis (AL) diagnosed during the same time period. All cases were diagnosed by kidney biopsy that showed Congo red-positive deposits. Amyloid typing was done by laser microdissection and mass spectrometry (LMD/MS) on 12 patients or by immunofluorescence on four patients. All patients with renal AH/AHL were Caucasians, with a male/female ratio of 2.2 and a median age at biopsy of 63 years. Compared with patients with renal AL, those with renal AH/AHL had less frequent concurrent cardiac involvement, higher likelihood of having circulating complete monoclonal immunoglobulin, lower sensitivity of fat pad biopsy and bone marrow biopsy for detecting amyloid, higher incidence of hematuria, and better patient survival. The hematological response to chemotherapy was comparable with renal AL. In 42% of patients, AH/AHL could not have been diagnosed without LMD/MS. Thus, renal AH/AHL is an uncommon and underrecognized form of amyloidosis, and its diagnosis is greatly enhanced by the use of LMD/MS for amyloid typing. The accurate histological diagnosis of renal AH/AHL and distinction from AL may have important clinical and prognostic implications. [ABSTRACT FROM AUTHOR]

C3 glomerulonephritis (C3GN) is a recently described disorder that typically results from abnormalities in the alternative pathway (AP) of complement. Here, we describe the clinical features, kidney biopsy findings, AP abnormalities, glomerular proteomic profile, and follow-up in 12 cases of C3GN. This disorder equally affected all ages, both genders, and typically presented with hematuria and proteinuria. In both the short and long term, renal function remained stable in the majority of patients with native kidney disease. In two patients, C3GN recurred within 1 year of transplantation and resulted in a decline in allograft function. Kidney biopsy mainly showed a membranoproliferative pattern, although both mesangial proliferative and diffuse endocapillary proliferative glomerulonephritis were noted. AP abnormalities were heterogeneous, both acquired and genetic. The most common acquired abnormality was the presence of C3 nephritic factors, while the most common genetic finding was the presence of H402 and V62 alleles of Factor H. In addition to these risk factors, other abnormalities included Factor H autoantibodies and mutations in CFH, CFI, and CFHR genes. Laser dissection and mass spectrometry of glomeruli from patients with C3GN showed accumulation of AP and terminal complement complex proteins. Thus, C3GN results from diverse abnormalities of the alternative complement pathway leading to subsequent glomerular injury. [ABSTRACT FROM AUTHOR]

Amyloidosis is caused by extracellular deposition of proteins in an insoluble manner within tissues. In hereditary forms of amyloidosis, transthyretin, fibrinogen A-α, lysozyme, gelsolin, apolipoprotein A-I, and A-II accumulate in the tissue plaques. Here we describe a 52-year-old man with no family history of renal disease who presented with increased urinary frequency, gradual loss of renal function but no significant proteinuria. Renal biopsy found large amounts of amyloid restricted to the medulla with no involvement of glomeruli or vessels. Immunohistochemical analysis for transthyretin or serum amyloid A and tests for an underlying monoclonal gammopathy were negative. Although initially suspected to be amyloid light chain amyloidosis, laser microdissection and mass spectrometry showed that the amyloid was composed of large amounts of apolipoprotein A-IV. This was based on mass spectrometry studies that showed 100, 96, and 73 spectra in three microdissected samples that matched to apolipoprotein A-IV with 100% probability. DNA analyses detected three sequence variants representing common polymorphisms of the apolipoprotein A-IV gene. Thus, in this case, apolipoprotein A-IV deposition and renal involvement appear to be restricted to the medulla. A high degree of suspicion is required for the diagnosis of apolipoprotein A-IV amyloidosis as it may be missed if a renal biopsy consists only of cortex. [ABSTRACT FROM AUTHOR]