Peter L. Goering

Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies

Kidney toxicity accounts both for the failure of many drug candidates as well as considerable patient morbidity. Whereas histopathology remains the gold standard for nephrotoxicity in animal systems, serum creatinine (SCr) and blood urea nitrogen (BUN) are the primary options for monitoring kidney dysfunction in humans. The transmembrane tubular protein kidney injury molecule-1 (Kim-1) was previously reported to be markedly induced in response to renal injury. Owing to the poor sensitivity and specificity of SCr and BUN, we used rat toxicology studies to compare the diagnostic performance of urinary Kim-1 to BUN, SCr and urinary N-acetyl-β-D-glucosaminidase (NAG) as predictors of kidney tubular damage scored by histopathology. Kim-1 outperforms SCr, BUN and urinary NAG in multiple rat models of kidney injury. Urinary Kim-1 measurements may facilitate sensitive, specific and accurate prediction of human nephrotoxicity in preclinical drug screens. This should enable early identification and elimination of compounds that are potentially nephrotoxic.

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortiums (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Meeting Report: Hazard Assessment for Nanoparticles—Report from an Interdisciplinary Workshop

In this report we present the findings from a nanotoxicology workshop held 6–7 April 2006 at the Woodrow Wilson International Center for Scholars in Washington, DC. Over 2 days, 26 scientists from government, academia, industry, and nonprofit organizations addressed two specific questions: what information is needed to understand the human health impact of engineered nanoparticles and how is this information best obtained? To assess hazards of nanoparticles in the near-term, most participants noted the need to use existing in vivo toxicologic tests because of their greater familiarity and interpretability. For all types of toxicology tests, the best measures of nanoparticle dose need to be determined. Most participants agreed that a standard set of nanoparticles should be validated by laboratories worldwide and made available for benchmarking tests of other newly created nanoparticles. The group concluded that a battery of tests should be developed to uncover particularly hazardous properties. Given the large number of diverse materials, most participants favored a tiered approach. Over the long term, research aimed at developing a mechanistic understanding of the numerous characteristics that influence nanoparticle toxicity was deemed essential. Predicting the potential toxicity of emerging nanoparticles will require hypothesis-driven research that elucidates how physicochemical parameters influence toxic effects on biological systems. Research needs should be determined in the context of the current availability of testing methods for nanoscale particles. Finally, the group identified general policy and strategic opportunities to accelerate the development and implementation of testing protocols and ensure that the information generated is translated effectively for all stakeholders.

Expression, Circulation, and Excretion Profile of MicroRNA-21, -155, and -18a Following Acute Kidney Injury

MicroRNAs (miRNAs) are endogenous noncoding RNA molecules that are involved in post-transcriptional gene silencing. Using global miRNA expression profiling, we found miR-21, -155, and 18a to be highly upregulated in rat kidneys following tubular injury induced by ischemia/reperfusion (I/R) or gentamicin administration. Mir-21 and -155 also showed decreased expression patterns in blood and urinary supernatants in both models of kidney injury. Furthermore, urinary levels of miR-21 increased 1.2-fold in patients with clinical diagnosis of acute kidney injury (AKI) (n = 22) as compared with healthy volunteers (n = 25) (p < 0.05), and miR-155 decreased 1.5-fold in patients with AKI (p < 0.01). We identified 29 messenger RNA core targets of these 3 miRNAs using the context likelihood of relatedness algorithm and found these predicted gene targets to be highly enriched for genes associated with apoptosis or cell proliferation. Taken together, these results suggest that miRNA-21 and -155 could potentially serve as translational biomarkers for detection of AKI and may play a critical role in the pathogenesis of kidney injury and tissue repair process.

Physicochemical Characterization and In Vitro Hemolysis Evaluation of Silver Nanoparticles

Silver nanomaterials are increasingly being used as antimicrobial agents in medical devices. This study assessed the in vitro hemolytic potential of unbound silver particles in human blood to determine which physical and chemical particle properties contribute to mechanisms of red blood cell (RBC) damage. Four silver particle powders (two nano-sized and two micron-sized) were dispersed in water and characterized using transmission electron microscopy, dynamic light scattering, surface-enhanced Raman spectroscopy, and zeta potential measurement. Particle size and agglomeration were dependent on the suspension media. Under similar conditions to the hemolysis assay, with the particles added to phosphate buffered saline (PBS) and plasma, the size of the nanoparticles increased compared with particles suspended in water alone due to interaction with chloride ions and plasma proteins. To determine hemolysis response, aqueous particle suspensions were mixed with heparinized human blood diluted in PBS for 3.5 h at 37°C. Both nanoparticle preparations were significantly more hemolytic than micron-sized particles at equivalent mass concentrations > 220 μg/ml and at estimated surface area concentrations > 10 cm(2)/ml. The presence or absence of surface citrate on nanoparticles showed no significant difference in hemolysis. However, the aqueous nanoparticle preparations released significantly more silver ions than micron-sized particles, which correlated with increased hemolysis. Although significant size changes occurred to the silver particles due to interaction with media components, the higher level of in vitro hemolysis observed with nanoparticles compared with micron-sized particles may be related to their greater surface area, increased silver ion release, and direct interaction with RBCs.

Interaction of copper with DNA and antagonism by other metals.

Copper [Cu(II)] has been shown to enhance DNA damage in several biological systems. Binding of copper to DNA may be a key step in producing these lesions. The results of this study indicate that the DNA double helix contains at least two kinds of binding sites for copper. One site is present once every four nucleotides, has high affinity, and shows a cooperative effect. The other is an intercalating site for copper that is present in every base pair. This site is saturable, has a dissociation constant (Kd) for Cu(II) of 41 microM. In single-stranded DNA, we found an average copper binding site every three nucleotides with lower affinity than in dsDNA. The binding of copper to DNA shows an unexpected high specificity when studied in the presence of other metallic ions. The relative efficacy of several divalent cations to antagonize Cu(II) binding was: Ni = Cd = Mg much much greater than Zn = Hg greater than Ca greater than Pb much much greater than Mn, while Cr(VI) enhanced Cu(II) binding to DNA. We hope this study will broaden the understanding of copper-DNA interactions, particularly as they relate to treatment modalities for diseases associated with disruption of copper homeostasis and potential development of copper antitumor agents.

Towards consensus practices to qualify safety biomarkers for use in early drug development

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Stereolithography in tissue engineering

Several recent research efforts have focused on use of computer-aided additive fabrication technologies, commonly referred to as additive manufacturing, rapid prototyping, solid freeform fabrication, or three-dimensional printing technologies, to create structures for tissue engineering. For example, scaffolds for tissue engineering may be processed using rapid prototyping technologies, which serve as matrices for cell ingrowth, vascularization, as well as transport of nutrients and waste. Stereolithography is a photopolymerization-based rapid prototyping technology that involves computer-driven and spatially controlled irradiation of liquid resin. This technology enables structures with precise microscale features to be prepared directly from a computer model. In this review, use of stereolithography for processing trimethylene carbonate, polycaprolactone, and poly(d,l-lactide) poly(propylene fumarate)-based materials is considered. In addition, incorporation of bioceramic fillers for fabrication of bioceramic scaffolds is reviewed. Use of stereolithography for processing of patient-specific implantable scaffolds is also discussed. In addition, use of photopolymerization-based rapid prototyping technology, known as two-photon polymerization, for production of tissue engineering scaffolds with smaller features than conventional stereolithography technology is considered.

Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophages

Photoluminescent silicon nanoparticles have a bright and stable fluorescence and are promising candidates for bio‐imaging, cell staining and drug delivery. With increasing development of nanotechnology applications for biomedicine, an understanding of the potential toxicity of nanoparticles is needed to assess safety concerns for clinical applications. The objective of this study was to compare biological responses of silicon nanoparticles (SNs, 3 nm diameter) with silicon microparticles (SMs, ∼100–3000 nm diameter) in cultured murine macrophages (RAW 264.7) using standard protocols for assessing cytotoxicity/cell viability and inflammatory responses developed for micron‐sized particles. SNs and SMs were exposed to macrophages with and without addition of endotoxin lipopolysaccharide (LPS), a positive inducer of tumor necrosis factor‐alpha (TNF‐α), interleukin 6 (IL‐6), and nitric oxide (NO). Cytotoxicity was assayed using the dye exclusion and MTT assays. Cell supernatants were assayed for production TNF‐α, IL‐6 and NO. SNs at concentrations ≤20 µg ml−1 exhibited no cytotoxicity or inflammatory responses; however, SNs and SMs >20 and 200 µg ml−1, respectively, increased cytotoxicity compared with controls. SMs induced concentration‐related increases in TNF‐α and IL‐6 production; in contrast, the production of these cytokines was shown to decrease with increasing concentrations of SNs. NO production was not induced by SNs or SMs alone. Fluorescence microscopy demonstrated that SNs were associated with the macrophages, either internalized or attached to cell membranes. In conclusion, evaluating differences in biological responses for nanoparticles compared with microparticles of the same material may help improve tests to assess biological responses of nanoparticles that may be used in biomedical applications. Copyright

Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity

Adverse environmental stimuli increase the synthesis of a class of proteins referred to as stress proteins. The effect of mercuric chloride, a model nephrotoxin, on protein synthesis in male rat kidney has been evaluated. Renal slices from exposed rats were incubated with [35S]methionine for 1 hr and subjected to SDS-PAGE, after which 35S-labeled proteins were detected by autoradiography. Enhanced de novo synthesis of 70- and 90-kDa relative molecular mass (M(r)) proteins were detected 2 hr after exposure to 1 mg Hg/kg, with maximum activity occurring at 4-8 hr. By 16 hr postinjection, synthesis of these two proteins had decreased. Dose-related increases in synthesis of these proteins, and of a 110-kDa protein, were observed 4 hr after i.v. injection of 0.25, 0.5, and 1.0 mg Hg/kg, with concomitant inhibition of synthesis of proteins of M(r) 38 and 68 kDa. At a dose of 1 mg/kg, kidney proximal tubules exhibited progressive degenerative changes from 4 to 24 hr. A functional deficit, decreased uptake of [para-3H]aminohippurate into renal slices, was not observed until 16 hr after i.v. injection of 1 mg/kg. No significant histopathologic changes were observed in kidneys 4 hr after treatment with 0.25 or 0.5 mg Hg/kg, iv. No changes in liver protein synthesis were apparent until 16-24 hr, where an increase in the 70- and 90-kDa proteins was observed. A concomitant increase in plasma sorbitol dehydrogenase activity occurred at 16-24 hr; however, there was no histopathological evidence of liver injury. The 72-kDa inducible member of the 70-kDa stress protein family and the 88-kDa member of the 90-kDa protein family were detected by immunoblotting techniques using monoclonal antibodies. The data demonstrate that Hg induces alterations in the expression of renal gene products in vivo as evidenced by enhanced stress protein synthesis and inhibition of synthesis of constitutive proteins. These changes in renal protein synthesis preceded overt renal injury, occurring in the early stages of nephropathy. Altered patterns of stress protein synthesis appeared to be target organ specific. The data suggest that altered protein synthesis patterns may serve as biomarkers of renal injury.