Biomedical subjects

Robert A Star

Publications and source records attributed to Robert A Star.

At least 19 recordsLinked to original sources

PubMed2007

Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10-30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80 degrees C or refrigerated overnight at 4 degrees C then stored at -80 degrees C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.

Biomarkers↗

PubMed2006

Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma.

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.

Animals↗

PubMed2006

Imaging acute renal failure with polyamine dendrimer-based MRI contrast agents.

Acute renal failure (ARF) induced by sepsis has a high mortality but lacks effective treatments. To develop novel therapies we must diagnose renal injury early and accurately in septic patients and identify any additional insults such as nephrotoxic drugs and ischemia. In this short review we describe our experience using MRI with dendrimer-based contrast agents in mouse models of ARF. This technique can diagnose early renal injury before serum creatinine is elevated, distinguish different ARF etiologies, track drug therapy and predict outcome. As an ARF biomarker, MRI with dendrimer-based contrast is a promising technique deserving further development.

Acute Kidney Injury↗

PubMed2006

Ischemic and nephrotoxic acute renal failure are distinguished by their broad transcriptomic responses.

Acute renal failure (ARF) has a high morbidity and mortality. In animal ARF models, effective treatments must be administered before or shortly after the insult, limiting their clinical potential. We used microarrays to identify early biomarkers that distinguish ischemic from nephrotoxic ARF or biomarkers that detect both injury types. We compared rat kidney transcriptomes at 2 and 8 h after ischemia/reperfusion and after mercuric chloride. Quality control and statistical analyses were necessary to normalize microarrays from different lots, eliminate outliers, and exclude unaltered genes. Principal component analysis revealed distinct ischemic and nephrotoxic trajectories and clear array groupings. Therefore, we used supervised analysis, t-tests, and fold changes to compile gene lists for each group, exclusive or nonexclusive, alone or in combination. There was little network connectivity, even in the largest group. Some microarray-identified genes were validated by TaqMan assay, ruling out artifacts. Western blotting confirmed that heme oxygenase-1 (HO-1) and activating transcription factor-3 (ATF3) proteins were upregulated; however, unexpectedly, their localization changed within the kidney. HO-1 staining shifted from cortical (early) to outer stripe of the outer medulla (late), primarily in detaching cells, after mercuric chloride but not ischemia/reperfusion. ATF3 staining was similar, but with additional early transient expression in the outer stripe after ischemia/reperfusion. We conclude that microarray-identified genes must be evaluated not only for protein levels but also for anatomical distribution among different zones, nephron segments, or cell types. Although protein detection reagents are limited, microarray data lay a rich foundation to explore biomarkers, therapeutics, and the pathophysiology of ARF.

Activating Transcription Factor 3↗

PubMed2006

Incidence and mortality of acute renal failure in Medicare beneficiaries, 1992 to 2001.

This study's objective was to determine the incidence and mortality of acute renal failure (ARF) in Medicare beneficiaries. Data were from hospitalized Medicare beneficiaries (5,403,015 discharges) between 1992 and 2001 from the 5% sample of Medicare claims. For 1992 to 2001, the overall incidence rate of ARF was 23.8 cases per 1000 discharges, with rates increasing by approximately 11% per year. Older age, male gender, and black race were strongly associated (P < 0.0001) with ARF. The overall in-hospital death rate was 4.6% in discharges without ARF, 15.2% in discharges with ARF coded as the principal diagnosis, and 32.6% in discharges with ARF as a secondary diagnosis. In-hospital death rates were 32.9% in discharges with ARF that required renal dialysis and 27.5% in those with ARF that did not require dialysis. Death within 90 d after hospital admission was 13.1% in discharges without ARF, 34.5% in discharges with ARF coded as the principal diagnosis, and 48.6% in discharges with ARF as a secondary diagnosis. Discharges with ARF were more (P < 0.0001) likely to have intensive care and other acute organ dysfunction than those without ARF. For discharges both with and without ARF, rates for death within 90 d after hospital admission showed a declining trend. In conclusion, the incidence rate of ARF in Medicare beneficiaries has been increasing. Those of older age, male gender, and black race are more likely to have ARF. These data show ARF to be a major contributor to morbidity and mortality in hospitalized patients.

Acute Kidney Injury↗

PubMed2005

Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin.

During intravascular hemolysis in human disease, vasomotor tone and organ perfusion may be impaired by the increased reactivity of cell-free plasma hemoglobin (Hb) with NO. We experimentally produced acute intravascular hemolysis in a canine model in order to test the hypothesis that low levels of decompartmentalized or cell-free plasma Hb will severely reduce NO bioavailability and produce vasomotor instability. Importantly, in this model the total intravascular Hb level is unchanged; only the compartmentalization of Hb within the erythrocyte membrane is disrupted. Using a full-factorial design, we demonstrate that free water-induced intravascular hemolysis produces dose-dependent systemic vasoconstriction and impairs renal function. We find that these physiologic changes are secondary to the stoichiometric oxidation of endogenous NO by cell-free plasma oxyhemoglobin. In this model, 80 ppm of inhaled NO gas oxidized 85-90% of plasma oxyhemoglobin to methemoglobin, thereby inhibiting endogenous NO scavenging by cell-free Hb. As a result, the vasoconstriction caused by acute hemolysis was attenuated and the responsiveness to systemically infused NO donors was restored. These observations confirm that the acute toxicity of intravascular hemolysis occurs secondarily to the accelerated dioxygenation reaction of plasma oxyhemoglobin with endothelium-derived NO to form bioinactive nitrate. These biochemical and physiological studies demonstrate a major role for the intact erythrocyte in NO homeostasis and provide mechanistic support for the existence of a human syndrome of hemolysis-associated NO dysregulation, which may contribute to the vasculopathy of hereditary, acquired, and iatrogenic hemolytic states.

Animals↗

PubMed2005

Prospects for urinary proteomics: exosomes as a source of urinary biomarkers.

Recent progress in biotechnology offers the promise of better medical care at lower costs. Among the techniques that show the greatest promise is mass spectrometry of proteins, which can identify proteins present in body fluids and tissue specimens at a large scale. Because urine can be collected in large amounts in a non-invasive fashion, the potential exists to use mass spectrometry to discover urinary biomarkers that are early predictors of renal disease, or useful in making therapeutic choices. Recently, the authors demonstrated that both membrane proteins and cytosolic proteins from renal epithelia are highly enriched in low-density urinary structures identified as exosomes. Exosomes were found to contain many disease-associated proteins including aquaporin-2, polycystin-1, podocin, non-muscle myosin II, angiotensin-converting enzyme, Na+ K+ 2Cl- cotransporter (NKCC2), thiazide-sensitive Na-Cl cotransporter (NCC), and epithelial sodium channel (ENaC). Potentially, other disease biomarkers could be discovered by mass spectrometry-based proteomic studies in well-defined patient populations. Herein is described the advantages of using urinary exosomes as a starting material for biomarker discovery. In addition, the purpose of this review is to present an overall strategy for biomarker discovery in urine using exosomes and for developing cost-effective clinical assays for these biomarkers, which can potentially be used for early detection of disease, as a means of differential diagnosis, or as a means of guiding therapy. Finally, potential barriers that need to be overcome before urinary proteomics can be applied clinically, are emphasized.

Biomarkers↗

PubMed2005

Dendrimer-enhanced MRI as a diagnostic and prognostic biomarker of sepsis-induced acute renal failure in aged mice.

BACKGROUND: Acute renal failure (ARF) induced by sepsis has a high mortality. In an aged mouse model of sepsis-induced ARF we have previously shown that renal injury occurs before serum creatinine is elevated. Development of a noninvasive biomarker that could diagnose renal dysfunction early in sepsis and monitor the response to therapy would be very valuable. METHODS: We performed magnetic resonance imaging (MRI) with gadolinium-based G4 dendrimer intravenous contrast in a fluid- and antibiotic-treated cecal ligation and puncture (CLP) sepsis model in aged mice. Imaging was also performed in a mouse volume depletion model and in models of ARF induced by ischemia/reperfusion (I/R) and cisplatin. RESULTS: Twenty hours post-CLP, aged mice had a distinct pattern of renal injury using dendrimer-enhanced MRI. This pattern was different from renal injury induced by either cisplatin or I/R. Prerenal azotemia induced by volume depletion was distinguished from sepsis by dendrimer-enhanced MRI. Dendrimer-enhanced MRI detected renal dysfunction 6 hours post-CLP, a time when serum creatinine was still normal. Ethyl pyruvate reversed the renal dysfunction detected by dendrimer-enhanced MRI at 20 hours, but not at 6 hours post-CLP. The appearance of renal dysfunction on dendrimer-enhanced MRI at 6 hours post-CLP predicted the length of survival. CONCLUSION: Dendrimer-enhanced MRI is a novel biomarker that provides information for the early diagnosis, drug responsiveness, and prognosis of sepsis-induced ARF.

Acute Kidney Injury↗

PubMed2005

Design of the VA/NIH Acute Renal Failure Trial Network (ATN) Study: intensive versus conventional renal support in acute renal failure.

The optimal management of renal replacement therapy (RRT) in acute renal failure (ARF) is uncertain. The VA/NIH Acute Renal Failure Trail Network Study (ATN Study) tests the hypothesis that a strategy of intensive RRT will decrease 60-day all-cause mortality in critically ill patients with ARF. Dose separation between the two treatment arms is achieved by increasing the frequency of intermittent hemodialysis (IHD) and sustained low efficiency dialysis (SLED) treatments from three times per week to six times per week, and by increasing continuous venovenous hemodiafiltration (CVVHDF) effluent volume from 20 mL/kg/hr to 35 mL/kg/hr. In both treatment arms, subjects convert between IHD and CVVHDF or SLED as hemodynamic status changes over time. This strategy attempts to replicate the conversion between modalities of RRT that occurs in clinical practice. However, in order to implement this strategy, flexible criteria needed to be developed to provide a balance between the need for uniformity of treatment between groups and practitioner discretion regarding modality of RRT to maintain patient safety. In order to address safety and ethical issues similar to those raised by the Office of Human Research Protections in its review of the ARDS Network studies, a survey of practitioner practices was performed and observational data on the management of RRT in comparable critically ill patients with ARF managed outside of the research context is being collected prospectively. These data will help inform the study's DSMB and site IRB's of the relationship between the study's treatment arms and concurrent clinical practice.

Acute Kidney Injury↗

PubMed2004

Lymphatic drainage imaging of breast cancer in mice by micro-magnetic resonance lymphangiography using a nano-size paramagnetic contrast agent.

BACKGROUND: The presence of lymph node metastases is an important factor in breast cancer patient prognosis. Therefore, the precise identification of sentinel lymph nodes in these patients is critical. Improving current magnetic resonance (MR) imaging methods using a newly synthesized nano-size paramagnetic molecule, G6, as a contrast agent, provides an attractive means toward attaining this goal. METHODS: A four-dimensional method of micro-MR lymphangiography using G6 (9 nm/240 kd) was developed to visualize the lymphatic ducts and lymph nodes draining mouse mammary tumors over time. The ability of micro-MR lymphangiography with the G6 contrast agent to visualize lymphatic drainage of normal mouse mammary tissue was compared with that of the conventional MR contrast agent, Gd-[DTPA]-dimeglumine (<1 kd). Lymphatic drainage in spontaneous and xenografted breast tumor models was visualized using the G6 contrast agent. RESULTS: Draining lymphatic ducts and lymph nodes were clearly visualized in the mammary tissue of normal mice and in spontaneous and xenografted breast tumor models after a direct mammary gland or peritumoral injection of G6. Gd-[DTPA]-dime-glumine, by contrast, failed to depict lymphatic flow from the mammary tissue in normal mice using the same method. Micro-MR lymphangiography using the G6 contrast agent revealed the absence of filling in the metastatic foci of affected lymph nodes. CONCLUSIONS: The superior temporal and spatial resolution of micro-MR lymphangiography using the contrast agent G6 may facilitate the study of tumor lymphatic drainage and lymphatic metastasis in both experimental animals and clinical medicine. In addition, this may be a powerful new method for sentinel lymph node localization in human breast cancer.

Animals↗

PubMed2004

A simplified method for HPLC determination of creatinine in mouse serum.

Mouse models are frequently used to study renal function. However, mouse serum contains chromagens that interfere with standard picric acid-based assays for serum creatinine. Several alternative methods exist for serum creatinine measurements, including assay by high-performance liquid chromatography (HPLC), but only one has been adapted to mouse serum. Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC. The HPLC method was compared with a standard alkaline picrate colorimetric assay, using serum from animals with low-to-moderate renal injury. Acidification of acetonitrile with HCl in the deproteinization step produced variable results, including an extra peak that interfered with integration of the creatinine peak or loss of the creatinine peak. Deproteinizing with acetonitrile alone resulted in a more reliable measurement of serum creatinine, which was validated by a series of known additions of creatinine standard. The HPLC assay was reproducible with coefficients of variation from 1.6 to 5.1%. The picric acid assay overestimated serum creatinine, when directly compared with the HPLC assay. The extent of overestimation, up to sixfold, was greatest at normal (0.1 to 0.2 mg/dl) to moderately elevated (0.5 mg/dl) serum creatinine levels. Mouse serum contains substances that interfere with standard picric acid assays for creatinine. Our new HPLC assay can accurately detect creatinine from 5 microl of mouse serum. These results support the widespread adoption of HPLC to accurately measure serum creatinine in mouse models of renal injury.

Animals↗

PubMed2004

Peripheral CD4 T cells rapidly accumulate at the host: parasite interface during an inflammatory Th2 memory response.

Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4(+), TCR-alphabeta(+) T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.

Administration, Oral↗

PubMed2004

Alpha-melanocyte-stimulating hormone inhibits lung injury after renal ischemia/reperfusion.

Combined acute renal and pulmonary failure has a very high mortality. In animals, lung injury develops after shock or visceral or renal ischemia. Alpha-melanocyte-stimulating hormone (alpha-MSH) is an antiinflammatory cytokine, which inhibits inflammatory, apoptotic, and cytotoxic pathways implicated in acute renal injury. We sought to determine if alpha-MSH inhibits acute lung injury after renal ischemia and to determine the early mechanisms of alpha-MSH action. Mice were subjected to renal ischemia treated with vehicle or alpha-MSH. At early time points, we measured organ histology, leukocyte accumulation, myeloperoxidase activity, activation of nuclear factor-kappaB, p38 mitogen-activated protein kinase, c-Jun, and activator protein-1 pathways, in addition to messenger RNA for intracellular adhesion molecule-1 and tumor necrosis factor-alpha. Renal ischemia rapidly activated kidney and lung nuclear factor-kappaB, p38 mitogen-activated protein kinase, c-Jun, and activator protein-1 pathways, and distant lung injury. Alpha-MSH administration immediately before reperfusion significantly decreased kidney and lung injury and prevented activation of kidney and lung transcription factors and stress response genes, and lung intracellular adhesion molecule-1 and tumor necrosis factor-alpha at early time points after renal ischemia/reperfusion. We conclude that distant lung injury occurs rapidly after renal ischemia. alpha-MSH protects against both kidney and lung damage after renal ischemia, in part, by inhibiting activation of transcription factors and stress genes early after renal injury.

Animals↗

PubMed2004

Polyamine dendrimer-based MRI contrast agents for functional kidney imaging to diagnose acute renal failure.

PURPOSE: To choose an efficacious renal functional MRI contrast agent to image early renal tubular damage. We synthesized and compared smaller polyamine dendrimer-based MRI contrast agents (<60 kD) that, unlike Gd-[DTPA], transiently accumulate in renal tubules and can be used to visualize renal structural and functional damage. MATERIALS AND METHODS: Six dendrimer-based MRI contrast agents smaller than 60 kD were studied by high resolution dynamic micro-MRI and compared to Gd-[DTPA]-dimeglumine and Gadomer-17. The best agent, DAB-G2, was further tested in a mouse ischemia/reperfusion model to validate its efficacy. RESULTS: Despite unequal renal clearance rates, all polyamine dendrimer agents visualized the renal functional anatomy of the mice better than Gd-[DTPA]-dimeglumine and Gadomer-17. DAB-G2 was excreted most rapidly, yet was able to visualize mild renal tubular injury very early after injury. CONCLUSION: DAB-G2 was found to be the best candidate for functional kidney imaging and enabled early diagnosis of acute renal injury.

Acute Kidney Injury↗

PubMed2004

Discovery of protein biomarkers for renal diseases.

Animal models and human studies have been useful in dissecting the molecular mechanisms of renal disease and finding new disease targets; however, translation of these findings to new clinical therapeutics remains challenging. Difficulties with detecting early disease, measuring drug effectiveness, and the daunting cost of clinical trials hampers the development of new therapeutics for renal diseases. Many existing laboratory tests were discovered because of inspired recognition that a particular protein might prove useful in clinical practice. New unbiased genomic and proteomic techniques identify many constituents present in biologic samples and thus may greatly accelerate biomarker research. This review focuses on the steps needed to develop new biomarkers that are useful in laboratory and clinical investigations, with particular focus on new proteomic screening technologies. New biomarkers will speed the laboratory and clinical development of new treatments for renal diseases through mechanistic insights, diagnoses that are more refined, early detection, and enhanced proof of concept testing.

Biomarkers↗

PubMed2004

Delayed DMSO administration protects the kidney from mercuric chloride-induced injury.

Reactive oxygen species are implicated as mediators of tissue damage in ischemic and toxic acute renal failure. Whereas many agents can inhibit renal ischemic injury, only hepatocyte growth factor, melatonin, N-acetylcysteine, and DMSO inhibit injury after mercuric chloride administration. Although it has been suggested that DMSO may chelate the mercuric ion, more recent studies suggest that it has anti-inflammatory and antioxidant effects. Acute renal failure was induced by 5 mg/kg subcutaneous injection of mercuric chloride in BALB/c mice. DMSO (3.8 ml/kg, 40% in PBS) or vehicle (PBS) was injected intraperitoneally at 0 and 24 h after mercuric chloride injection, or DMSO treatment was delayed 3 or 5 h. DMSO prevented increases in serum creatinine and tubular damage at 24 and 48 h. When DMSO treatment was delayed by 3 h, it was still beneficial; however, with a 5-h delay, the histology score and serum creatinine were not significantly decreased. DMSO partially prevented a mercuric chloride-induced decrease in glutathione peroxidase activity and completely prevented the transient decrease in superoxide dismutase activity. Neither mercuric chloride nor DMSO affected catalase activity significantly. For investigating possible effects of DMSO on cellular mercuric ion uptake, MDCK cells that were transfected with human organic anion transporter-1 were used. 203Hg uptake was inhibited 90% by N-acetylcysteine but only 5% by DMSO, indicating that the effect of DMSO is not related to chelating mercuric ion or inhibiting its uptake. It is concluded that DMSO acts in part as an antioxidant to inhibit mercuric chloride-induced acute renal injury.

Acute Kidney Injury↗

PubMed2004

Micro-MRI methods to detect renal cysts in mice.

BACKGROUND: Mouse models of disease, especially using transgenic and knockout technologies, are powerful tools to analyze the molecular basis of disease. We recently reported that a new dynamic micro-MRI method with dendrimer-based contrast agents can visualize renal structure and function in normal living mice and mice with acute renal failure. While MRI contrast enhancement is useful for detecting functional impairment of the kidneys, this technology has limitations in assessing morphologic changes, particularly cystic disease, because contrast-enhanced micro-MRI depicts cysts as low-intensity areas that cannot be distinguished from fibrotic foci. METHODS: In the current study, we evaluated if micro-MRI employing a new three-dimensional MR hydrography signal sequence [three-dimensional fast imaging employing steady-state acquisition (3D-FIESTA)] can visualize chronic cystic changes without any contrast agents. RESULTS: We were able to positively depict multiple renal cortical cysts of approximately 0.2 mm diameter in a mouse model of sickle cell disease and observe serial changes of renal cysts (>0.2 mm diameter) in cyclooxygenase-2 (COX-2) knockout mice during a 21/2-month period. Some cysts decreased in size over time. CONCLUSIONS: Micro-MRI with 3D-FIESTA can depict cyst formation in the diseased kidneys of living mice without injection of contrast agents.

Anemia, Sickle Cell↗

PubMed2003

Database for renal collecting duct regulatory and transporter proteins.

The mammalian kidney collecting duct plays an important role in the fine regulation of Na, K, water, and acid-base balance. Functional genomic and proteomic studies of the kidney offer new opportunities in the understanding of renal physiology and pathophysiology, and the collecting duct is an appropriate target tissue because of the relative simplicity of its cells and the ease of isolating or culturing large numbers of collecting duct cells. Study of the collecting duct includes assessment of gene expression and protein regulation and abundance. For example, DNA and protein microarrays can be used to quantitate gene expression and protein regulation and abundance under varying physiological conditions. An Internet-accessible database has been devised for major collecting duct proteins involved in transport and regulation of cellular processes. The individual proteins included in this database are those culled from literature searches and from previously published studies involving cDNA arrays and serial analysis of gene expression (SAGE). Design of microarray targets for the study of kidney collecting duct tissues is facilitated by the database, which includes links to curated base pair and amino acid sequence data, relevant literature, and related databases. Use of the database is illustrated by a search for water channel proteins, aquaporins, and by a subsequent search for vasopressin receptors. Links are shown to the literature and to sequence data for human, rat, and mouse, as well as to relevant web-based resources. Extension of the database is dynamic and is done through a maintenance interface. This permits creation of new categories, updating of existing entries, and addition of new ones.

Animals↗