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Bruce A Molitoris

Publications and source records attributed to Bruce A Molitoris.

At least 19 recordsLinked to original sources
PubMed2006

A non-nephrotoxic gentamicin congener that retains antimicrobial efficacy.

Aminoglycoside antibiotics, although of major clinical importance in the treatment of serious Gram- negative infections and a potential therapeutic agent in the amelioration of diseases that are characterized by premature stop mutations, are associated with a high incidence of acute renal failure. With the use of HPLC techniques, the four components (congeners) of gentamicin, the most commonly used aminoglycoside, were isolated and characterized. Described here is a congener with minimal cytotoxicity in cell culture and animal studies that retained normal bactericidal properties in both Bacillus subtilis and a multidrug-resistant form of Klebsiella pneumoniae. Furthermore, in animal studies, this congener failed to induce the functional and pathologic changes that are characteristic of gentamicin nephrotoxicity that is seen with the native compound. Finally, internalization of this non-nephrotoxic component was unaltered, but the subcellular distribution was different from native gentamicin or the other three cytotoxic congeners. These studies have identified a component of the native gentamicin congener mixture that retains its bactericidal properties with minimal or no apparent nephrotoxicity.

Animals↗
PubMed2006

Pharmacophotonics: utilizing multi-photon microscopy to quantify drug delivery and intracellular trafficking in the kidney.

The recent introduction of multi-photon microscopy coupled with advances in optics, computer sciences and the available fluorophores used to label molecules of interest have empowered investigators to study the dynamic events within the functioning kidney at cellular and subcellular levels. This emerging technique, with improved spatial and temporal resolution and sensitivity, enables investigators to follow the cell specific uptake of large and small molecules, determine the mode of cellular uptake, intracellular trafficking and drug metabolism in complex heterogeneous organs such as the kidney over time. Repeat determinations over seconds to hours to days allow for multiple observations within the same animal, thereby minimizing animal use and inter-animal variability. This can be particularly useful for preclinical studies. Furthermore, the ability to obtain volumetric data (3-D) makes quantitative 4-D (time) analysis possible. Finally, up to three fluorophores can be visualized simultaneously allowing for three different or interactive processes to be observed and resolved.

Animals↗
PubMed2006

Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals.

By combining intravital multiphoton microscopy and bacterial genetics we have developed a technique enabling real-time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP(+)-expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP(+) was introduced in the clinical UPEC strain CFT073 as a single-copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.

Animals↗
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↗
PubMed2006

Cofilin mediates ATP depletion-induced endothelial cell actin alterations.

Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.

Actin Depolymerizing Factors↗
PubMed2006

Loss of the homotypic fusion and vacuole protein sorting or golgi-associated retrograde protein vesicle tethering complexes results in gentamicin sensitivity in the yeast Saccharomyces cerevisiae.

Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity.

Anti-Bacterial Agents↗
PubMed2005

Adenoviral vectors expressing human endostatin-angiostatin and soluble Tie2: enhanced suppression of tumor growth and antiangiogenic effects in a prostate tumor model.

Angiogenesis is essential for prostate cancer development and metastasis. Antiangiogenic therapy targeting tumor neovasculature, therefore, represents a promising approach for prostate cancer treatment. We hypothesized that adenoviral-mediated delivery of a combination of antiangiogenic factors might have an enhanced antitumor response. We developed the adenoviral vectors Ad-hEndo-angio, expressing a unique, chimeric human endostatin-angiostatin fusion protein, and Ad-sTie2, expressing a soluble form of endothelium-specific receptor tyrosine kinase Tie2. Matrigel angiogenesis assays using Ad-hEndo-angio revealed significant inhibition of tubular network formation and endothelial sprouting compared to Ad-sTie2. In vivo studies in a bilateral PC-3 tumor xenograft model following either intratumoral or systemic administration of Ad-hEndo-angio led to enhanced tumor growth suppression compared to Ad-sTie2. A novel finding is that an intratumoral, combination therapy employing one-half the dose of Ad-hEndo-angio as well as Ad-sTie2 led to a complete regression of the injected, as well as the contralateral uninjected, tumor and prolonged the tumor-free survival in 80% of the animals. In addition, a novel, real-time, intravital imaging modality was used to monitor antiangiogenic responses following adenoviral-mediated gene transfer. These results suggest that a combinatorial antiangiogenic gene therapy approach involving Ad-hEndo-angio and Ad-sTie2 could become a novel form of treatment for localized human prostate cancer.

Adenoviridae↗
PubMed2005

Quantitative intravital microscopy using a Generalized Polarity concept for kidney studies.

In this article, we describe a ratiometric intravital two-photon microscopy technique for studying glomerular permeability and differences in proximal tubule cell reabsorption. This quantitative approach is based on the Generalized Polarity (GP) concept, in which the intensity difference between two fluorescent molecules is normalized to the total intensity produced by the two dyes. After an initial intravenous injection of a mixture of 3-, 40-, and 70-kDa fluorescently labeled dextrans into live Munich-Wistar-Frömter (MWF) rats, we were able to monitor changes in the GP values between any two dyes within local regions of the kidney, including the glomerulus, Bowman's capsule, proximal tubule lumens and proximal tubule cells, and individual capillary vessels. We were able to quantify accumulations of different dextrans in the Bowman's space and in tubular lumens as well as reabsorption by proximal tubular cells at different time points in the same rat. We found that for 6- to 8-wk-old MWF rats that developed spontaneous albuminuria, the 40- and 70-kDa dextrans, with hydrodynamic radii larger than albumin, were differentially filtered, but both were able to pass the glomerular filtration barrier and enter into the urinary space of the Bowman's capsule within a few seconds after intravenous infusion. Using GP image analysis, we found that negatively charged dextrans of both 40 and 70 kDa were better reabsorbed by the proximal tubule cells than the neutrally charged 40-kDa dextran. These results demonstrate the potential power of the GP imaging technique for quantitative studies of glomerular filtration and tubular reabsorption.

Albuminuria↗
PubMed2005

Renal blood flow in sepsis: a complex issue.

The clinical complexity of sepsis and the regional variability in renal blood flow present a difficult challenge for the clinician or investigator in understanding the role and clinical importance of reduced blood flow in the pathophysiology of sepsis-induced acute renal failure. Understanding the role of regional microvasculature flow and interactions between endothelium and white blood cells in the local delivery of oxygen and substrates is of critical importance. Therefore, measuring total renal blood flow may not permit an adequate understanding of the role of altered hemodynamics in septic patients who develop acute renal failure.

Animals↗
PubMed2005

Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney.

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.

Adenoviridae↗
PubMed2005

Intravital multiphoton microscopy of dynamic renal processes.

Recent advances in microscopy and optics, computer sciences, and the available fluorophores used to label molecules of interest have empowered investigators to utilize intravital two-photon microscopy to study the dynamic events within the functioning kidney. This emerging technique enables investigators to follow functional and structural alterations with subcellular resolution within the same field of view over seconds to weeks. This approach invigorates the validity of data and facilitates analysis and interpretation as trends are more readily determined when one is more closely monitoring indicative physiological parameters. Therefore, in this review we emphasize how specific approaches will enable studies into glomerular permeability, proximal tubule endocytosis, and microvascular function within the kidney. We attempt to show how visual data can be quantified, thus allowing enhanced understanding of the process under study. Finally, emphasis is given to the possible future opportunities of this technology and its present limitations.

Animals↗
PubMed2004

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy.

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments.

Animals↗
PubMed2004

Endothelial injury and dysfunction: role in the extension phase of acute renal failure.

The pathophysiology of ischemic acute renal failure (ARF) involves a complex interplay between renal hemodynamics, tubular and endothelial cell injury, and inflammatory processes. A growing body of evidence supports the contribution of altered renal vascular function, especially at the microvascular level, in initiating and subsequently extending the initial tubular injury. The extension phase of ischemic ARF involves continued reduction in renal perfusion, ongoing hypoxia, and inflammatory processes that occur during reperfusion and contribute to continued tubular cell injury. Vascular endothelial cell injury and dysfunction play a vital part in this extension phase. With injury, the endothelial cell loses its ability to regulate vascular tone, perfusion, permeability and inflammation/adhesion. This loss of regulatory function has a detrimental impact upon renal function. Vascular congestion, edema formation, diminished blood flow, and infiltration of inflammatory cells have been documented in the corticomedullary junction of the kidney. However, linking their genesis to microvascular endothelial injury and dysfunction has been difficult. New diagnostic and therapeutic approaches to ischemic ARF must incorporate these finding to devise early recognition strategies and therapeutic approaches.

Acute Kidney Injury↗
PubMed2003

Renal ischemia induces tropomyosin dissociation-destabilizing microvilli microfilaments.

Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.

Actin Cytoskeleton↗
PubMed2003

Gentamicin traffics retrograde through the secretory pathway and is released in the cytosol via the endoplasmic reticulum.

Previous mechanisms describing how aminoglycosides exert their cellular toxicity, including lysosomal accumulation, rupture, and release, cannot account for the rapidity and extent of the observed subcellular and organ effects. Using immunoamplification techniques and colocalization with epitopes of the endoplasmic reticulum (ER), we report rapid retrograde transport of gentamicin to the ER. Additionally, exposure times of 2 and 4 h in LLC-PK1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight cationic compounds, in a retrograde manner through the Golgi complex and to the ER. From there, these compounds move into the cytosol for delivery throughout the cell. To quantify the potential toxic effects of cytosolic aminoglycoside release, experiments examining mitochondrial membrane potential in the continued presence of extracellular gentamicin were undertaken and demonstrated a significant reduction after 4 and 8 h. These observations provide a mechanism for the rapidly induced known cellular alterations, including aberrant vesicle fusion, mitochondrial toxicity/free radical generation, and decreased protein synthesis either by reduced transcription or translation after aminoglycoside exposure.

Animals↗
PubMed2003

Mechanism of actin polymerization in cellular ATP depletion.

Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin beta4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin beta4 from 4 to 11 microm. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.

Actins↗