Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracell... more Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracellular kidney injury biomarkers. To study whether they contain transcription factors, we collected urine from two acute kidney injury models (cisplatin or ischemia-reperfusion), two podocyte ...
Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We a... more Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-β1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-β1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunt...
Journal of Pharmacology and Experimental Therapeutics
Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a... more Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a transient increase in c-fos proto-oncogene mRNA levels (Introna et al., 1986). After 2 h from the initial treatment, c-fos mRNA could no longer be detected and its expression could not be restimulated either by LPS or by other signals including colony stimulating factor-1 (CSF-1) and phorbol myristate acetate (PMA), both of which are able to induce expression of the c-fos gene in unstimulated macrophages. When LPS was removed after an initial 30 min incubation, responsiveness to a second exposure to LPS began to reappear after 3 h and was completely restored by 20 h. The same pattern of desensitization of c-fos induction was observed when CSF-1 stimulated macrophages were subsequently exposed to LPS. The loss of sensitivity to PMA following pretreatment with LPS was selective for c-fos expression as LPS treated macrophages remained responsive to PMA with respect to the ability to stimulate secretion of H2O2. The mechanism of desensitization was localized, at least in part, at the level of transcription as demonstrated by analysis of c-fos transcripts in nuclei isolated from macrophages pretreated and restimulated with LPS.
cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with ³Â... more cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with ³²P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties
Dominant negative mutants are unique tools to define functions of a protein, not only within comp... more Dominant negative mutants are unique tools to define functions of a protein, not only within complex cellular and organismal contexts, but also mechanistically within a protein. Guanylyl cyclases are amenable to studies with dominant negative mutants, with their own sets of opportunities for insight and pitfalls to overcome. Membrane and soluble forms of guanylyl cyclase represent self-contained signal transduction modules that recognize, transduce, and amplify an external signal to give a carefully controlled response. Beginning with recognition of peptide hormones versus nitric oxide, membrane and soluble guanylyl cyclases are considerably different, except that their catalytic domains are closely related. Studies on these catalytic domains and their counterparts in adenylyl cyclases have raised an integral question of whether one or two domains form a catalytic site, which remains unresolved. Regardless of which model is correct, guanylyl cyclases appear to require an oligomeric ...
Light, in the presence of ATP, has been reported to stimulate cGMP binding to a 58 kDa protein in... more Light, in the presence of ATP, has been reported to stimulate cGMP binding to a 58 kDa protein in ROS (rod outer segments, Fesenko and Krapivinsky, 1986b, Photobiochem. Photobiophys. 13 345-58). This apparent light-related redistribution of ROS cGMP has been suggested to eliminate any requirement for phosphodiesterase-promoted hydrolysis of cGMP in the mechanism subserving phototransduction. Using conditions identical to those previously reported, this effect of light and ATP was examined further by characterizing the metabolic products that arise and the nucleotides that become liganded. The increased binding of radiolabeled guanine nucleotide upon illumination of ROS in the presence of ATP was confirmed, but the species of guanine nucleotide that were stimulated to bind under these conditions were identified as [32P]GDP and [32P]GTP rather than [32P]cGMP. The precautions to prevent enzymic hydrolysis of cGMP, which included conducting the reactions at 0 degrees and the addition of 3-isobutyl-l-methylxanthine (250 microM) to the reaction mixture did not prevent about a 20-fold increase in the rate of phosphodiesterase-catalyzed hydrolysis of radiolabeled cGMP by light when ATP was also present. This stimulation of phosphodiesterase activity is undoubtedly related to transphosphorylation by exogenous ATP of endogenous GMP and GDP involving catalytic actions of guanylate kinase and nucleoside diphosphate kinase in isolated ROS. These enzymes can also serve to generate [32P]GDP and [32P]GTP, which subsequently bind to ROS components. Such a mechanism involving ATP as phosphoryl donor was supported by observing that an analog of ATP (beta,gamma-methyleneadenosine 5'-triphosphate), which cannot serve as a phosphoryl donor, did not increase radiolabeled guanine nucleotide binding. Although several ROS proteins can form filter-retainable complexes with GDP and GTP, the properties of the 58 kDa protein found to be photoaffinity labeled with radioactive guanine nucleotide are most characteristic of those attributable to tubulin. The previous report that illumination in the presence of ATP stimulates the binding of cGMP to ROS components finds no support from the data obtained in the present studies.
Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppr... more Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increas...
Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely use... more Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely used, with semiquantitative scores subjectively assigned by trained operators. We have developed an objective technique combining Sirius Red staining, polarization contrast microscopy, and automated analysis. Repeated analysis of the same sections by the same operator (r = 0.99) or by different operators (r = 0.98) was highly consistent for Sirius Red, while Masson trichrome performed less consistently (r = 0.61 and 0.72, respectively). These techniques performed equally well when comparing sections from the left and right kidneys of mice. Poor correlation between Sirius Red and Masson trichrome may reflect different specificities, as enhanced birefringence with Sirius Red staining is specific for collagen type I and III fibrils. Combining whole-section imaging and automated image analysis with Sirius Red/polarization contrast is a rapid, reproducible, and precise technique that is compleme...
American journal of physiology. Renal physiology, Jan 15, 2014
Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed w... more Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed when based on serum creatinine (SCr) changes, due in part, to decreased creatinine production. During experimental sepsis, we compared serum cystatin C (sCysC), SCr, and blood urea nitrogen (BUN) to inulin glomerular filtration rate (iGFR) before or 3-18 h after cecal ligation and puncture (CLP)-induced sepsis in CD-1 mice. sCysC had a faster increase and reached peak levels more rapidly than SCr in both sepsis and bilateral nephrectomy (BiNx) models. sCysC was a better surrogate of iGFR than SCr during sepsis. Combining sCysC with SCr values into a composite biomarker improved correlation with iGFR better than any biomarker alone or any other combination. We determined the renal contribution to sCysC handling with BiNx. sCysC and SCr were lower post-BiNx/CLP than post-BiNx alone, despite increased inflammatory and nonrenal organ damage biomarkers. Sepsis decreased CysC production in neph...
Animal studies have shown that mesenchymal stem cell (MSC) infusions improve acute kidney injury ... more Animal studies have shown that mesenchymal stem cell (MSC) infusions improve acute kidney injury (AKI) outcomes when administered early after ischemic/reperfusion injury or within 24hr after cisplatin administration. These findings have spurred several human clinical trials to prevent AKI. However, no specific therapy effectively treats clinically-obvious AKI or rescues renal function once advanced injury is established. We investigated if noninvasive image-guided pulsed focused ultrasound (pFUS) could alter the kidney microenvironment to enhance homing of subsequently-infused MSC. To examine the efficacy of pFUS-enhanced cell homing in disease, we targeted pFUS to kidneys to enhance MSC homing after cisplatin-induced AKI. We found that pFUS enhanced MSC homing at 1 day post-cisplatin, prior to renal functional deficits, and that enhanced homing improved outcomes of renal function, tubular cell death, and regeneration at 5 days post-cisplatin compared to MSC alone. We then investiga...
Sepsis is a severe and complex syndrome that lacks effective prevention or therapeutics. The effe... more Sepsis is a severe and complex syndrome that lacks effective prevention or therapeutics. The effects of sepsis on the microvasculature have become an attractive area for possible new targets and therapeutics. Microparticles (MPs) are cell membrane-derived particles that can promote coagulation, inflammation, and angiogenesis, and they can participate in cell-to-cell communication. MPs retain cell membrane and cytoplasmic constituents of their parental cells, including two procoagulants: phosphatidylserine and tissue factor. We highlight the role of microparticles released by endothelial and circulating cells after sepsis-induced microvascular injury, and we discuss possible mechanisms by which microparticles can contribute to endothelial dysfunction, immunosuppression, and multiorgan dysfunction-including sepsis-AKI. Once viewed as cellular byproducts, microparticles are emerging as a new class of markers and mediators in the pathogenesis of sepsis.Kidney International advance online publication, 18 February 2015; doi:10.1038/ki.2015.26.
Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracell... more Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracellular kidney injury biomarkers. To study whether they contain transcription factors, we collected urine from two acute kidney injury models (cisplatin or ischemia-reperfusion), two podocyte ...
Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We a... more Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-β1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-β1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunt...
Journal of Pharmacology and Experimental Therapeutics
Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a... more Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a transient increase in c-fos proto-oncogene mRNA levels (Introna et al., 1986). After 2 h from the initial treatment, c-fos mRNA could no longer be detected and its expression could not be restimulated either by LPS or by other signals including colony stimulating factor-1 (CSF-1) and phorbol myristate acetate (PMA), both of which are able to induce expression of the c-fos gene in unstimulated macrophages. When LPS was removed after an initial 30 min incubation, responsiveness to a second exposure to LPS began to reappear after 3 h and was completely restored by 20 h. The same pattern of desensitization of c-fos induction was observed when CSF-1 stimulated macrophages were subsequently exposed to LPS. The loss of sensitivity to PMA following pretreatment with LPS was selective for c-fos expression as LPS treated macrophages remained responsive to PMA with respect to the ability to stimulate secretion of H2O2. The mechanism of desensitization was localized, at least in part, at the level of transcription as demonstrated by analysis of c-fos transcripts in nuclei isolated from macrophages pretreated and restimulated with LPS.
cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with ³Â... more cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with ³²P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties
Dominant negative mutants are unique tools to define functions of a protein, not only within comp... more Dominant negative mutants are unique tools to define functions of a protein, not only within complex cellular and organismal contexts, but also mechanistically within a protein. Guanylyl cyclases are amenable to studies with dominant negative mutants, with their own sets of opportunities for insight and pitfalls to overcome. Membrane and soluble forms of guanylyl cyclase represent self-contained signal transduction modules that recognize, transduce, and amplify an external signal to give a carefully controlled response. Beginning with recognition of peptide hormones versus nitric oxide, membrane and soluble guanylyl cyclases are considerably different, except that their catalytic domains are closely related. Studies on these catalytic domains and their counterparts in adenylyl cyclases have raised an integral question of whether one or two domains form a catalytic site, which remains unresolved. Regardless of which model is correct, guanylyl cyclases appear to require an oligomeric ...
Light, in the presence of ATP, has been reported to stimulate cGMP binding to a 58 kDa protein in... more Light, in the presence of ATP, has been reported to stimulate cGMP binding to a 58 kDa protein in ROS (rod outer segments, Fesenko and Krapivinsky, 1986b, Photobiochem. Photobiophys. 13 345-58). This apparent light-related redistribution of ROS cGMP has been suggested to eliminate any requirement for phosphodiesterase-promoted hydrolysis of cGMP in the mechanism subserving phototransduction. Using conditions identical to those previously reported, this effect of light and ATP was examined further by characterizing the metabolic products that arise and the nucleotides that become liganded. The increased binding of radiolabeled guanine nucleotide upon illumination of ROS in the presence of ATP was confirmed, but the species of guanine nucleotide that were stimulated to bind under these conditions were identified as [32P]GDP and [32P]GTP rather than [32P]cGMP. The precautions to prevent enzymic hydrolysis of cGMP, which included conducting the reactions at 0 degrees and the addition of 3-isobutyl-l-methylxanthine (250 microM) to the reaction mixture did not prevent about a 20-fold increase in the rate of phosphodiesterase-catalyzed hydrolysis of radiolabeled cGMP by light when ATP was also present. This stimulation of phosphodiesterase activity is undoubtedly related to transphosphorylation by exogenous ATP of endogenous GMP and GDP involving catalytic actions of guanylate kinase and nucleoside diphosphate kinase in isolated ROS. These enzymes can also serve to generate [32P]GDP and [32P]GTP, which subsequently bind to ROS components. Such a mechanism involving ATP as phosphoryl donor was supported by observing that an analog of ATP (beta,gamma-methyleneadenosine 5'-triphosphate), which cannot serve as a phosphoryl donor, did not increase radiolabeled guanine nucleotide binding. Although several ROS proteins can form filter-retainable complexes with GDP and GTP, the properties of the 58 kDa protein found to be photoaffinity labeled with radioactive guanine nucleotide are most characteristic of those attributable to tubulin. The previous report that illumination in the presence of ATP stimulates the binding of cGMP to ROS components finds no support from the data obtained in the present studies.
Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppr... more Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increas...
Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely use... more Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely used, with semiquantitative scores subjectively assigned by trained operators. We have developed an objective technique combining Sirius Red staining, polarization contrast microscopy, and automated analysis. Repeated analysis of the same sections by the same operator (r = 0.99) or by different operators (r = 0.98) was highly consistent for Sirius Red, while Masson trichrome performed less consistently (r = 0.61 and 0.72, respectively). These techniques performed equally well when comparing sections from the left and right kidneys of mice. Poor correlation between Sirius Red and Masson trichrome may reflect different specificities, as enhanced birefringence with Sirius Red staining is specific for collagen type I and III fibrils. Combining whole-section imaging and automated image analysis with Sirius Red/polarization contrast is a rapid, reproducible, and precise technique that is compleme...
American journal of physiology. Renal physiology, Jan 15, 2014
Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed w... more Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed when based on serum creatinine (SCr) changes, due in part, to decreased creatinine production. During experimental sepsis, we compared serum cystatin C (sCysC), SCr, and blood urea nitrogen (BUN) to inulin glomerular filtration rate (iGFR) before or 3-18 h after cecal ligation and puncture (CLP)-induced sepsis in CD-1 mice. sCysC had a faster increase and reached peak levels more rapidly than SCr in both sepsis and bilateral nephrectomy (BiNx) models. sCysC was a better surrogate of iGFR than SCr during sepsis. Combining sCysC with SCr values into a composite biomarker improved correlation with iGFR better than any biomarker alone or any other combination. We determined the renal contribution to sCysC handling with BiNx. sCysC and SCr were lower post-BiNx/CLP than post-BiNx alone, despite increased inflammatory and nonrenal organ damage biomarkers. Sepsis decreased CysC production in neph...
Animal studies have shown that mesenchymal stem cell (MSC) infusions improve acute kidney injury ... more Animal studies have shown that mesenchymal stem cell (MSC) infusions improve acute kidney injury (AKI) outcomes when administered early after ischemic/reperfusion injury or within 24hr after cisplatin administration. These findings have spurred several human clinical trials to prevent AKI. However, no specific therapy effectively treats clinically-obvious AKI or rescues renal function once advanced injury is established. We investigated if noninvasive image-guided pulsed focused ultrasound (pFUS) could alter the kidney microenvironment to enhance homing of subsequently-infused MSC. To examine the efficacy of pFUS-enhanced cell homing in disease, we targeted pFUS to kidneys to enhance MSC homing after cisplatin-induced AKI. We found that pFUS enhanced MSC homing at 1 day post-cisplatin, prior to renal functional deficits, and that enhanced homing improved outcomes of renal function, tubular cell death, and regeneration at 5 days post-cisplatin compared to MSC alone. We then investiga...
Sepsis is a severe and complex syndrome that lacks effective prevention or therapeutics. The effe... more Sepsis is a severe and complex syndrome that lacks effective prevention or therapeutics. The effects of sepsis on the microvasculature have become an attractive area for possible new targets and therapeutics. Microparticles (MPs) are cell membrane-derived particles that can promote coagulation, inflammation, and angiogenesis, and they can participate in cell-to-cell communication. MPs retain cell membrane and cytoplasmic constituents of their parental cells, including two procoagulants: phosphatidylserine and tissue factor. We highlight the role of microparticles released by endothelial and circulating cells after sepsis-induced microvascular injury, and we discuss possible mechanisms by which microparticles can contribute to endothelial dysfunction, immunosuppression, and multiorgan dysfunction-including sepsis-AKI. Once viewed as cellular byproducts, microparticles are emerging as a new class of markers and mediators in the pathogenesis of sepsis.Kidney International advance online publication, 18 February 2015; doi:10.1038/ki.2015.26.
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Papers by Peter Yuen