Background Hyponatremia associated with a low serum osmolality is a common and confounding electr... more Background Hyponatremia associated with a low serum osmolality is a common and confounding electrolyte disorder. Correcting hyponatremia is also complicated, especially in the setting of chronic hyponatremia. Here, we provide a rational approach to accurately detecting and safely treating acute on chronic euvolemic hyponatremia in the setting of acute polydipsia with a chronic reset osmostat. Case presentation A 71-year-old hispanic gentleman with chronic hyponatremia presented with hiccups, polydipsia, and a serum sodium concentration of 120 mEq/L associated with diffuse weakness, inattentiveness, and suicidal ideation. Symptomatic euvolemic hyponatremia warranted hypertonic saline treatment in the acute phase and water restriction in the chronic phase. Both interventions resulted in improvement in symptoms and/or the serum sodium concentration, but to a serum sodium level that persistently remained below the normal range. Remarkably, the urine osmolality appropriately fell when th...
Journal of the American Society of Nephrology, 2018
Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Diff... more Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target.Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target.Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that inte...
Effective lecturing stimulates learning, creates a verbal history for our profession, and is a ce... more Effective lecturing stimulates learning, creates a verbal history for our profession, and is a central basis for evaluating academic promotion. Unfortunately, few resources exist in the medical literature to guide the academician toward success as an effective lecturer. Using evidence-based principles, this review fosters adult learning in academic venues by incorporating the latest innovations in educational theory for both online and traditional teaching. The novice or advanced academic teacher will be guided toward critical self-evaluation of current teaching practices and encouraged to replace ineffective methods with ones more likely to be both rewarding and rewarded. By introducing literature-based learning techniques, emphasizing audience targeting, truncating content to an appropriate level of detail, effectively linking images and text, and accepting the brevity of learners' attentiveness, we show that the audience, not the speaker, is the primary educational focus.
in vivo and exposure to metabolic inhibitors in vitro cause mitochondrial membrane injury associa... more in vivo and exposure to metabolic inhibitors in vitro cause mitochondrial membrane injury associated with bax activation and apoptosis in proximal tubule epithelial. Hsp27, an inducible heat stress protein, reduces apoptosis in diverse cells types by an unknown mechanism. To evaluate the hypothesis that hsp27 inhibits Bax activation and apoptosis, renal epithelial cells (REC) were infected with adenovirus (AdV) containing human hsp27 prior to pharmacologic ischemia, an insult that activates bax and induces apoptosis. Hsp27 over-expression inhibited bax activation detected by a conformation specific antibody, reduced mitochondrial leakage of both cytochrome c and apoptosis inducing factor (AIF) and improved survival by >50% after 1-2 hr pharmacologic ischemia compared to empty vector. Immunoprecipitation (IP) did not detect hsp27-bax interaction before, during or after ischemia, suggesting that hsp27 does not directly regulate bax. To determine whether hsp27 indirectly antagonizes bax via Akt, a pro-survival serine-threonine kinase that phosphorylates and inactivates bax, p-ser 473 Akt (active) and total Akt content were compared at baseline and after ischemia in hsp27 over-expressing and in control cells. Hsp27 over-expression increased p-Akt content both at baseline and after ischemia compared to empty vector without altering total Akt. Hsp27 over-expression and ischemia selectively increased the interaction between hsp27 and Akt detected by IP. Hsp27 overexpression did not appear to alter the apparent half-life of p-ser 473 Akt during metabolic stress, an insult, which inactivates Akt, suggesting that hsp27 does not inhibit PP2-mediated Akt dephosphorylation. Ly294002 and wortmannin (two distinct Akt/PI3 kinase inhibitors) completely abrogated the pro-survival effect of hsp27, showing that Akt activation and bax inactivation are central to improved REC survival after injury. These data support the hypothesis that hsp27 indirectly inhibits bax-mediated mitochondrial injury and apoptosis after in vitro ischemia by facilitating Akt activation via a PI3 kinasedependent pathway. Both Akt and hsp27 represent potential new targets for improving REC survival after an ischemic insult.
Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeti... more Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeting the proximal tubule epithelial cell. The development of an effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we propose that RNAi signal pathway screening could identify a unifying mechanism of gentamicin-induced cell injury and suggest a therapeutic strategy to ameliorate it. Computational analysis of RNAi signal screens in gentamicin-exposed human proximal tubule cells suggested the cross-organelle stress response (CORE), the unfolded protein response (UPR), and cell chaperones as key targets of gentamicin-induced injury. To test this hypothesis, we assessed the effect of gentamicin on the CORE, UPR, and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-s...
We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediate... more We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPM-Bax complexes formed, and both NPM and Bax accumulated in mitochondria. Expression of a cytosol-restricted NPMmutant (NPM-NLS), but not a nucleus-restricted NPMmutant, increased NPM-Bax complex formation, mitochondrial NPM and Bax accumulation, mitochondrial membrane injury, caspase 3 activation, and ischemia-induced cell death. Coexpression of NPM-NLS with constitutively active Bax mutants caused nearly universal cell death in the absence of metabolic stress, whereas ex-pression of active Bax or NPM-NLS alone did not. A Bax peptide that disrupts NPM-Bax interaction significantly reduced cell death caused by exposure to metabolic inhibitors in vitro and preserved kidney function after ischemia in vivo. Thus, NPM-Bax interaction enhances mi...
Clinical Diabetes : A Publication of the American Diabetes Association, 2016
A 47-year-old woman with a self-reported 11-year history of diabetes mellitus presented with 2 da... more A 47-year-old woman with a self-reported 11-year history of diabetes mellitus presented with 2 days of nausea, vomiting, decreased oral intake, and back pain radiating to the neck. Her review of systems was remarkable for a “throbbing” headache of 1 day’s duration. Her medications included levothyroxine, subcutaneous long-acting insulin (glargine), topiramate, and canagliflozin, a selective sodium–glucose cotransporter 2 (SGLT2) inhibitor of the gloflozin class, which had been initiated 2 weeks earlier. Her medical history was remarkable for post-thyroidectomy Graves’ disease, cholecystectomy for multiple cholelithiasis, depression, fibromyalgia, and hyperlipidemia. She also had a history of spinal fusion surgery. In the emergency department, she appeared volume depleted. Her vital signs were temperature 98.9° F, blood pressure 118/76 mmHg, and a regular heart rate of 91 bpm. Her BMI was 27.45 kg/m2. Physical examination was remarkable for dry mucosal membranes, the absence of axill...
The mechanism by which the serine-threonine kinase glycogen synthase kinase-3 (GSK3 ) affects sur... more The mechanism by which the serine-threonine kinase glycogen synthase kinase-3 (GSK3 ) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3 promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3 , Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3 mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference–mediated knockdown of GSK3 promoted cell survival. Furthermore, RNA interference–mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3 . In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine site targeted by stress-activated ...
Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after... more Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton colla...
The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transien... more The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transient renal ischemia in vivo induces HSP-72, it is not known whether prior heat stress protects renal epithelial cells from injury mediated by ATP depletion. To evaluate this hypothesis, opossum kidney (OK) cells were exposed to sodium cyanide and 2-deoxy-D-glucose in the absence of medium glucose, a maneuver that reduced cell ATP content to < 10% of the control value within 10 min and decreased cell survival. One day after 2 h of ATP depletion, OK cells previously exposed to heat stress (to induce accumulation of HSP-72) exhibited marked improvement in survival (a > 4-fold increase in total DNA), less uptake of vital dye, and less release of lactate dehydrogenase (LDH) than cells subjected to ATP depletion alone (23.0 +/- 1.6 vs. 34.1 +/- 1.2% of total LDH, respectively). Enhanced clonogenicity post-heat stress was completely prevented by cycloheximide and positively correlated with t...
American journal of physiology. Renal physiology, Jan 31, 2016
Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure ... more Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure of proximal tubular epithelial cells to excess albumin promotes tubular atrophy and fibrosis, key predictors of progressive organ dysfunction. However, the link between proteinuria and tubular damage is unclear. We propose that pathologic albumin exposure impairs proximal tubular autophagy, an essential process for recycling damaged organelles and toxic intracellular macromolecules. In both mouse primary proximal tubule and immortalized human kidney cells, albumin exposure decreased the number of autophagosomes, visualized by the autophagosome-specific fluorescent markers monodansylcadaverine and GFP-LC3, respectively. Similarly, renal cortical tissue harvested from proteinuric mice contained reduced numbers of autophagosomes on electron micrographs and immunoblot showed reduced steady state LC3-II content. Albumin exposure decreased autophagic flux in vitro in a concentration-dependent ...
American journal of physiology. Renal physiology, Jan 21, 2016
Protein mimotopes or blocking peptides are small therapeutic peptides that prevent protein-protei... more Protein mimotopes or blocking peptides are small therapeutic peptides that prevent protein-protein interactions by selectively mimicking a native binding domain. Inexpensive technology facilitates straightforward design and production of blocking peptides in sufficient quantities to undertake preventive and therapeutic trials in both in vitro and in vivo experimental disease models. The kidney is an ideal peptide target since small molecules undergo rapid filtration and efficient bulk absorption by tubular epithelial cells. Compared to the blood stream, the half-life of peptides in the kidney is markedly prolonged, making blocking peptides an attractive tool for treating diverse renal diseases including ischemia, proteinuric states such as membranous glomerulonephritis (MGN) and focal and segmental glomerulosclerosis (FSGS) or renal cell carcinoma. Therapeutic peptides represent one of the fastest growing reagents classes for novel drug development in human disease partly due their ...
Intoxications frequently perturb acid-base and electrolyte status, intravascular volume, and rena... more Intoxications frequently perturb acid-base and electrolyte status, intravascular volume, and renal function. In selected cases, extracorporeal techniques effectively restore homeostasis and augment intoxicant removal. The use of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, is a new and effective treatment for patients exposed to toxic alcohols. In this section, practical approaches to commonly encountered intoxicants and the use of extracorporeal techniques are critically reviewed.
Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (... more Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.
Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt ... more Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress. Furthermore, active beta-catenin decreased Bax activation, oligomerization, and translocation to mitochondria, and reduced both organelle membrane injury and apoptosis. Dominant negative beta-catenin had the opposite effects. Because Akt regulates Bax, we examined the effects of the beta-catenin mutants on Akt expression and activation. Constitutively active beta-catenin increased Akt-1 expression and activation before and after stress, and treatment with a phosphatidylinositol-3 kinase inhibitor antagonized the protective effects of beta-catenin on Akt activation, Bax inhibition, and cell survival. In addition, beta-catenin significantly increased the rate of phosphorylation at Bax serine(184), an Akt-specific target. Taken together, these results suggest that beta-catenin/Wnt signaling promotes survival of renal epithelial cells after metabolic stress, in part by inhibiting Bax in a phosphatidylinositol-3 kinase/Akt-dependent manner.
Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic ... more Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, howe...
Background Hyponatremia associated with a low serum osmolality is a common and confounding electr... more Background Hyponatremia associated with a low serum osmolality is a common and confounding electrolyte disorder. Correcting hyponatremia is also complicated, especially in the setting of chronic hyponatremia. Here, we provide a rational approach to accurately detecting and safely treating acute on chronic euvolemic hyponatremia in the setting of acute polydipsia with a chronic reset osmostat. Case presentation A 71-year-old hispanic gentleman with chronic hyponatremia presented with hiccups, polydipsia, and a serum sodium concentration of 120 mEq/L associated with diffuse weakness, inattentiveness, and suicidal ideation. Symptomatic euvolemic hyponatremia warranted hypertonic saline treatment in the acute phase and water restriction in the chronic phase. Both interventions resulted in improvement in symptoms and/or the serum sodium concentration, but to a serum sodium level that persistently remained below the normal range. Remarkably, the urine osmolality appropriately fell when th...
Journal of the American Society of Nephrology, 2018
Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Diff... more Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target.Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target.Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that inte...
Effective lecturing stimulates learning, creates a verbal history for our profession, and is a ce... more Effective lecturing stimulates learning, creates a verbal history for our profession, and is a central basis for evaluating academic promotion. Unfortunately, few resources exist in the medical literature to guide the academician toward success as an effective lecturer. Using evidence-based principles, this review fosters adult learning in academic venues by incorporating the latest innovations in educational theory for both online and traditional teaching. The novice or advanced academic teacher will be guided toward critical self-evaluation of current teaching practices and encouraged to replace ineffective methods with ones more likely to be both rewarding and rewarded. By introducing literature-based learning techniques, emphasizing audience targeting, truncating content to an appropriate level of detail, effectively linking images and text, and accepting the brevity of learners' attentiveness, we show that the audience, not the speaker, is the primary educational focus.
in vivo and exposure to metabolic inhibitors in vitro cause mitochondrial membrane injury associa... more in vivo and exposure to metabolic inhibitors in vitro cause mitochondrial membrane injury associated with bax activation and apoptosis in proximal tubule epithelial. Hsp27, an inducible heat stress protein, reduces apoptosis in diverse cells types by an unknown mechanism. To evaluate the hypothesis that hsp27 inhibits Bax activation and apoptosis, renal epithelial cells (REC) were infected with adenovirus (AdV) containing human hsp27 prior to pharmacologic ischemia, an insult that activates bax and induces apoptosis. Hsp27 over-expression inhibited bax activation detected by a conformation specific antibody, reduced mitochondrial leakage of both cytochrome c and apoptosis inducing factor (AIF) and improved survival by >50% after 1-2 hr pharmacologic ischemia compared to empty vector. Immunoprecipitation (IP) did not detect hsp27-bax interaction before, during or after ischemia, suggesting that hsp27 does not directly regulate bax. To determine whether hsp27 indirectly antagonizes bax via Akt, a pro-survival serine-threonine kinase that phosphorylates and inactivates bax, p-ser 473 Akt (active) and total Akt content were compared at baseline and after ischemia in hsp27 over-expressing and in control cells. Hsp27 over-expression increased p-Akt content both at baseline and after ischemia compared to empty vector without altering total Akt. Hsp27 over-expression and ischemia selectively increased the interaction between hsp27 and Akt detected by IP. Hsp27 overexpression did not appear to alter the apparent half-life of p-ser 473 Akt during metabolic stress, an insult, which inactivates Akt, suggesting that hsp27 does not inhibit PP2-mediated Akt dephosphorylation. Ly294002 and wortmannin (two distinct Akt/PI3 kinase inhibitors) completely abrogated the pro-survival effect of hsp27, showing that Akt activation and bax inactivation are central to improved REC survival after injury. These data support the hypothesis that hsp27 indirectly inhibits bax-mediated mitochondrial injury and apoptosis after in vitro ischemia by facilitating Akt activation via a PI3 kinasedependent pathway. Both Akt and hsp27 represent potential new targets for improving REC survival after an ischemic insult.
Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeti... more Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeting the proximal tubule epithelial cell. The development of an effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we propose that RNAi signal pathway screening could identify a unifying mechanism of gentamicin-induced cell injury and suggest a therapeutic strategy to ameliorate it. Computational analysis of RNAi signal screens in gentamicin-exposed human proximal tubule cells suggested the cross-organelle stress response (CORE), the unfolded protein response (UPR), and cell chaperones as key targets of gentamicin-induced injury. To test this hypothesis, we assessed the effect of gentamicin on the CORE, UPR, and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-s...
We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediate... more We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPM-Bax complexes formed, and both NPM and Bax accumulated in mitochondria. Expression of a cytosol-restricted NPMmutant (NPM-NLS), but not a nucleus-restricted NPMmutant, increased NPM-Bax complex formation, mitochondrial NPM and Bax accumulation, mitochondrial membrane injury, caspase 3 activation, and ischemia-induced cell death. Coexpression of NPM-NLS with constitutively active Bax mutants caused nearly universal cell death in the absence of metabolic stress, whereas ex-pression of active Bax or NPM-NLS alone did not. A Bax peptide that disrupts NPM-Bax interaction significantly reduced cell death caused by exposure to metabolic inhibitors in vitro and preserved kidney function after ischemia in vivo. Thus, NPM-Bax interaction enhances mi...
Clinical Diabetes : A Publication of the American Diabetes Association, 2016
A 47-year-old woman with a self-reported 11-year history of diabetes mellitus presented with 2 da... more A 47-year-old woman with a self-reported 11-year history of diabetes mellitus presented with 2 days of nausea, vomiting, decreased oral intake, and back pain radiating to the neck. Her review of systems was remarkable for a “throbbing” headache of 1 day’s duration. Her medications included levothyroxine, subcutaneous long-acting insulin (glargine), topiramate, and canagliflozin, a selective sodium–glucose cotransporter 2 (SGLT2) inhibitor of the gloflozin class, which had been initiated 2 weeks earlier. Her medical history was remarkable for post-thyroidectomy Graves’ disease, cholecystectomy for multiple cholelithiasis, depression, fibromyalgia, and hyperlipidemia. She also had a history of spinal fusion surgery. In the emergency department, she appeared volume depleted. Her vital signs were temperature 98.9° F, blood pressure 118/76 mmHg, and a regular heart rate of 91 bpm. Her BMI was 27.45 kg/m2. Physical examination was remarkable for dry mucosal membranes, the absence of axill...
The mechanism by which the serine-threonine kinase glycogen synthase kinase-3 (GSK3 ) affects sur... more The mechanism by which the serine-threonine kinase glycogen synthase kinase-3 (GSK3 ) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3 promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3 , Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3 mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference–mediated knockdown of GSK3 promoted cell survival. Furthermore, RNA interference–mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3 . In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine site targeted by stress-activated ...
Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after... more Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton colla...
The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transien... more The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transient renal ischemia in vivo induces HSP-72, it is not known whether prior heat stress protects renal epithelial cells from injury mediated by ATP depletion. To evaluate this hypothesis, opossum kidney (OK) cells were exposed to sodium cyanide and 2-deoxy-D-glucose in the absence of medium glucose, a maneuver that reduced cell ATP content to < 10% of the control value within 10 min and decreased cell survival. One day after 2 h of ATP depletion, OK cells previously exposed to heat stress (to induce accumulation of HSP-72) exhibited marked improvement in survival (a > 4-fold increase in total DNA), less uptake of vital dye, and less release of lactate dehydrogenase (LDH) than cells subjected to ATP depletion alone (23.0 +/- 1.6 vs. 34.1 +/- 1.2% of total LDH, respectively). Enhanced clonogenicity post-heat stress was completely prevented by cycloheximide and positively correlated with t...
American journal of physiology. Renal physiology, Jan 31, 2016
Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure ... more Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure of proximal tubular epithelial cells to excess albumin promotes tubular atrophy and fibrosis, key predictors of progressive organ dysfunction. However, the link between proteinuria and tubular damage is unclear. We propose that pathologic albumin exposure impairs proximal tubular autophagy, an essential process for recycling damaged organelles and toxic intracellular macromolecules. In both mouse primary proximal tubule and immortalized human kidney cells, albumin exposure decreased the number of autophagosomes, visualized by the autophagosome-specific fluorescent markers monodansylcadaverine and GFP-LC3, respectively. Similarly, renal cortical tissue harvested from proteinuric mice contained reduced numbers of autophagosomes on electron micrographs and immunoblot showed reduced steady state LC3-II content. Albumin exposure decreased autophagic flux in vitro in a concentration-dependent ...
American journal of physiology. Renal physiology, Jan 21, 2016
Protein mimotopes or blocking peptides are small therapeutic peptides that prevent protein-protei... more Protein mimotopes or blocking peptides are small therapeutic peptides that prevent protein-protein interactions by selectively mimicking a native binding domain. Inexpensive technology facilitates straightforward design and production of blocking peptides in sufficient quantities to undertake preventive and therapeutic trials in both in vitro and in vivo experimental disease models. The kidney is an ideal peptide target since small molecules undergo rapid filtration and efficient bulk absorption by tubular epithelial cells. Compared to the blood stream, the half-life of peptides in the kidney is markedly prolonged, making blocking peptides an attractive tool for treating diverse renal diseases including ischemia, proteinuric states such as membranous glomerulonephritis (MGN) and focal and segmental glomerulosclerosis (FSGS) or renal cell carcinoma. Therapeutic peptides represent one of the fastest growing reagents classes for novel drug development in human disease partly due their ...
Intoxications frequently perturb acid-base and electrolyte status, intravascular volume, and rena... more Intoxications frequently perturb acid-base and electrolyte status, intravascular volume, and renal function. In selected cases, extracorporeal techniques effectively restore homeostasis and augment intoxicant removal. The use of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, is a new and effective treatment for patients exposed to toxic alcohols. In this section, practical approaches to commonly encountered intoxicants and the use of extracorporeal techniques are critically reviewed.
Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (... more Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.
Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt ... more Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress. Furthermore, active beta-catenin decreased Bax activation, oligomerization, and translocation to mitochondria, and reduced both organelle membrane injury and apoptosis. Dominant negative beta-catenin had the opposite effects. Because Akt regulates Bax, we examined the effects of the beta-catenin mutants on Akt expression and activation. Constitutively active beta-catenin increased Akt-1 expression and activation before and after stress, and treatment with a phosphatidylinositol-3 kinase inhibitor antagonized the protective effects of beta-catenin on Akt activation, Bax inhibition, and cell survival. In addition, beta-catenin significantly increased the rate of phosphorylation at Bax serine(184), an Akt-specific target. Taken together, these results suggest that beta-catenin/Wnt signaling promotes survival of renal epithelial cells after metabolic stress, in part by inhibiting Bax in a phosphatidylinositol-3 kinase/Akt-dependent manner.
Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic ... more Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, howe...
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Papers by Steven Borkan