Richard V. Paul

A protocol-based treatment for intradialytic hypotension in hospitalized hemodialysis patients

Human serum albumin is used in hemodialysis (HD) units as treatment for hypotension despite its high cost and undetermined efficacy. During a 4-month period in 1995, albumin was used in 22% of 1,296 consecutive HD treatments in the HD unit or intensive care units (ICUs) at our tertiary-care hospital. We evaluated the safety and efficacy of a protocol designed to minimize albumin use for treating HD-associated hypotension (HDAH). The protocol consisted of the stepwise use of saline, mannitol, and albumin for the purpose of achieving physician-determined ultrafiltration goals. Patients were exempted from receiving the protocol for age younger than 18 years, freshly declotted angioaccess, or cardiovascular instability. The protocol was evaluated prospectively in 2,559 consecutive dialysis sessions (15% in ICUs) in 442 patients. Hypotension occurred during 608 sessions (24%), and attending nephrologists elected to initiate the protocol in 71% of these cases. Of the 433 instances in which the protocol was begun, reversal of hypotension was achieved without the need for albumin in 91% and with the addition of albumin in an additional 2%. Protocol treatment was not completed because of nursing error in 1% or clotting of filter or angioaccess in 4%. Use of the protocol failed to reverse hypotension in only 2% of the cases in which it was completed. Albumin was administered in only 6% of the 2,559 HD treatments. In summary, our protocol-based approach to HDAH was effective, easy for nurses to use, albumin sparing, and cost reducing.

Regulation of vascular angiotensin II receptors by EGF

After vascular endothelial injury, angiotensin II (ANG II) plays a role in the resulting hypertrophic response, and expression of epidermal growth factor (EGF) is enhanced. Therefore, we tested the possibility that EGF regulates vascular ANG II action and receptor expression. Incubation of cultured aortic vascular smooth muscle cells (VSMC) with EGF (or basic fibroblast growth factor but not platelet-derived growth factor isoforms) resulted in concentration-dependent (1-50 ng/ml EGF), time-dependent (>8 h), and reversible decreases in ANG II surface receptor density. For example, a 50% reduction was observed after exposure to 50 ng/ml EGF for 24 h. Incubation of cultured VSMC with 50 ng/ml EGF for 24 h resulted in a 77% reduction in ANG II-stimulated inositol phosphate formation. EGF not only prevented but also reversed ANG II receptor upregulation by 100 nM corticosterone. The specific tyrosine kinase inhibitor tyrphostin A48 (50 microM) reduced EGF-stimulated thymidine incorporation and EGF-stimulated phosphorylation of mitogen-activated protein kinase but did not prevent EGF from reducing ANG II receptor density. Neither pertussis toxin (100 ng/ml) nor downregulation of protein kinase C by phorbol myristate acetate (100 nM for 24 h) prevented EGF from reducing ANG II receptor density. In summary, EGF is a potent negative regulator of vascular ANG II surface receptor density and ANG II action by mechanisms that do not appear to include tyrosine phorphorylation, pertussis toxin-sensitive G proteins, or phorbol ester-sensitive protein kinase C. The possibility that EGF shifts the cell culture phenotype to one that exhibits reduced surface ANG II density cannot be eliminated by the present studies.After vascular endothelial injury, angiotensin II (ANG II) plays a role in the resulting hypertrophic response, and expression of epidermal growth factor (EGF) is enhanced. Therefore, we tested the possibility that EGF regulates vascular ANG II action and receptor expression. Incubation of cultured aortic vascular smooth muscle cells (VSMC) with EGF (or basic fibroblast growth factor but not platelet-derived growth factor isoforms) resulted in concentration-dependent (1-50 ng/ml EGF), time-dependent (>8 h), and reversible decreases in ANG II surface receptor density. For example, a 50% reduction was observed after exposure to 50 ng/ml EGF for 24 h. Incubation of cultured VSMC with 50 ng/ml EGF for 24 h resulted in a 77% reduction in ANG II-stimulated inositol phosphate formation. EGF not only prevented but also reversed ANG II receptor upregulation by 100 nM corticosterone. The specific tyrosine kinase inhibitor tyrphostin A48 (50 μM) reduced EGF-stimulated thymidine incorporation and EGF-stimulated phosphorylation of mitogen-activated protein kinase but did not prevent EGF from reducing ANG II receptor density. Neither pertussis toxin (100 ng/ml) nor downregulation of protein kinase C by phorbol myristate acetate (100 nM for 24 h) prevented EGF from reducing ANG II receptor density. In summary, EGF is a potent negative regulator of vascular ANG II surface receptor density and ANG II action by mechanisms that do not appear to include tyrosine phorphorylation, pertussis toxin-sensitive G proteins, or phorbol ester-sensitive protein kinase C. The possibility that EGF shifts the cell culture phenotype to one that exhibits reduced surface ANG II density cannot be eliminated by the present studies.

Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP

Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a β-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable β-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.

Mechanisms of vascular angiotensin II surface receptor regulation by epidermal growth factor

We investigated mechanisms by which epidermal growth factor (EGF) reduces angiotensin II (AngII) surface receptor density and stimulated actions in vascular smooth muscle cells (VSMC). EGF downregulated specific AngII radioligand binding in intact cultured rat aortic smooth muscle cells but not in cell membranes and also inhibited AngII‐stimulated contractions of aortic segments. Inhibitors of cAMP‐dependent kinases, PI‐3 kinase, MAP kinase, cyclooxygenase, and calmodulin did not prevent EGF‐mediated downregulation of AngII receptor binding, whereas the EGF receptor kinase inhibitor AG1478 did. Total cell AngII AT1a receptor protein content of EGF‐treated and untreated cells, measured by immunoblotting, did not differ. Actinomycin D or cytochalasin D, which interacts with the cytoskeleton, but not the protein synthesis inhibitor cycloheximide, prevented EGF from downregulating AngII receptor binding. Consistently, EGF inhibited AngII‐stimulated formation of inositol phosphates in the presence of cycloheximide but not in the presence of actinomycin D or cytochalasin D. In conclusion, EGF needs an intact signal transduction pathway to downregulate AngII surface receptor binding, possibly by altering cellular location of the receptors. Published 2004 Wiley‐Liss, Inc.

Correction of metabolic alkalosis by potassium chloride in ectopic adrenocorticotropic hormone syndrome

A 57-year-old white man presented with metabolic alkalosis, hypokalemia (pH 7.58, HCO3 >50 mEq/L, serum K 1.8 mEq/L) and hypertension. The initial evaluation was significant for markedly elevated serum cortisol and adrenocorticotropic hormone (ACTH) level; neither hormone showed circadian rhythm or suppression with high-dose dexamethasone. Perihilar and supraclavicular masses were found to consist of undifferentiated small cell carcinoma. Ectopic ACTH syndrome was diagnosed. In spite of progressively rising hormone levels (ACTH, 723 pg/dL; and cortisol, 212 microgram/dL), his severe metabolic alkalosis was largely corrected by aggressive treatment with potassium chloride alone. Possible mechanisms of these clinical findings are discussed.

Clinical Disorders of Fluid and Electrolyte Metabolism

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