Systematic Quality Improvement in Medicine: Everyone Can Do It

In this brief review, written from the perspective of a physician-leader who has fostered the development of comprehensive quality improvement efforts at two academic medical centers, I review the need for improvement, some conceptual barriers that must be overcome, the goals of a comprehensive quality improvement (QI) effort, some of the results we have obtained, and some observations on how to develop a culture of continuous improvement in an academic medical center. The mandate for quality improvement is clear; current healthcare is wasteful and error-prone, leading to excessive morbidity and mortality and unsustainably high costs. Successful quality improvement requires the abandonment of two paradigms: the craft model of medical practice and the notion that many forms of harm to patients are not preventable. I will describe how dramatic improvement has been achieved in reducing, by up to 10-fold, rates of central line infections, ventilator-associated pneumonias, peritonitis in peritoneal dialysis patients, and mortality due to cardiac arrest in hospital. I will describe as well how these methods can improve access to out-patient clinics dramatically and enhance the reliability and safety of hand-offs between covering physicians. To develop and maintain systematic quality improvement in all phases of medical care we must articulate a culture in which: everyone working at the medical center makes improvements every day; front-line staff, who know best how the work is done, are empowered to improve the processes of care; and multidisciplinary teams create the protocols that reduce variation that is due to physician preference, leaving only the variation required by the individual needs of patients. I will review as well the crucial elements of education of trainees and faculty members needed to guide and sustain a culture of quality. Finally, I will add some observations on how oversight boards and medical center leaders can help create systematic quality improvement in their medical centers

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (\u3cem\u3eSqualus acanthias\u3c/em\u3e) Shark

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver \u3e gill \u3e intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species

Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells

Abstract Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pH, was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pH, of MCD cells was 7.19±0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pH, recovery to the resting pH, over a 5-min timecourse. Depletion of >95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pH, recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pH, recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pH, recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/ H' exchanger, plays a role in proton transport and pH, regulation in rabbit MCD

Water and Deuterium Oxide Permeability through Aquaporin 1: MD Predictions and Experimental Verification

Determining the mechanisms of flux through protein channels requires a combination of structural data, permeability measurement, and molecular dynamics (MD) simulations. To further clarify the mechanism of flux through aquaporin 1 (AQP1), osmotic pf (cm3/s/pore) and diffusion pd (cm3/s/pore) permeability coefficients per pore of H2O and D2O in AQP1 were calculated using MD simulations. We then compared the simulation results with experimental measurements of the osmotic AQP1 permeabilities of H2O and D2O. In this manner we evaluated the ability of MD simulations to predict actual flux results. For the MD simulations, the force field parameters of the D2O model were reparameterized from the TIP3P water model to reproduce the experimentally observed difference in the bulk self diffusion constants of H2O vs. D2O. Two MD systems (one for each solvent) were constructed, each containing explicit palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) phospholipid molecules, solvent, and AQP1. It was found that the calculated value of pf for D2O is ∼15% smaller than for H2O. Bovine AQP1 was reconstituted into palmitoyl-oleoyl-phosphatidylcholine (POPC) liposomes, and it was found that the measured macroscopic osmotic permeability coefficient Pf (cm/s) of D2O is ∼21% lower than for H2O. The combined computational and experimental results suggest that deuterium oxide permeability through AQP1 is similar to that of water. The slightly lower observed osmotic permeability of D2O compared to H2O in AQP1 is most likely due to the lower self diffusion constant of D2O

Structural Determinants of Water Permeability through the Lipid Membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility

Salt Tolerance of Archaeal Extremely Halophilic Lipid Membranes

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments. Large unilamellar vesicles (LUV) prepared from the polar lipids of extreme halophiles, Halobacterium halobium and Halobacterium salinarum, retain entrapped carboxyfluorescein and resist aggregation in the whole range 0-4 m NaCl, similarly to LUV prepared from purified PGP-Me. By contrast, LUV made of polar lipid extracts from moderately halophilic and non-halophilic Archaea (Methanococcus jannaschii, Methanosarcina mazei, Methanobrevibacter smithii) are leaky and aggregate at high salt concentrations. However, adding PGP-Me to M. mazei lipids results in gradual enhancement of LUV stability, correlating with the PGP-Me content. The LUV data are substantiated by the x-ray results, which show that H. halobium and M. mazei lipids have dissimilar phase behavior and form different structures at high NaCl concentrations. H. halobium lipids maintain an expanded lamellar structure with spacing of 8.5-9 nm, which is stable up to at least 100 degrees C in 2 m NaCl and up to approximately 60 degrees C in 4 m NaCl. However, M. mazei lipids form non-lamellar structures, represented by the Pn3m cubic phase and the inverted hexagonal H(II) phase. From these data, the forces preventing membrane aggregation in halophilic Archaea appear to be steric repulsion, because of the large head group of PGP-Me, or possibly out-of-plane bilayer undulations, rather than electrostatic repulsion attributed to the doubly charged PGP-Me head group

Annexin A4 Reduces Water and Proton Permeability of Model Membranes but Does Not Alter Aquaporin 2–mediated Water Transport in Isolated Endosomes

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25°C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25°C, 31% at 37°C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25°C, 19.5% at 37°C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4

Biologically active two-dimensional crystals of aquaporin CHIP

Plasma membranes of several mammalian tissues are highly permeable to water due to the presence of CHIP, the 28-kDa channel-forming integral protein which is the archetypal member of the aquaporin family of water channel proteins. To define its native structure, purified red cell CHIP protein was reconstituted into lipid bilayers at a high protein-to-lipid ratio, and the resulting 3-microns diameter membrane vesicles were examined by high resolution electron microscopy. The reconstituted membranes contained highly ordered two-dimensional crystalline lattices of p422(1) symmetry in which each CHIP tetramer contained a central depression extending from the outer and inner surfaces of the membrane into the transbilayer domain of the molecule. The reconstituted membranes also exhibited extremely high osmotic water permeability, Pf = 0.472 cm/s, corresponding to the sum of activities of all incorporated CHIP molecules. These studies report the first two-dimensional crystallization of a biologically active water channel and provide direct evidence of the structure responsible for its pore-like behavior

SLE serum deposits C4d on red blood cells, decreases red blood cell membrane deformability, and promotes nitric oxide production

Objective Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBC). The aim of this study was to address whether this process affects the biophysical properties of RBC. Methods Serum and red blood cells were isolated from patients with SLE, and healthy controls. RBC from healthy O Rh negative individuals were incubated with SLE or control serum. We used flow cytometry to assess complement fragment deposition on RBC. RBC membrane deformability was measured using 2D microchannel arrays. Protein phosphorylation levels were quantified by western blot. Results Incubation of healthy donor RBC with sera from patients with SLE but not control sera led to deposition of C4 fragments on the RBC. Complement decorated RBC exhibited significant decrease in both membrane deformability and flickering. Sera from SLE patients triggered a transitory Ca++ influx in RBC that was associated with decreased phosphorylation of ?-spectrin, and increased phosphorylation of band 3, two key proteins of RBC cytoskeleton. Finally, SLE but not control sera led to the production of nitric oxide (NO) by RBC. Conclusion Our data suggest that complement activation in patients with SLE leads to calcium dependent cytosketeletal changes in RBC that render them less deformable, likely impairing their flow through capillaries. This phenomenon may negatively impact the delivery of oxygen to the tissues