Use of renin angiotensin system inhibitors in patients with chronic kidney disease.

Current guidelines recommend renin angiotensin system inhibitors (RASI) as key components of treatment of hypertension in patients with chronic kidney disease (CKD), because of their effect on reducing the future rate of loss of glomerular filtration rate (GFR). A common risk of RASI in CKD is a haemodynamically mediated, and reversible, fall in GFR of varying severity and duration, any time after commencement of the Inhibitors. A benefit of the acute reduction in filtration rate with RASI may be a reduction in the future rate of loss in GFR: the greatest benefit likely to be in those patients with a greater rate of loss of GFR prior to, and a lesser acute loss of GFR after, introduction of RASI; and in those patients with significant proteinuria. An acute loss of GFR of >25% following the introduction of RASI is an indication to cease the RASI. An acute loss of GFR < 25% requires consideration of the likely risks of the lower GFR and benefits of any future reduced rate of loss of GFR. A fall in GFR in patients while on RASI is usually associated with a remediable cause. When the cause for the fall in GFR is not revealed, and the fall is less than 25%, hopeful expectancy is recommended. Hyperkalaemia in patients with CKD on RASI is more common with more severe disease, potassium retaining diuretics and hypoaldosteronism. Treatment should be modified to maintain a plasma potassium <6 mmol/L.

A justification for less restrictive guidelines on the use of metformin in stable chronic renal failure.

AIM: The aim was to justify less restrictive use of metformin in stable chronic renal failure, because a literature review reveals metformin is associated with a significantly lower incidence of cardiovascular events and mortality compared with other hypoglycaemic agents, and metformin-associated lactic acidosis is rare and causation uncertain. Studies on intentional metformin overdose and metformin bioavailability, renal clearance and plasma metformin in renal impairment provide evidence in support of a less restrictive use of metformin. METHODS: In metformin overdose (n = 22), lactic acidosis was not inevitable with a plasma metformin > 40 mg/l (therapeutic level c. 1 mg/l): Severe lactic acidosis (pH ≤ 7.21, plasma lactate ≥ 11 mmol/l, n = 8) did not occur unless plasma metformin was > 40 mg/l. Plasma lactate was a more consistent predictor of pH than plasma metformin, with plasma lactate ≤ 4.7 being associated with a pH ≥ 7.34. A likely 'safe' plasma lactate is < 3.5 mmol/l and plasma metformin < 10 mg/l. RESULTS: Plasma metformin can be predicted from estimated glomerular filtration rate and metformin dose. Reported plasma metformin in renal failure was always less than predicted plasma metformin. Predicted plasma metformin (mg/l), with an estimated glomerular filtration rate of 30 ml/min and metformin 2000 mg/day was 6.8; an estimated glomerular filtration rate of 20 ml/min and metformin 1500 mg/day was 5.1; an estimated glomerular filtration rate of 10 ml/min and metformin 500 mg/day was 4.4. CONCLUSION: Metformin accumulates in renal failure and, although accumulation does not always lead to lactic acidosis, dose modification to achieve a predicted plasma metformin < 10 mg/l is suggested. As plasma metformin is not routinely available, plasma lactate should be useful in monitoring the use of metformin in renal failure.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTPγS or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.

A small ribozyme with dual-site kinase activity.

Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-(32)P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5' target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5' mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation.

Utility, or not, of estimates of glomerular filtration rate in modifying drug dosage, with particular reference to enoxaparin.

A reduced clearance of some drugs in renal failure is a problem, particularly with drugs that are excreted by the kidney substantially unmetabolised and also have significant toxicity and a low therapeutic ratio. The problem is compounded by the significant inaccuracy of estimated glomerular filtration rate (eGFR). The aim was to develop general recommendations to reduce the risk of drug toxicity in renal failure, with particular reference to enoxaparin. The substantial inaccuracies in eGFR (eGFR in 32% of patients is different from measured GFR by 20-30%) are compounded when using a dichotomous decision tree (renal failure or not). As the eGFR approaches the GFR decision boundary, for classification as renal failure or not, misclassification approaches 50%. Recommendations, when patients are at risk, include the following: acknowledge inaccuracies of eGFR, particularly in anthropometrically diverse populations; measure drug levels wherever possible; realise that drug levels after early doses relate more to volume of distribution, rather than renal function, allowing time for modification of the drug dose; where accurate urine collection is feasible, use creatinine clearance as an estimate of GFR; and use eGFR as a (more) continuous, rather than dichotomous, variable to adjust dosage, exampled by enoxaparin.

Simulation of medical emergencies in dental practice: development and evaluation of an undergraduate training programme.

BACKGROUND: The evidence available suggests that many dentists on graduation do not feel competent managing medical emergencies; a problem requiring improved undergraduate training. This study developed a comprehensive simulation based training programme for final year undergraduate dental students and assessed student attitudes towards training. METHODS: Final year dental students (n = 52) from The University of Melbourne were required to complete simulation training incorporating an interactive tutorial and realistic, simulated emergency scenarios conducted in the students' real clinical environment. A post-participation questionnaire utilizing a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree) assessed student attitudes. RESULTS: Student responses supported simulation training, evidenced by the following selected questionnaire responses: achieved greater confidence in managing emergencies 4.65 ± 0.48 (n = 52); prefer lecture to simulation 1.46 ± 0.74 (n = 52); simulation training is important in undergraduate teaching 4.86 ± 0.35 (n = 43). CONCLUSIONS: Realistic simulation training in management of medical emergencies for dental students is an effective adjunct to traditional lecture style teaching. Given the importance of this subject, this mode of training would benefit students if incorporated into undergraduate dental courses.

Fibromuscular dysplasia presenting with bilateral renal infarction.

Fibromuscular dysplasia (FMD) describes a group of conditions which cause nonatheromatous arterial stenoses, most commonly of the renal and carotid arteries, typically in young women. We report a rare case of bilateral segmental renal infarction secondary to FMD in a young male patient. His initial presentation with loin pain and pyrexia resulted in a delay in the definitive diagnosis of FMD. He was successfully treated with bilateral balloon angioplasty. The delayed diagnosis in this patient until the condition had progressed to bilateral renal infarcts highlights the need for prompt investigation and diagnosis of suspected cases of FMD.

Functional hepatocyte cation compartmentation demonstrated with 133Cs NMR.

This study utilized the large intrinsic chemical shift range of (133)Cs, a potassium congener, in an NMR study of intracellular cation distribution. It demonstrates two distinct intracellular environments in isolated perfused hepatocytes from cesium-fed rats, evident as compartments with different (133)Cs chemical shifts and containing different proportions of total detected cesium. The chemical shifts of the two intracellular compartments were 2.44 +/- 0.07 and 1.21 +/- 0.18 ppm, relative to the cesium signal from the perfusate. The observation of two distinct intracellular cesium signals suggests slow exchange on an NMR chemical shift time-scale (k exchange > 0.02 s). The area of the high-frequency component represented 62 +/- 10% (N = 12) of the total intracellular cesium signal. Manipulation of the intracellular environment using anoxia with aglycemia or digitonin produced changes in the distribution between the two intracellular compartments, showing their dynamic nature. Changes measured in association with metabolic manipulation suggest cytoplasm and mitochondria as the origin of the high and low-frequency intracellular peaks, respectively.

Comorbidities and prediction of length of hospital stay.

BACKGROUND: Variability in length of stay (LOS) within Australian National Diagnosis Related Groups (AN-DRGs) reflects clinical heterogeneity in age, severity of illness, complications and comorbidities. AIM: To develop a clinically based score which measures patient morbidity and which will better predict LOS compared to existing methods. METHODS: ICD-9-CM codes of diseases and procedures were allocated to one of 23 body system categories to calculate the body burden of disease (BBD) score. Evaluation of BBD in predicting LOS was performed using multiple regression and analysis of variance with a data set of 34,079 cases from 75 AN-DRGs from three Victorian hospitals. RESULTS: Adding BBD and age improved prediction of LOS by 27.2% in AN-DRG version 1.0 and by 17.5% in AN-DRG version 3.1. When using average inlier LOS for AN-DRG, BBD and age improved prediction of LOS by 44.6% and by 14.8% in AN-DRG version 1.0 and version 3.1 respectively. Deaths were positively related to BBD. CONCLUSIONS: BBD is a simple quantitative measure of extent of disease that improves current methods in accounting for variability in LOS.

Involvement of both the classical and alternate pathways of complement in an ex vivo model of xenograft rejection.

BACKGROUND: It is now generally accepted that complement activation is critical for the hyperacute rejection of xenografts. Activation of the classical pathway as the result of the interaction of xenoreactive IgM xenoantibodies with the vascular endothelium has been observed in all species combinations examined to date. A number of studies using a variety of species combinations have also implicated alternate pathway involvement; however, these studies do not enable a conclusion to be drawn as to whether the alternate pathway can be activated in the complete absence of classical pathway activation. METHODS: In this study, human plasma was depleted of both Clq and factor D and then reconstituted with purified Clq or factor D to restore the classical and alternate complement pathways, respectively. The ability of these modified plasmas to prosecute hyperacute rejection was then examined using an ex vivo isolated mouse heart perfusion model based on the Langendorff system. RESULTS AND CONCLUSIONS: In the mouse to human species combination, both the classical and alternate pathways of complement are independently capable of initiating complement activation and mediating xenograft rejection.

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.

133Cs relaxation times in rat tissues.

133Cs relaxation-time studies of tissues from rats into which cesium has been incorporated by dietary loading have been carried out in vivo and in vitro. Whereas tissue T1 values are on the order of seconds, T2 values are as low as a few tens of milliseconds. 133Cs tissue relaxation times are analogous to those of 39K in the same tissues, but are more readily measured because of the greater sensitivity of 133Cs compared with 39K. T1 and T2 data of excised tissue at two resonance frequencies (65.60 and 39.37 MHz) and temperatures (302 and 278K) have been analyzed in terms of a general description of spin-7/2 relaxation. The results are consistent with most of the cesium ions being in a free state, undergoing fast exchange with bound ions having long correlation times located in one or more intracellular compartments.

Clusterin depletion enhances immune glomerular injury in the isolated perfused kidney.

Clusterin is a normal plasma protein, shown to be an inhibitor of reactive complement hemolysis and a component of the fluid phase SC5b-9 terminal complement complexes. It is a component of glomerular immune deposits in human and experimental glomerulonephritis. Using the complement-dependent isolated perfused rat kidney model of autologous phase passive Heymann nephritis, we have studied the effect of clusterin depletion of perfused plasma on the development of glomerular injury. Kidneys with planted glomerular sheep anti-rat Fx1A antibody were perfused with human plasma either depleted of clusterin to < or = 30%, or control plasma depleted of plasma fibronectin. Glomerular injury was then initiated by the addition of guinea pig anti-sheep immunoglobulins to the perfusate. Kidneys perfused with clusterin depleted plasma developed significantly greater proteinuria at all time points when compared to control kidneys. Glomerular antibody binding and C3 deposition were similar in the two groups, but terminal complement components were deposited in larger amounts in the clusterin depleted group. These data support a possible role for clusterin in vivo in the protection of complement-induced glomerular injury.

The use of dietary loading of 133Cs as a potassium substitute in NMR studies of tissues.

133Cs NMR chemical shifts and relaxation times have been measured for tissue samples in vitro and in vivo from rats which have been fed on a high cesium, low potassium diet, which leads to a predominantly intracellular distribution of this ion, similar to that of K+. The high sensitivity, large chemical shift range, and narrow linewidths of 133Cs, compared with 39K, allow chemical shift differences to be observed between tissues, and in subcellular organelles such as mitochondria. For example, in vitro tissue chemical shifts, relative to 150 mM CsCl, are 1.06 +/- 0.11 ppm for liver, 0.02 +/- 0.05 ppm for brain, 1.76 +/- 0.20 ppm for erythrocytes, and -0.13 +/- 0.02 ppm for plasma. T1 and spin-echo T2 values range from 1.26 +/- 0.05 s (T1), and 0.028 +/- 0.006 s (T2) for liver, to 6.49 +/- 0.19 s and 1.12 +/- 0.03 s for plasma. 133Cs relaxation times show the same relative trends between tissues as are observed in 39K tissue studies.

Altered blood pressure, renal tubular function and levels of circulating inhibitors of Na-K-ATPase in mature SHR exposed to low salt diet in the perinatal period.

1. It has been shown previously that low salt diet, confined to the perinatal period only, reduces blood pressure (BP) and sodium retention in spontaneously hypertensive rat (SHR) offspring at maturity when compared with those given high salt diet. 2. In this study it is shown that such high salt diets are associated with an increased level of circulating Na-K-ATPase inhibitor (CINK) activity. 3. Animals given perinatal high salt diet have a significantly greater tubular reabsorptive capacity when compared with those given low salt diet. 4. The finding of a high level of circulating Na-K-ATPase inhibitory material in the face of increased renal tubular capacity and blood pressure suggests that while this inhibitory material may play a role in the elevated blood pressure of animals given high salt diet, it cannot cause the elevated rate of fluid reabsorption.

NMR measurement of 39K detectability and relaxation constants in rat tissue.

Differences in the NMR detectability of 39K in various excised rat tissues (liver, brain, kidney, muscle, and testes) have been observed. The lowest NMR detectability occurs for liver (61 +/- 3% of potassium as measured by flame photometry) and highest for erythrocytes (100 +/- 7%). These differences in detectability correlate with differences in the measured 39K NMR relaxation constants in the same tissues. 39K detectabilities were also found to correlate inversely with the mitochondrial content of the tissues. Mitochondria prepared from liver showed greatly reduced 39K NMR detectability when compared with the tissue from which it was derived, 31.6 +/- 9% of potassium measured by flame photometry compared to 61 +/- 3%. The detectability of potassium in mitochondria was too low to enable the measurement of relaxation constants. This study indicates that differences in tissue structure, particularly mitochondrial content are important in determining 39K detectability and measured relaxation rates.

Effects of opiates on sodium excretion in the isolated perfused rat kidney.

1. A rat isolated perfused kidney preparation was utilized to define clearly a renal site of action. The variables measured were perfusate pressure and flow, glomerular filtration rate, urine volume, sodium excretion and potassium excretion. 2. Dextromethorphan (3 nmol/L) and dextrorphan (10 nmol/L) reduced sodium excretion in kidneys from rats on either control or high K+ diet, in the absence of any other measured renal effects. Dextromethorphan (10 nmol/L) produced a decrease in glomerular filtration rate as well as a decrease in sodium excretion. Naloxone (1 mumol/L) inhibited the effect of dextromethorphan on sodium excretion but had no effect when administered alone. 3. The levorotatory opiates levorphanol and levomethorphan, the kappa agonist ketocyclazocine and a range of other opiates had no effect on sodium excretion. 4. The results suggest a renal action specific for dextrorotatory opiates. This renal action is consistent with earlier binding studies suggesting preferential recognition of dextrorotatory opiates.