David Roth

A More Accurate Method To Estimate Glomerular Filtration Rate from Serum Creatinine: A New Prediction Equation

The glomerular filtration rate (GFR) is traditionally considered the best overall index of renal function in health and disease (1). Because GFR is difficult to measure in clinical practice, most clinicians estimate the GFR from the serum creatinine concentration. However, the accuracy of this estimate is limited because the serum creatinine concentration is affected by factors other than creatinine filtration (2, 3). To circumvent these limitations, several formulas have been developed to estimate creatinine clearance from serum creatinine concentration, age, sex, and body size (4-12). Despite more recent studies that have related serum creatinine concentration to GFR (13-24), no formula is more widely used to predict creatinine clearance than that proposed by Cockcroft and Gault (4). This formula is used to detect the onset of renal insufficiency, to adjust the dose of drugs excreted by the kidney, and to evaluate the effectiveness of therapy for progressive renal disease. More recently, it has been used to document eligibility for reimbursement from the Medicare End Stage Renal Disease Program (25) and for accrual of points for patients on the waiting list for cadaveric renal transplantation (26). Major clinical decisions in general medicine, geriatrics, and oncology (as well as nephrology) are made by using the Cockcroft-Gault formula and other formulas to predict the level of renal function. Therefore, these formulas must predict GFR as accurately as possible. The Modification of Diet in Renal Disease (MDRD) Study, a multicenter, controlled trial, evaluated the effect of dietary protein restriction and strict blood pressure control on the progression of renal disease (27-30). During the baseline period, GFR, serum creatinine, and several variables that affect the relation between them were measured in patients with chronic renal disease. The purpose of our study was to develop an equation from MDRD Study data that could improve the prediction of GFR from serum creatinine concentration. Methods Baseline Cohort and Measurement Methods in the Modification of Diet in Renal Disease Study The overall study design and methods of recruitment for the MDRD Study have been described elsewhere (31, 32). A total of 1785 patients entered the baseline period. Of these patients, 1628 (91%) also underwent measurement of GFR and the other variables described below; these patients constitute the study group for these analyses. Glomerular filtration rate was measured as the renal clearance of 125I-iothalamate (33, 34). Creatinine clearance was computed from creatinine excretion in a 24-hour urine collection and a single measurement of serum creatinine. Serum and urine creatinine were measured by using a kinetic alkaline picrate assay with a normal range in serum of 62 to 124 mol/L (0.7 to 1.4 mg/dL) (35). Glomerular filtration rate and creatinine clearance were expressed per 1.73 m2 of body surface area by multiplying measured values by 1.73/body surface area (36). The serum and urine specimens were also used for other measurements, including serum albumin (bromcresol green method [35]), serum urea nitrogen (urease method [35]), and urine urea nitrogen (urease method [35]). Protein intake (g/d) was estimated as 6.25 [UUN (g/d) + 0.031 (g/kg per day) SBW (kg)], where UUN is urine urea nitrogen, SBW is standard body weight, and 0.031 g/kg per day is a constant reflecting the rate of excretion of nitrogen in compounds other than urine urea (37, 38). The diagnosis of diabetes and the cause of renal disease were assigned on the basis of chart review at the clinical center (39). Statistical Analysis Descriptive Statistics The relation of renal function measurements to other baseline characteristics was assessed by using contingency tables, t-tests, analysis of variance, and linear regression, as appropriate. Nonparametric tests (Wilcoxon rank-sum tests and Kruskal-Wallis tests) gave consistent results. A P value less than 0.01 was considered statistically significant. Multivariable Analysis of Glomerular Filtration Rate We used stepwise multiple regression to determine a set of variables that jointly predicted GFR. The stepwise regression models were developed by using a training sample consisting of a random sample of 1070 of the 1628 patients. We found that the variability of the difference between the observed and predicted GFR values was greater for higher GFR values. This increase was eliminated by performing multiple regressions on log-transformed data. To facilitate clinical interpretation, the results were re-expressed in terms of the original units. Consequently, the prediction equation is a multiplicative model; regression coefficients refer to the change in geometric mean GFR associated with unit changes in the independent variable. Predicted GFR is expressed in mL/min per 1.73 m2. The following variables were considered for possible inclusion in the regression model: weight, height, sex, ethnicity, age, diagnosis of diabetes, serum creatinine concentration, serum urea nitrogen level, serum albumin level, serum phosphorus level, serum calcium level, mean arterial pressure, urine creatinine level, urine urea nitrogen level, urine protein level, and urine phosphorus level. The cause of renal disease was not included because in clinical practice, the cause may be unknown or clinicians may not use the same classification method as the investigators in the MDRD Study. A P value less than 0.001 was used as the criterion for entry of a variable into the model. Because of the difficulty in collecting complete 24-hour urine samples in clinical practice, an additional stepwise regression was performed to develop a prediction model that did not include urine biochemistry variables. Finally, because of the interest in developing a prediction equation to assess eligibility for Medicare reimbursement and listing for cadaveric renal transplantation, we repeated the analysis restricting the population to the subgroup of patients with higher serum creatinine concentrations (>221 mol/L [2.5 mg/dL]; n=509 in the training sample). Methods for Comparing Equations To Predict Glomerular Filtration Rate We first developed coefficients for each prediction equation (including the selection of the predictor variables for the stepwise regressions) using the data from the training sample to predict log GFR. Each prediction equation also included a multiplicative constant to account for any consistent bias in the application of that equation in the MDRD Study Group. This was particularly important for equations that are intended to estimate creatinine clearance, which is known to be higher than GFR. The regression coefficients determined in the training sample were then applied to obtain predicted GFRs in a separate validation sample consisting of the remaining 558 patients (172 patients with serum creatinine concentration>221 mol/L [2.5 mg/dL]). These predicted GFR values were compared with the actual GFRs in the validation sample to evaluate the performance of each prediction equation. In this way, separate data sets were used to construct the equations and assess their accuracy after removal of systematic bias. For each equation, we computed overall R 2 (percentage of variability in log GFR explained by the regression model) and the 50th, 75th, and 90th percentiles of the distribution of the percentage absolute difference between measured and predicted GFRs in the validation sample. The 50th percentiles indicate the typical size of the errors in prediction of GFR, and the 75th and 90th percentiles assess the sizes of the larger errors that occurred for each model. Development of Final Prediction Equations To improve the accuracy of the final MDRD Study prediction equations, the regression coefficients derived from the training sample were updated on the basis of data from all 1628 patients. As a result, the standard errors of the regression coefficients in the final MDRD Study prediction equations are slightly smaller than those derived from the training sample; thus, the accuracy of the final prediction equations may be slightly better (by about 0.1% to 0.2%) than their accuracy as assessed in the validation sample. Results Demographic and Clinical Characteristics The mean age ( SD) of the cohort was 50.6 12.7 years. Sixty percent of patients were male, 88% were white, and 6% were diabetic. Causes of renal disease were glomerular disease (32%), polycystic kidney disease (22%), tubulointerstitial disease (7%), and other or unknown renal diseases (40%). Mean protein intake was 0.99 0.24 g/kg of body weight per day and mean arterial pressure was 99.4 12.2 mm Hg. Mean weight was 79.6 16.8 kg, body surface area was 1.91 0.23 m2, serum urea nitrogen concentration was 11.4 5.7 mmol/L [32 16 mg/dL], and serum albumin concentration was 40.0 4.0 g/L [4.0 0.4 g/dL], respectively. Glomerular Filtration Rate, Creatinine Clearance, and Serum Creatinine Concentration Renal function measurements for the study group and for various subgroups are shown in Table 1. Mean GFR for the population was 0.38 mL s 2 m 2 (39.8 mL/min per 1.73 m2), with lower values in patients with lower protein intake, white patients compared with black patients, and older patients ( 55 years) compared with younger patients (P<0.01). The mean value of creatinine clearance was 0.81 mL s 2 m 2 (48.6 mL/min per 1.73 m2) and was lower in older patients and patients with lower protein intake (P 0.01). The mean serum creatinine concentration was 203 mol/L (2.3 mg/dL) and was higher in men, patients with lower protein intake, and patients with higher mean arterial pressure (P 0.01). Figure 1 shows the well-known reciprocal relation of serum creatinine concentration to GFR for subgroups based on sex and ethnicity. At any given GFR, the serum creatinine concentration is significantly higher in men than in women and in black persons than in white persons (P<0.001). Table 1. Association of Renal Fu

Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte β3 integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte β3 integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR–β3 integrin interaction through antibodies and small molecules targeting either uPAR or β3 integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS.

Grazoprevir plus elbasvir in treatment-naive and treatment-experienced patients with hepatitis C virus genotype 1 infection and stage 4–5 chronic kidney disease (the C-SURFER study): a combination phase 3 study

BACKGROUND Chronic hepatitis C virus (HCV) infection in patients with stage 4-5 chronic kidney disease increases the risk of death and renal graft failure, yet patients with hepatitis C and chronic kidney disease have few treatment options. This study assesses an all-oral, ribavirin-free regimen in patients with HCV genotype 1 infection and stage 4-5 chronic kidney disease. METHODS In this phase 3 randomised study of safety and observational study of efficacy, patients with HCV genotype 1 infection and chronic kidney disease (stage 4-5 with or without haemodialysis dependence) were randomly assigned to receive grazoprevir (100 mg, NS3/4A protease inhibitor) and elbasvir (50 mg, NS5A inhibitor; immediate treatment group) or placebo (deferred treatment group) once daily for 12 weeks. Randomisation was done centrally with an interactive voice response system. An additional cohort of patients who were not randomised received the same regimen open-label and underwent intensive pharmacokinetic sampling. The primary efficacy outcome was a non-randomised comparison of sustained virological response at 12 weeks (SVR12) after the end of therapy for the combined immediate treatment group and the pharmacokinetic population with a historical control. The primary safety outcome was a randomised comparison between the immediate treatment group and the deferred treatment group. After 4 weeks of follow-up (study week 16), unmasking occurred and patients in the deferred treatment group received grazoprevir and elbasvir. The primary efficacy hypothesis was tested at a two-sided significance level (type I error) of 0·05 using an exact test for a binomial proportion. Safety event rates were compared between immediate treatment and deferred treatment groups using the stratified Miettinen and Nurminen method with baseline dialysis status as the strata. The study is registered at ClinicalTrials.gov, number NCT02092350. FINDINGS 224 patients were randomly assigned to the immediate treatment group with grazoprevir and elbasvir (n=111) or the deferred treatment group (n=113), and 11 were assigned to the intensive pharmacokinetic population. Overall, 179 (76%) were haemodialysis-dependent, 122 (52%) had HCV genotype 1a infection, 189 (80%) were HCV treatment-naive, 14 (6%) were cirrhotic, and 108 (46%) were African American. Of the 122 patients receiving grazoprevir and elbasvir, six were excluded from the primary efficacy analysis for non-virological reasons (death, lost-to-follow-up [n=2], non-compliance, patient withdrawal, and withdrawal by physician for violent behaviour). No patients in the combined immediate treatment group and intensive pharmacokinetic population and five (4%) in the deferred treatment group discontinued because of an adverse event. Most common adverse events were headache, nausea, and fatigue, occurring at similar frequencies in patients receiving active and placebo drugs. SVR12 in the combined immediate treatment group and intensive pharmacokinetic population was 99% (95% CI 95·3-100·0; 115/116), with one relapse 12 weeks after end of treatment when compared with a historical control of 45%, based on meta-analyses of interferon-based regimens used in clinical trials of patients infected with HCV who are on haemodialysis. INTERPRETATION Once-daily grazoprevir and elbasvir for 12 weeks had a low rate of adverse events and was effective in patients infected with HCV genotype 1 and stage 4-5 chronic kidney disease. FUNDING Merck Sharp & Dohme Corp.

Unexpected augmentation of mycophenolic acid pharmacokinetics in renal transplant patients receiving tacrolimus and mycophenolate mofetil in combination therapy, and analogous in vitro findings

Mycophenolate mofetil (MMF) a potent immunosuppressive agent, has recently been approved for clinical use (CellCept) in renal transplant patients in combination with cyclosporine (CsA). With the expanded use of tacrolimus (Prograf) as well in renal transplant patients, there is a lack of pharmacokinetic studies clarifying drug interactions between the three agents. A pharmacokinetic study was performed on 18 stable renal transplant patients receiving MMF and tacrolimus together, and four control groups, one receiving tacrolimus alone, two receiving CsA, in combination with MMF (1.0 or 1.5 g bid), and one receiving CsA microemulsion (Neoral). Area-under-the-curve values were calculated for each drug to assess if there was a reciprocal effect on the respective bioavailability of each. In vitro, the immunosuppressive effect of trough level plasma from each patient group was studied using mixed lymphocyte culture (MLC), as well as MLC reactions spiked with various combinations of each drug. There was a minimal effect of MMF on tacrolimus pharmacokinetics. However, patients receiving tacrolimus and MMF displayed significantly higher levels (Cmin and area under the curve) of mycophenolic acid (MPA) than those receiving CsA (Sandimmune or Neoral) and the same dose of MMF (50.2 +/- 16.5 vs 32.1 +/- 16.7 micrograms h/ml AUC, p < 0.02). Equivalent MPA levels could be attained in patients receiving CsA if the MMF dose was increased by 50% (1.5 g bid). There were also significantly lower levels of the glucuronide metabolite of MPA (MPAG) (755 +/- 280 vs 1230 +/- 250 micrograms h/ml AUC, p = 0.02), suggesting a specific inhibition (either direct or indirect) of the conversion of MPA to MPAG in tacrolimus patients, as opposed to those receiving CsA. For each drug combination, there was a positive correlation between the plasma immunosuppressive effect seen in MLC assays and the MMF dose. In addition, trough plasma from patients receiving tacrolimus and MMF was significantly more MLC inhibitory than from those receiving CsA or CsA microemulsion and equivalent-dose MMF. Culture media containing MPA and tacrolimus equal to clinical therapeutic trough concentrations (10 ng/ml) were significantly more MLC inhibitory than CsA at equivalent clinical therapeutic trough concentrations (200 ng/ml) with equivalent MPA levels. These studies in renal transplant patients suggest that tacrolimus in combination with MMF may result in a greater degree of immunosuppression than may be anticipated.

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, β1-adrenergic receptors (β1-AR), β2-AR, Gαs, and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (β-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and α-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/μm of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Gαs from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-Gs-AC in lipid rafts/caveolae.

A randomized trial of three renal transplant induction antibodies: early comparison of tacrolimus, mycophenolate mofetil, and steroid dosing, and newer immune-monitoring.

Background. New trends in immunosuppression in clinical transplantation include the use of antibody induction agents in protocols that emphasize reduction or avoidance of steroids and calcineurin inhibitors. Methods. In a randomized trial using three different antibody induction agents in 90 first renal transplant recipients from cadaver donors, group A received Thymoglobulin, group B received Alemtuzumab, and group C received Daclizumab. Maintenance immunosuppression included tacrolimus and mycophenolate in all three arms, and methylprednisolone in groups A and C only (standard clinical institutional practice). The targeted trough level of tacrolimus was between 8 and 10 ng/mL for groups A and C, respectively, with a targeted mycophenolate dose of 1 g twice daily. However, in group B, the target tacrolimus trough level was 4 to 7 ng/mL to reduce long-term nephrotoxicity, with 500 mg twice-daily doses of mycophenolate, without steroid maintenance. Results. In this 15-month median postoperative interval report, there were no notable differences in demographics and patient and graft survivals. Acute rejection rates at 1 year were equivalent, that is, 5 of 30 in all three groups (16.6%). In group B, there was slightly lower renal function at 1 month, but no difference at 1 year. There was also significantly more leukopenia, but a greater percentage of T-regulatory cells and number of Fox-P3 mRNA copies by flow cytometry and semiquantitative polymerase chain reaction analysis, respectively, in group B. Conclusions. This preliminary analysis indicates that 80% of the patients in group B remained steroid-free 1 year postoperatively, with lower tacrolimus trough levels and no difference in other adverse events.

Adenylylcyclase Increases Responsiveness to Catecholamine Stimulation in Transgenic Mice

BACKGROUND The cellular content of cAMP generated by activation of adenylylcyclase (AC) through the beta-adrenergic receptor (betaAR) is a key determinant of a cells response to catecholamine stimulation. We tested the hypothesis that increased AC content, independently of betaAR number, increases responsiveness to catecholamine stimulation in vivo. METHODS AND RESULTS Transgenic mice with cardiac-directed expression of ACVI showed increased transgene AC expression but no change in myocardial betaAR number or G-protein content. When stimulated through the betaAR, cardiac function was increased, and cardiac myocytes showed increased cAMP production. In contrast, basal cAMP and cardiac function were normal, and long-term transgene expression was not associated with abnormal histological findings or deleterious changes in cardiac function. CONCLUSIONS The amount of AC sets a limit on cardiac beta-adrenergic signaling in vivo, and increased AC, independent of betaAR number and G-protein content, provides a means to regulate cardiac responsiveness to betaAR stimulation. Overexpressing an effector (AC) does not alter transmembrane signaling except when receptors are activated, in contrast to receptor/G-protein overexpression, which yields continuous activation and has detrimental consequences. Our findings establish the importance of AC content in modulating beta-adrenergic signaling in the heart, suggesting a new target for safely increasing cardiac responsiveness to betaAR stimulation.

Inhibition of p75 Neurotrophin Receptor Attenuates Isoflurane-mediated Neuronal Apoptosis in the Neonatal Central Nervous System

Background:Exposure to anesthetics during synaptogenesis results in apoptosis and subsequent cognitive dysfunction in adulthood. Probrain-derived neurotrophic factor (proBDNF) is involved in synaptogenesis and can induce neuronal apoptosis via p75 neurotrophic receptors (p75NTR). proBDNF is cleaved into mature BDNF (mBDNF) by plasmin, a protease converted from plasminogen by tissue plasminogen activator (tPA) that is released with neuronal activity; mBDNF supports survival and stabilizes synapses through tropomyosin receptor kinase B. The authors hypothesized that anesthetics suppress tPA release from neurons, enhance p75NTR signaling, and reduce synapses, resulting in apoptosis. Methods:Primary neurons (DIV5) and postnatal day 5-7 (PND5-7) mice were exposed to isoflurane (1.4%, 4 h) in 5% CO2, 95% air. Apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Dendritic spine changes were evaluated with the neuronal spine marker, drebrin. Changes in synapses in PND5-7 mouse hippocampi were assessed by electron microscopy. Primary neurons were exposed to tPA, plasmin, or pharmacologic inhibitors of p75NTR (Fc-p75NTR or TAT-Pep5) 15 min before isoflurane. TAT-Pep5 was administered by intraperitoneal injection to PND5-7 mice 15 min before isoflurane. Results:Exposure of neurons in vitro (DIV5) to isoflurane decreased tPA in the culture medium, reduced drebrin expression (marker of dendritic filopodial spines), and enhanced Cl-Csp3. tPA, plasmin, or TAT-Pep5 stabilized dendritic filopodial spines and decreased Cl-Csp3 in neurons. TAT-Pep5 blocked isoflurane-mediated increase in Cl-Csp3 and reduced synapses in PND5-7 mouse hippocampi. Conclusion:tPA, plasmin, or p75NTR inhibition blocked isoflurane-mediated reduction in dendritic filopodial spines and neuronal apoptosis in vitro. Isoflurane reduced synapses and enhanced Cl-Csp3 in the hippocampus of PND5-7 mice, the latter effect being mitigated by p75NTR inhibition in vivo. These data support the hypothesis that isoflurane neurotoxicity in the developing rodent brain is mediated by reduced synaptic tPA release and enhanced proBDNF/p75NTR-mediated apoptosis.

Cardiac-Directed Adenylyl Cyclase Expression Improves Heart Function in Murine Cardiomyopathy

BACKGROUND We tested the hypothesis that increased cardiac myocyte adenylyl cyclase (AC) content increases cardiac function and response to catecholamines in cardiomyopathy. METHODS AND RESULTS Transgenic mice with cardiac-directed expression of AC type VI (ACVI) were crossbred with mice with cardiomyopathy induced by cardiac-directed Gq expression. Gq mice had dilated left ventricles, reduced heart function, decreased cardiac responsiveness to catecholamine stimulation, and impaired beta-adrenergic receptor (betaAR)-dependent and AC-dependent cAMP production. Gq/AC mice showed improved basal cardiac function in vivo (P=0.01) and ex vivo (P<0.0005). When stimulated through the betaAR, cardiac responsiveness was increased (P=0.02), and cardiac myocytes showed increased cAMP production in response to isoproterenol (P=0.03) and forskolin (P<0.0001). CONCLUSIONS Increasing myocardial ACVI content in cardiomyopathy restores cAMP-generating capacity and improves cardiac function and responsiveness to betaAR stimulation.

Posttransplant hyperglycemia. Increased incidence in cyclosporine-treated renal allograft recipients

The incidence of posttransplant diabetes mellitus (PTDM) was compared in two groups of renal allograft recipients. These were all nondiabetic patients who had been transplanted between 1979 and 1987 and received either azathioprine-methylprednisolone (group 1) or cyclosporine-methylprednisolone (group 2) therapy as maintenance immunosuppression. The incidence of PTDM in group 1 was 9.1% vs. 18.6% in group 2 (P less than .05). The mean daily dose of methylprednisolone during the initial 2 months posttransplant was not greater among the PTDM patients of groups 1 or 2. Cyclosporine levels and mean daily CsA doses during the initial 2 posttransplant months were also not different among the CsA-PTDM and euglycemic CsA patients. Posttransplant diabetes mellitus occurred rapidly (less than 2 months) and required insulin therapy in the majority of cases. Increased age (greater than 40 years) was associated with a higher risk for PTDM, however, the greater incidence accompanying increased body weight only approached significance. Patient gender and donor source were not associated with significant risk for PTDM. The development of PTDM was accompanied by a significant decrease in graft survival at 3 years in the entire PTDM population and at 4 years in the CsA-PTDM subgroup. Actuarial patient survival was not adversely affected. The current study suggests that CsA may be diabetogenic when administered with methylprednisolone to renal allograft recipients. The adverse effect on allograft survival requires further investigation. These results may also have important implications for pancreatic and islet cell transplantation.