Structural Basis for Enzymatic Off-Loading of Hybrid Polyketides by Dieckmann Condensation.

While several bioactive natural products that contain tetramate or pyridone heterocycles have been described, information on the enzymology underpinning these functionalities has been limited. Here we biochemically characterize an off-loading Dieckmann cyclase, NcmC, that installs the tetramate headgroup in nocamycin, a hybrid polyketide/nonribosomal peptide natural product. Crystal structures of the enzyme (1.6 Å) and its covalent complex with the epoxide cerulenin (1.6 Å) guide additional structure-based mutagenesis and product-profile analyses. Our results offer mechanistic insights into how the conserved thioesterase-like scaffold has been adapted to perform a new chemical reaction, namely, heterocyclization. Additional bioinformatics combined with docking and modeling identifies likely candidates for heterocycle formation in underexplored gene clusters and uncovers a modular basis of substrate recognition by the two subdomains of these Dieckmann cyclases.

Mouse models to study kidney development, function and disease.

PURPOSE OF REVIEW: The mouse is the most widely used model organism to study gene function in the kidney in vivo. Here we review recent advances in technologies to manipulate the mouse genome and gene function to study renal biology. We discuss strengths and weaknesses of the approaches and provide examples in which they have been used to address renal questions. In addition, we provide a summary of the available resources of mouse tools and gene-targeting consortia. RECENT FINDINGS: Although conventional gene-targeting and spontaneous genetic mutations in mice have provided great insights into kidney function over several decades, the addition of powerful renal-specific gene-targeting tools and the advent of RNA technologies to manipulate gene function in vivo allow investigators to address research questions more precisely in the laboratory. Together with the establishment of multiple international consortia to target all the genes in the mouse genome and the development of gene trap and N-ethyl-N-nitrosourea resources, genetic manipulation in mice has become more efficient. SUMMARY: The availability of newer technologies and tremendous resources for mouse strains and reagents ensures that the mouse will remain a key model organism to study renal function.

The Sweet Pee model for Sglt2 mutation.

Inhibiting renal glucose transport is a potential pharmacologic approach to treat diabetes. The renal tubular sodium-glucose transporter 2 (SGLT2) reabsorbs approximately 90% of the filtered glucose load. An animal model with sglt2 dysfunction could provide information regarding the potential long-term safety and efficacy of SGLT2 inhibitors, which are currently under clinical investigation. Here, we describe Sweet Pee, a mouse model that carries a nonsense mutation in the Slc5a2 gene, which results in the loss of sglt2 protein function. The phenotype of Sweet Pee mutants was remarkably similar to patients with mutations in the Scl5a2 gene. The Sweet Pee mutants had improved glucose tolerance, higher urinary excretion of calcium and magnesium, and growth retardation. Renal physiologic studies demonstrated a prominent distal osmotic diuresis without enhanced natriuresis. Sweet Pee mutants did not exhibit increased KIM-1 or NGAL, markers of acute tubular injury. After induction of diabetes, Sweet Pee mice had better overall glycemic control than wild-type control mice, but had a higher risk for infection and an increased mortality rate (70% in homozygous mutants versus 10% in controls at 20 weeks). In summary, the Sweet Pee model allows study of the long-term benefits and risks associated with inhibition of SGLT2 for the management of diabetes. Our model suggests that inhibiting SGLT2 may improve glucose control but may confer increased risks for infection, malnutrition, volume contraction, and mortality.

Impact of augmenting dialysis frequency and duration on cardiovascular function.

Conventional hemodialysis (CHD) only delivers 10% to 15% of renal function in a nonphysiological intermittent mode. Because it occurs nightly and is sustained over a longer dialysis time, the uremic clearance provided by nocturnal hemodialysis (NHD) far exceeds that of CHD. Increasing the dose and frequency of dialysis by NHD has been demonstrated, in both short- and long-term studies, to reverse several important risk factors for adverse cardiovascular events in patients with end-stage renal disease such as hypertension, left ventricular hypertrophy, systolic dysfunction, conduit artery stiffness, attenuated baroreflex regulation of heart rate, disturbed heart rate variability, sleep apnea, and endothelium-dependent vasodilation. In addition, the Toronto NHD experience has reported an emerging body of evidence demonstrating the benefits of NHD on anemia management, inflammation, and endothelial progenitor cell biology. The mechanism(s) by which nocturnal hemodialysis improves cardiovascular outcomes are under active investigation by our group. It is tempting to speculate that NHD has the potential to decrease endothelial/myocardial injury and restore simultaneously endothelial repair, thereby improving cardiovascular function in patients with end-stage renal disease. The objectives of the present document are (1) to review the mechanisms underlying dialysis-associated cardiovascular morbidity and (2) to describe the restorative potential of NHD on the cardiovascular system.

Red blood cell survival in chronic renal failure.

BACKGROUND: A decrease in the lifespan of erythrocytes has been accepted universally as one of the contributory factors to anemia in patients with chronic renal failure. This observation was made in the 1950s and 1960s when continuous renal replacement therapy was at its infancy. Based on the premise that a reduced red blood cell (RBC) lifespan in renal disease is primarily caused by the toxic uremic milieu, the purpose of this study is 2-fold: to compare the RBC survival in today's renal patients with that in the existing literature and to explore if there are differential RBC survival benefits with various dialysis dosages. METHODS: This is an observational study. Patients with end-stage renal disease were recruited from the dialysis program at the University Health Network and St Michael's Hospital in Toronto. The patients were stratified into 3 groups including conventional thrice-weekly, nocturnal, and short-daily hemodialysis. Healthy subjects were recruited to validate the normal range for RBC lifespan. Red cell survival was assessed using radiolabeled sodium chromate (Na2 51CrO4). RESULTS: Twenty-two patients and 2 healthy control subjects were recruited. The average red cell half-lives in thrice-weekly, nocturnal, short-daily, and healthy subjects were 14.5 +/- 1.6, 17.1 +/- 4.7, 15.9 +/- 2.2, and 23.5 days, respectively. All 3 patient groups exhibited reduced RBC lifespan (P < 0.05). Further, the overall RBC lifespan was not different from that reported half a century ago. Despite better urea clearances among the nocturnal and short-daily dialysis groups, no RBC survival benefit was observed. CONCLUSION: A reduced RBC lifespan continues to contribute to renal anemia despite technologic advancements and improved uremic environment.