Kidney Membranes Research Articles

Hepatic and renal expression of Oatp1 in obstructive uropathy. First detection of Oatp1 in urine, a potential biomarker.

Obstructive renal diseases affect renal function and kidney integrity. Nevertheless, little is known about its systemic or extra-renal effects. The organic anion transporting polypeptide 1 (Oatp1) is a carrier expressed in liver and kidneys. In this study, the hepatic and renal expression of Oatp1 was evaluated in rats with obstructive nephropathy. Moreover, the urinary excretion of Oatp1 (Oatp1u ) was evaluated as a potential biomarker for this pathology. Male Wistar rats with bilateral ureteral obstruction for 5hours (BUO5), 24hours (BUO24) or sham operated were used. After 24hours of ureteral releasing, liver and kidney functional parameters, histopathology, Oatp1 tissular expression and Oatp1u were evaluated. For Oatp1u evaluation two groups were added; BUO1 and BUO2 (1 and 2hours of ureteral obstruction, respectively). Both liver and kidney functional parameters and histopathological studies showed alterations in BUO5 and BUO24. In hepatic homogenates, Oatp1 significantly decreased in BUO groups and in total liver membranes no modifications were observed. In renal homogenates, Oatp1 significantly decreased in BUO groups, but in apical kidney membranes, its expression was increased. Oatp1u was only detected in BUO groups, even in those (BUO1, BUO2) in which no alterations in the traditional parameters of renal function were observed. Modulations in liver and renal expression of Oatp1 could be an organism strategy to attenuate the effects of the disease and an attempt to maintain the complex organ cross-talk between liver and kidneys. Oatp1u could be a new, early and specific biomarker of obstructive nephropathy.

Monoclonal and polyclonal immunoglobulin G deposits on tubular basement membranes of native and pretransplant kidneys: A retrospective study

Monoclonal tubular basement membrane immune deposits (TBMID) are associated with progression of interstitial injury in renal allograft. However, the significance of monoclonal and polyclonal TBMID in the native kidney remains unclear. We retrospectively analyzed 1894 native kidney biopsies and 1724 zero-hour biopsies performed between 2008 and 2018 in our institution. The rate of immunoglobulin G (IgG) TBMID was found to be 8.4% among native kidney biopsies and 0.4% among zero-hour biopsies. Polyclonal TBMID is common in IgG4-related tubulointerstitial nephritis (37.5%), diabetic nephropathy (31.3%) and lupus nephritis (25.5%). Monoclonal IgG TBMID was identified in seven cases, including three zero-hour biopsies. The combination of IgG1κ was observed in two cases, IgG1λ in three, and IgG2κ in two. Electron microscopy revealed powdery electron-dense deposits in all cases. Monoclonal gammopathy of undetermined significance was diagnosed in one case. Although one patient with focal segmental glomerulosclerosis developed renal failure, all others exhibited stable renal function. Monoclonal IgG TBMID in the native kidney is not associated with renal prognosis. However, this may be an interesting immunopathological finding that would help clarify the pathogenesis of TBM immune deposits. Further study for both monoclonal and polyclonal TBMID is required in the future.

Mechanical Strength Enhancement of Porous Nanocrystalline-Silicon (pnc-Si) Membrane via Titanium-oxide (Ti-O) Coating

Porous nanocrystalline silicon (pnc-Si) membrane is mainly studied as a blood filtration membrane, mimicking the glomerulus filtration membrane of a human kidney. However, the pnc-Si material itself is not hemocompatible and enormous membrane area to thickness ratio makes the membrane to be easily fractured. Silicon surface modification via titanium-oxide (Ti-O) thin film layer deposition has been proven to be hemocompatible and the presence of Ti-O layer has been numerically studied to give higher membrane flexural strength. In this work, square pnc-Si membranes of 2 mm × 2 mm × 20 nm size have been fabricated with and without Ti-O layer. Point loading-unloading nanoindentation method has been performed and the membranes’ displacement behaviour subjected to point loads is studied. The pnc-Si membranes with Ti-O layer were found to attain higher fracture strength, membrane bending stiffness and average hardness with the increase of ~20, ~11 and ~24%, respectively, compared to bare pnc-Si membranes. The mechanical strength of a free-standing pnc-Si membrane is improved by depositing a Ti-O thin film layer on the membrane structure.

Newly Developed System for the Robust Detection of Listeria monocytogenes Based on a Bioelectric Cell Biosensor.

Human food-borne diseases caused by pathogenic bacteria have been significantly increased in the last few decades causing numerous deaths worldwide. The standard analyses used for their detection have significant limitations regarding cost, special facilities and equipment, highly trained staff, and a long procedural time that can be crucial for foodborne pathogens with high hospitalization and mortality rates, such as Listeria monocytogenes. This study aimed to develop a biosensor that could detect L. monocytogenes rapidly and robustly. For this purpose, a cell-based biosensor technology based on the Bioelectric Recognition Assay (BERA) and a portable device developed by EMBIO Diagnostics, called B.EL.D (Bio Electric Diagnostics), were used. Membrane engineering was performed by electroinsertion of Listeria monocytogenes homologous antibodies into the membrane of African green monkey kidney (Vero) cells. The newly developed biosensor was able to detect the pathogen’s presence rapidly (3 min) at concentrations as low as 102 CFU mL−1, demonstrating a higher sensitivity than most existing biosensor-based methods. In addition, lack of cross-reactivity with other Listeria species, as well as with Escherichia coli, was shown, thus, indicating biosensor’s significant specificity against L. monocytogenes.

Peroxidasin-mediated bromine enrichment of basement membranes

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain.

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1–10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the α2 Na,K-ATPase isozyme and without changes in α1 and α2 Na,K-ATPase protein content. Ouabain (10–20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the α1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the α2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment.

Estimation of Serum Cystatin-C as early marker of Kidney Dysfunction in correlation with Serum Ferritin among ?- Thalassemia Major Patients

The Cystatin C is early predictor for evaluation the kidney functions. The Serum Cystatin C is a cysteine proteinase inhibitor, that the nucleated cells produce it at stable rate. It is filtered through the glomerular filtration membranes of kidneys, and the filtration rate will be unaffected by external factors. A group of recessively inherited hemoglobin disorders called Major Beta-thalassemia and considered the most common genetic disorder all over the world, which could be noticed by the reduction in of ?-globin chain synthesis.Objective: To study Serum Cystatin-C estimation as early marker of renal function in relation with other parameters in beta thalassemia patients.Materials and Method: A case-control study was executed on thalassemia patients between February and April 2019 at Al-Zahra’a Teaching Hospital in Najaf/Iraq.Results: the results in this study showed there are significant differences (p< 0.05) between major beta thalassemia patients and control group in regarding to hematological parameters. The result of biochemical parameters showed there are variations between patients and control where there were significant statistical increases in our thalassemic patients comparing to the control about Cystatin-C (1.27±0.33and 0.7±0.24 ng\ml respectively).Conclusion: the cystatin –C is useful marker at major beta thalassemia patients that suffering from renal dysfunction

Estimation of Serum Cystatin-C as early marker of Kidney Dysfunction in correlation with Serum Ferritin among β- Thalassemia Major Patients

The Cystatin C is early predictor for evaluation the kidney functions. The Serum Cystatin C is a cysteine proteinase inhibitor, that the nucleated cells produce it at stable rate. It is filtered through the glomerular filtration membranes of kidneys, and the filtration rate will be unaffected by external factors. A group of recessively inherited hemoglobin disorders called Major Beta-thalassemia and considered the most common genetic disorder all over the world, which could be noticed by the reduction in of β-globin chain synthesis.Objective: To study Serum Cystatin-C estimation as early marker of renal function in relation with other parameters in beta thalassemia patients.Materials and Method: A case-control study was executed on thalassemia patients between February and April 2019 at Al-Zahra’a Teaching Hospital in Najaf/Iraq.Results: the results in this study showed there are significant differences (p< 0.05) between major beta thalassemia patients and control group in regarding to hematological parameters. The result of biochemical parameters showed there are variations between patients and control where there were significant statistical increases in our thalassemic patients comparing to the control about Cystatin-C (1.27±0.33and 0.7±0.24 ng\ml respectively).Conclusion: the cystatin –C is useful marker at major beta thalassemia patients that suffering from renal dysfunction

Localization of aquaporin-3 proteins in the bovine rumen

Urea nitrogen salvaging is a crucial mechanism that ruminants have evolved to conserve nitrogen. Facilitative urea transporter-B proteins are known to be involved in urea transport across the rumen epithelium and thus efficiently facilitate the urea nitrogen salvaging process. Recently, functional studies have suggested that aquaglyceroporin transporters might also play a significant role in ruminal urea transport and aquaporin-3 (AQP3) protein has previously been detected in rumen tissue. In this current study, we investigated the specific localization of AQP3 transporters in the bovine rumen. First, end-point reverse-transcription PCR experiments confirmed strong AQP3 expression in both bovine rumen and kidney. Immunoblotting analysis using 2 separate anti-AQP3 antibodies detected AQP3 protein signals at 25, 32, and 42-45 kDa. Further immunolocalization studies showed AQP3 protein located in all the layers of rumen epithelium, especially in the stratum basale, and in the basolateral membranes of kidney collecting duct cells. These data confirm that AQP3 transporters are highly abundant within the bovine rumen and appear to be located throughout the ruminal epithelial layers. The physiological significance of the multiple AQP3 proteins detected and their location is not yet clear, hence further investigation is required to determine their exact contribution to ruminal urea transport.

Activity-Based Training Reverses Spinal Cord Injury-Induced Changes in Kidney Receptor Densities and Membrane Proteins.

Complications in upper and lower urinary function arise after spinal cord injury (SCI), which creates a significant impact on quality of life for those affected. One upper urinary complication is SCI-induced polyuria, or the overproduction of urine, of which the underlying mechanisms have yet to be elucidated. Activity-based training (ABT) has been utilized in both animal and clinical settings as a rehabilitative therapy to improve many issues that arise after SCI, including more recently urogenital function. The goal of the current study was to identify potential mechanisms contributing to previously identified improvements in polyuria with ABT, using a male rat moderate-severe spinal contusion model. Although ABT had no significant effect on reversing injury-induced alterations of serum arginine vasopressin and urinary atrial natriuretic peptide levels, there was a dramatic effect upon the receptors of these fluid balance hormones (vasopressin receptor 2 and natriuretic peptide A receptor), as well as kidney aquaporin 2 and sodium channels. ABT changes in densities of key receptors and kidney membrane proteins involved in fluid balance after chronic SCI support the likelihood of multiple mechanisms through which exercise can positively influence urinary tract function after SCI. By understanding the mechanisms, amount, and timing regarding how ABT improves different aspects of urinary function, more targeted training strategies can be developed to optimize the functional gains within the SCI population.

Proton Nuclear Magnetic Resonance (1H NMR) profiling of isolated organs in the snail Helix aspersa maxima

• The 1 H NMR spectra showed characteristic metabolic profiles for each tissue. • Tissues contained LMW metabolites of metabolic pathways affected by toxic compounds. • Metabolic activity, cellular hypoxia and oxidative stress increased with Methomyl. • Kidney and pulmonary membrane were the main target organs of Methomyl exposure. Helix aspersa maxima is a terrestrial snail living at the air-ground interface, which makes it an ideal bioindicator candidate to evaluate environmental pollution. In this study, Proton Nuclear Magnetic Resonance ( 1 H NMR) based metabolomics approach was applied on diverse tissue extracts isolated from Helix aspersa individuals. This omics technique allows to characterize in one single spectroscopic measurement the global metabolic changes caused by pollution on the organism and to assess the potential of this species as a pollution indicator. In a first set of experiments conducted on naïve individuals, the main endogenous metabolites were identified. Those included amino and organic acids, Krebs cycle intermediates and a series of osmolytes. Next, a second group of animals was exposed to methomyl, a pesticide used in agriculture, in order to reveal what metabolic pathways were potentially affected by the pollutant. Our results indicate important fluctuations in energetic metabolism and several biomarkers of oxidative stress. In conclusion, this work demonstrates the potential of combining invertebrates models to metabonomics as a powerful tool to predict adverse effects of chemicals.

Patent Highlights December 2018–January 2019

Patent Highlights December 2018–January 2019

Towards Identifying the Binding Site for 125I‐Angiotensin 1‐7

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system (RAS) that exhibits central actions to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been considered the receptor for Ang 1‐7, however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has demonstrated high affinity (low nanomolar KD) binding of a preparation of 125/127I‐Ang 1‐7 in liver membranes, however, this binding is not pharmacologically specific in that the IC50 of Ang 1‐7 is in the micromolar range and all angiotensin peptides compete for 125/127I‐Ang 1‐7 binding equivalently. 125/127I‐Ang 1‐7 binding to liver, kidney, brain and testes membrane preparations from both rats and mice has also failed to demonstrate a high affinity, pharmacologically specific binding site for 125/127I‐Ang 1‐7. Considering that rapid degradation of Ang 1‐7 could be adversely affecting the binding assay, we tested different peptidase inhibitors (JMV‐390, O‐phenanthroline and a proprietary combination of peptidase inhibitors Biotools®) to prevent degradation of the radioligand. Although we were able to reduce Ang 1‐7 degradation with these inhibitors (~80% preservation), we also reduced the total and specific binding of 125I‐Ang 1‐7. Indeed, competition binding assays for 125I‐Ang‐(1–7) with JMV‐390, O‐phenanthroline and Biotools® showed that 125I‐Ang‐(1–7) and all the inhibitors compete for the same binding site with JMV‐390 having a nanomolar IC50. It is important to note that the radioligand used for receptor binding studies contains an iodine125 on the tyrosine in position four of Ang 1‐7, and may have different characteristics for enzymatic metabolism. To assess this possibility, we determined the metabolism of 127I‐Ang 1‐7 (non‐radioactive iodine isotope) and indeed, the metabolism of Ang 1‐7 and 127I‐Ang 1‐7 in liver appear to differ, with 127I‐Ang 1‐7 being more resistant to metabolism. In addition, competition binding assays showed that 127I‐Ang 1‐7 is ~1000‐fold more potent than Ang 1‐7 in competing for 125I‐Ang 1‐7 binding. In conclusion, our results suggest that 125I‐Ang‐(1–7) may be binding to a peptidase rather than a receptor and the addition of the iodine molecule to the tyrosine in position four attenuates its degradation. Studies are underway to identify this peptidase and determine its potential therapeutic importance as a druggable target.Support or Funding InformationCardiovascular Neuroscience Research Fund 337667 Speth (PI) 03/31/15‐12/31/18 ‐ Nova Southeastern Univ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Superimposing a high-fat diet on Schistosoma mansoni infection affects renin-angiotensin system components in the mouse kidney.

The levels of the full-length form of the (pro)renin receptor (PRR), a component of the renin-angiotensin system (RAS), may be reduced in the membranes of kidneys in renal diseases. This study aimed to investigate the RAS components in the kidneys of mice submitted to a combination of a high-fat diet and Schistosoma mansoni infection. Female BALB/c mice were maintained on a control or high-fat diet from 3 weeks of age. After 10 weeks on the designated diets, half the mice in each group were infected with S. mansoni cercariae. The blood and kidneys were harvested 8 weeks after infection. The high-fat diet increased the number of eggs in the feces and the number of adult worms in the mesenteric bed. Schistosoma mansoni infection reduced the plasma levels of glucose, triglycerides, and HDL cholesterol in the control and high-fat diet groups. In mice on the control diet, S. mansoni infection resulted in increased expression of IL-6 in the kidneys; however, in mice on the high-fat diet, the levels of IL-6 were reduced and those of superoxide anions were increased. The RAS components evaluated were ACE2, renin, PRR, AT1R, and AT2R, and the levels of PRR were found to be reduced in the kidneys of infected mice on the high-fat diet. The finding regarding PRR is not yet clear. However, combining a high-fat diet and S. mansoni infection resulted in increased oxidative stress in the kidney that can aggravate hypertension as well as its associated complications.

New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity.

A scientific session entitled "New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity" focused on novel biomarkers to monitor kidney injury both preclinically and clinically, as well as providing mechanistic insight of the induced injury. Further, the role and impact of kidney membrane transporters in drug-induced kidney toxicity provided additional considerations when understanding kidney injury and the complex role of drug transporters in either sensitivity or resistance to drug-induced injury. The onset of nephropathy in diabetic patients was also presented, focusing on the quest to discover novel biomarkers that would differentiate diabetic populations more susceptible to nephropathy and renal failure. The session highlighted exciting new research areas and novel biomarkers that will enhance our understanding of kidney injury and provide tools for ensuring patient safety clinically.

Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1–7

It is well established that Mas (NP_002368) is the receptor for angiotensin 1–7 (Ang 1–7), however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has repeatedly failed to reliably demonstrate high affinity, saturable binding of 125I‐Ang 1–7 in liver, kidney, brain and testes membrane preparations from both rats and mice. While the presence of a single iodine‐125 molecule on the tyrosine of Ang II does not impair its binding to the AT1 and AT2 receptors, it is not known whether the presence of an iodine‐125 molecule on the tyrosine of Ang 1–7 is equally innocuous. To begin to address this question, we prepared a novel analog of Ang 1–7: Tyr0‐Ang 1–7, to provide an alternate site for radioiodination of Ang 1–7. Tyr0‐Ang 1–7 was radioiodinated using the chloramine T procedure of Hunter and Greenwood (1962) with a 7 times excess of peptide to iodine to minimize di‐iodination of the peptide. Two radioiodinated peaks, presumed to be mono125I‐Y0‐Ang 1–7 and mono125I‐Y4‐Ang 1–7, were resolved from the uniodinated peptide peak by HPLC (C18 reverse phase column, 14.5% acetonitrile: triethylamine phosphate, pH 3.0 isocratic mobile phase). Saturation and competition radioligand binding assays were run for each peak using rat liver membrane preparations in the presence and absence of 10 μM Ang 1–7. While the identity of the early and late 125I‐Y0‐ Ang 1–7 peaks has not yet been established, preliminary studies indicate that both 125I‐Y0‐Ang 1–7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: KD = 7.7±2.3 nM, BMAX = 3.3±0.72 fmol/mg wet weight; and late peak KD = 11±2, BMAX = 3.6±0.4 fmol/mg wet weight. Competition binding assays run using the late peak 125I‐Y0‐Ang 1–7 revealed IC50's of 32±15 nM for Ang 1–7 and 15 ± 9.6 nM for Y0‐Ang 1–7. Comparison of the ability of a variety of Ang peptides to compete for late peak 125I‐Y0‐Ang 1–7 binding at a 10 μM concentration gave a rank order of Ang 1–7&gt;Ang 3–7&gt;Ang IV&gt;Ang III&gt;Ang 2–7&gt;Ang II&gt;Ang I. These results suggest that addition of a tyrosine at the amino terminus of Ang 1–7 may increase its binding affinity for Mas and that the presence of an iodine‐125 on either Y0 or Y4 of Ang 1–7 does not preclude high affinity saturable binding to Mas.Support or Funding InformationThis work was supported by the Cardiovascular Neuroscience Fund, Nova Southeastern University and the Peptide Radioiodination Shared Resource, Georgetown University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic acidosis inhibits AMPK function in kidney cells

The metabolic sensor AMP‐activated protein kinase (AMPK) is stimulated by cellular metabolic depletion. We have shown that AMPK activation inhibits kidney membrane transport proteins. AMPK‐activated processes protect kidney cells from further metabolic depletion and damage. AMPK is tightly regulated, and its pharmacological activation can protect kidneys from superimposed injury, such as acute kidney injury (AKI). Conversely, AMPK is inhibited in chronic kidney disease (CKD). Therefore, pharmacologic AMPK activators are proposed as therapies to slow CKD progression. However, the mechanisms by which AMPK may promote kidney survival are unclear. CKD results in metabolic acidosis (MA) due to the inability of kidneys to excrete non‐volatile acid. MA, if untreated, has severe health sequelae such as worsening of kidney function. We hypothesized that there is cross‐talk between acid‐base status and AMPK signaling, and that dysregulation of AMPK function by MA disrupts kidney regeneration in the setting of CKD. We used kidney epithelial cell lines in culture and adult male C57BL/6 mice (WT) and AMPKβ1‐KO mice for our experiments. Cells were exposed to media at either pH 7.3–7.4 (control) or 6.9–7.0 (acidic) for 3 d ± an AMPK activator (last 16 h). We found that, as compared to cells grown at pH 7.4, kidney cells grown in acidic media have reduced baseline AMPK activity and blunted pharmacologic AMPK activation. We then evaluated changes in AMPK function in WT mice fed a normal diet vs. WT mice given 1.5% saccharin ± 0.28 M NH4Cl in drinking water for 2 mo. To evaluate the AMPK pathway in kidney tissue, we examined levels of phosphorylation of acetyl CoA carboxylase (pACC) by immunoblot. Mice on the acidogenic diet developed MA without significant weight loss compared to mice on the control diet (blood pH 7.2 vs 7.3 ± 0.01). In mice after UNX, the superimposed MA induced a statistically significant decrease in pACC, compared to mice with UNX on a control diet. We also evaluated differences in BUN in WT vs. AMPKβ1‐KO mice 3 mos. after sham surgery or UNX, a model for reduced kidney mass. Both WT and KO mice post‐UNX had significant worsening of kidney function (higher BUN) compared to sham‐operated mice of the same genotype. However, we did not observe any significant differences in BUN between WT and KO mice following sham or UNX. Our results point to worsening in kidney function post‐UNX and to a direct inhibition of the AMPK pathway in the setting of reduced kidney mass and MA. This AMPK inhibition may be detrimental to kidney regeneration in these settings.Support or Funding InformationKeck School of Medicine of USC; USC/UKRO Kidney Research CenterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Glutathione Peroxidase-1 (Gpx1) Genotype on Selenoenzyme and Transcript Expression When Repleting Selenium-Deficient Mice.

Glutathione peroxidase (Gpx1) is the major selenoprotein in most tissues in animals. Knockout (KO) of Gpx1 decreases Gpx1 activity to near zero and substantially reduces liver selenium (Se) levels, but has no overt effects in otherwise healthy mice. To investigate the impact of deletion of Gpx1 on Se metabolism, Se flux, and apparent Se requirements, KO, Gpx1 heterozygous (Het), and Gpx1 wild-type (WT) mice were fed Se-deficient diet for 17weeks, then repleted with graded levels of Se (0-0.3μg Se/g as Na2SeO3) for 7days, and selenoprotein activities and transcripts were determined in blood, liver, and kidney. Se deficiency decreased the activities of plasma Gpx3, liver Gpx1, liver Txnrd, and liver Gpx4 to 3, 0.3, 11, and 50% of WT Se-adequate levels, respectively, but the Gpx1 genotype had no effect on growth or changes in activity or expression of selenoproteins other than Gpx1. Se repletion increased selenoprotein transcripts to Se-adequate levels after 7days; Se response curves and apparent Se requirements for selenoprotein transcripts were similar to those observed in studies starting with Se-adequate mice. With short-term Se repletion, selenoenzyme activities resulted in three Se response curve patterns: (1) liver and kidney Gpx1, Gpx4, and Txnrd activities were sigmoidal or hyperbolic with breakpoints (0.08-0.19μg Se/g) that were double those observed in studies starting with Se-adequate mice; (2) red blood cell Gpx1 activity was not significantly changed; and (3) plasma Gpx3 activity only increased substantially with 0.3μg Se/g. Plasma Gpx3 is secreted from kidney. In this short-term study, kidney Gpx3 mRNA reached plateau levels at 0.1μg Se/g, and other kidney selenoenzyme activities reached plateau levels at ≤0.2μg Se/g, so sufficient Se should have been present in kidney. Thus, the delayed increase in plasma Gpx3 activity suggests that newly synthesized and secreted kidney Gpx3 is preferentially retained in kidney or rapidly cleared by binding to basement membranes in kidney or in other tissues. This repletion study shows that loss of capacity to incorporate Se into Gpx1 in Gpx1 KO mice does not dramatically alter expression of other Se biomarkers, nor the short-term flux of Se from intestine to liver to kidney.

Membrane Cholesterol Reduces Polymyxin B Nephrotoxicity in Renal Membrane Analogs

Polymyxin B (PmB) is a “last-line” antibiotic scarcely used due to its nephrotoxicity. However, the molecular basis for antibiotic nephrotoxicity is not clearly understood. We prepared kidney membrane analogs of detergent-susceptible membranes, depleted of cholesterol, and cholesterol enriched, resistant membranes. In both analogs, PmB led to membrane damage. By combining x-ray diffraction, molecular dynamics simulations, and electrochemistry, we present evidence for two populations of PmB molecules: peptides that lie flat on the membranes, and an inserted state. In cholesterol depleted membranes, PmB forms clusters on the membranes leading to an indentation of the bilayers and increase in water permeation. The inserted peptides formed aggregates in the membrane core leading to further structural instabilities and increased water intake. The presence of cholesterol in the resistant membrane analogs led to a significant decrease in membrane damage. Although cholesterol did not inhibit peptide insertion, it minimized peptide clustering and water intake through stabilization of the bilayer structure and suppression of lipid and peptide mobility.

Matrix Gelatinases in Atherosclerosis and Diabetic Nephropathy: Progress and Challenges.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases that degrade components of the extracellular matrix (ECM). In glomerular disease, MMPs are major regulators of ECM degradation as well as structural and functional integrity in the glomerulus. In altered matrix composition diseases, glomerular damage is due to increased degradation of kidney and vessel basement membranes (BMs) by MMPs. MMP -2 and -9 are both considered as the main enzymes that degrade collagen type-IV (coll-IV), which represents the key collagenous component of ECM and constitutes the architectural structure of vessels and glomerular BM. There is growing evidence implicating MMPs in atherosclerosis as well as in cardiovascular disease (CVD) and chronic kidney disease (CKD). Specific endogenous tissue inhibitors of MMPs (TIMPs) are also implicated in CKD, CVD and diabetic nephropathy (DN). The present review discusses the role of MMPs -2 and -9 in DN, as a leading cause of endstage renal disease and as a model of the link between progressive glomerulosclerosis and MMP expression.