Kidney Brush Border Membranes Research Articles

Proteomics profiling of kidney brush border membrane from rats using LC-MS/MS analysis.

Chronic kidney disease (CKD) is defined by a reduced renal function, that is, glomerular filtration rate, and the extent of kidney damage is assessed by determining serum creatinine levels and proteins in urine, diagnosed as proteinuria/albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush border membranes (BBMs) on PT cells are important in maintaining the stability of PT functions. An LC-MS/MS bottom-up proteomics analysis of BBMs from four groups of rat models was applied to investigate protein abundance alterations associated with CKD progression. Moreover, systems biology analyses were used to identify key proteins that can provide insight into the different regulated molecular pathways and processes associated with CKD. Our results indicated that 303 proteins showed significantly altered expressions from the severe CKD BBM group when compared to the control. Focusing on renal diseases, several proteins including Ctnnb1, Fah, and Icam1 were annotated to kidney damage and urination disorder. The up-regulation of Ctnnb1 (β-catenin) could contribute to CKD through the regulation of the WNT signaling pathway. Overall, the study of protein abundance changes in BBMs from rat models helps to reveal protein corrections with important pathways and regulator effects involved in CKD. Although this study is focused on rat models, the results provided more information for a deeper insight into possible CKD mechanisms in humans.

NHERF1 loss results in metabolic stress and increased susceptibility to cisplatin‐induced acute kidney injury

BackgroundAcute kidney injury (AKI) is associated with a high mortality rate and develops in 30% of patients who receive cisplatin. The sodium hydrogen regulatory factor isoform 1 (NHERF1) is a scaffolding protein that anchors multiple membrane proteins, in renal proximal tubules. We have recently demonstrated that NHERF1 loss results in changes in mitochondrial protein abundance and function.HypothesisWe hypothesize that NHERF1 loss increases susceptibility to AKI through an underlying metabolic stress.Materials and MethodsWe treated 2 month old male and female wild type (WT) C57BL/6 and NHERF1 knock out (KO) mice with vehicle (VEH) or cisplatin (CIS) (20 mg/kg dose IP) and euthanized after 72 hours. Blood was collected for blood urea nitrogen (BUN) levels. Urine was collected for NGAL. Kidneys were harvested for histology, TUNEL assay, and Western Blot for cleaved caspase 3. Proteomic analysis was conducted on kidney brush border membrane (BBM) of WT and KO kidneys. Metabolic measurements were conducted via the XF Flux analyzer on non‐treated primary proximal tubule cell cultures. Three‐way ANOVA was used to compare the different treatment groups. Ordinal regression was performed for the semi‐quantitative scoring. All statistical analysis was conducted with SPSS software. P values of <0.05 were considered statistically significant.ResultsCIS caused significantly greater severity of kidney injury in KO compared to WT mice by semi‐quantitative injury score (WT: 1.89, KO: 2.8), BUN levels (WT: 97.8 mg/dL +/− 10.1, KO: 151.8 mg/dL +/− 17.2) and NGAL protein (WT: 2.7 μg/mL +/− 0.53, KO: 55.6 μg/mL +/−21.3). Apoptosis markers were significantly increased in CIS treated KO and WT mice compared to respective controls. Proteomic comparison of BBM protein expression in WT and KO showed marked changes in the expression of mitochondrial proteins in KO mice, suggesting alterations in cell metabolism. KO proximal tubule cells also exhibited decreases of 35% in glycolytic reserve capacity, 40% in baseline mitochondrial respiration rate, 37% in ATP‐linked mitochondrial respiration rate, 40% in mitochondrial reserve capacity, and 40% in maximum mitochondrial capacity.ConclusionsWe conclude that the KO mice have increased susceptibility to CIS‐induced AKI, which may be due to altered metabolism and/or mitochondrial dysfunction.Support or Funding InformationFunding for this research was provided by VA and UofL School of Medicine (to EDL).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of ¹¹¹in-labelled exendin-4 derivatives containing different meprin β-specific cleavable linkers.

BackgroundCleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.ResultsThe insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the 111In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound 111In-Ex4NOD40.ConclusionIn conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.

Meprin Metalloproteases Inactivate Interleukin 6

Meprins have been implicated in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, in which the cytokine IL-6 is a prominent effector molecule. Because IL-6 levels are elevated markedly in meprin α and α/β knockout mice in an experimental model of inflammatory bowel disease, the interaction between meprins and IL-6 was studied. The results demonstrate that rodent and human meprin A and B cleave IL-6 to a smaller product and, subsequently, are capable of extensive degradation of the cytokine. Analysis of the limited degradation product formed by meprin A indicated that three to five amino acids are removed from the C terminus of the cytokine. Meprin A and meprin B cleaved IL-6 with micromolar affinities (Km of 4.7 and 12.0 μM, respectively) and with high efficiencies (kcat/Km of 0.2 and 2.5 (M(-1)/s(-1)) × 10(6), respectively). These efficiency constants are among the highest for known meprin substrates. Madin-Darby canine kidney cells transiently transfected with meprin α or meprin β constructs also cleave exogenous IL-6. Both human and murine IL-6 cleaved by meprin A or B are inactivated, as demonstrated by their decreased capability to stimulate proliferation of B9 cells. These results are consistent with the proposition that one function of meprin metalloproteases is to modulate inflammation by inactivating IL-6.

Characterization of simvastatin‐induced structural and functional alterations in the molecules of kidney brush border membrane by ATR‐FTIR spectroscopy and chemometric approaches

Simvastatin, lipophilic statin, is the most commonly used drugs to treat hyperlipidaemia. Recent studies reported that they have both adverse and beneficial effects on kidney. However these results are contradictory. In this study, the aim was to investigate the effects of simvastatin on concentration, structure and function of molecules of kidney brush border membrane (BBM) by Attenuated Total Reflectance‐Fourier Transform Infrared (ATR‐FTIR) spectroscopy and chemometric analysis.During 30 days, control and simvastatin treated rats were taken serum physiologic and 50 mg/kg/day simvastatin intragastrically, respectively. Simvastatin‐induced changes in the structure and function of molecules in kidney BBM were determined from the frequency, area and width analysis of spectral bands. Based on these alterations, cluster and principal component analysis (PCA) were applied to discriminate these groups. Moreover, artificial neural network (ANN) analysis was used to determine the secondary structure alterations in proteinsSpectral analysis indicated a decrease in the amount of saturated and unsaturated lipids and proteins with simvastatin treatment. Control and simvastatin groups were successfully distinguished. A significant decrease in beta sheet and increase in random coil content were observed in simvastatin treated group. This study was supported by BAP‐08–11‐DPT‐2011K121010.

Inhibition of the OCTN2 carnitine transporter by HgCl2 and methylmercury in the proteoliposome experimental model: insights in the mechanism of toxicity

Mercury causes toxic effects in many tissues interacting with protein cysteine (Cys) thiols. Transport systems represent critical targets of mercurials. Indeed, the majority of transport systems of higher eukaryotes contain several Cys residues. One of the most up to date method of studying transport is the reconstitution of transporters in proteoliposomes. This method has been used as a useful approach to test the effect of HgCl2 and methylmercury (MeHg) on the carnitine (OCTN2) transporter. OCTN2, extracted from kidney brush border membranes with 3% octaethylene glycol monododecyl ether (C12E8), was reconstituted in liposomes by removing the detergent with hydrophobic chromatography columns. Transport was measured as [3H]carnitine uptake into proteoliposomes containing carnitine (antiport reaction). Mercurials strongly inhibited the antiport. Inhibition was reversed by 1,4-dithioerythritol, L-cysteine (Cys), and N-acetyl-L-cysteine (NAC) indicating that it was caused by covalent reaction of mercurials with Cys residue(s). The IC50 for HgCl2, and MeHg were 2.5 and 7.4 µM, respectively. Kinetic studies showed non competitive or mixed inhibition for HgCl2 or MeHg with Ki of 4.2 and 13 µM, respectively. The presence of substrate prevented the inhibition indicating that the mercurial binding residue (Cys) is in the substrate binding site. Efflux of carnitine from proteoliposomes was trans-stimulated, not inhibited, by higher concentrations (500 µM) of extraliposomal MeHg and HgCl2. Differently, no effects on uptake of carnitine were exerted by mercurials present in the internal compartment of the proteoliposomes. The results allowed gaining new insights in the molecular mechanism of inhibition and of mercurial toxicity.

N-Methylnicotinamide Is an Endogenous Probe for Evaluation of Drug–Drug Interactions Involving Multidrug and Toxin Extrusions (MATE1 and MATE2-K)

Multidrug and toxin extrusion 1 (MATE1) and MATE2-K are H(+)/organic cation exchangers mediating the efflux of cationic drugs into the urine. N-methylnicotinamide (NMN) was found to be an endogenous substrate of MATE1 (Michaelis constant (K(m)) 301 ± 18 µmol/l) and MATE2-K (K(m) 422 ± 63 µmol/l) as well as a basolateral influx transporter, organic cation transporter 2 (K(m) 318 ± 29 µmol/l). A potent MATE inhibitor, pyrimethamine, competitively inhibited the uptake by MATE1 and MATE2-K with inhibition constant (K(i)) values of 83 ± 15 and 56 ± 11 nmol/l, respectively. The uptake of NMN by human kidney brush border membrane vesicles with a H(+) gradient was saturable (K(m) 360 ± 55 µmol/l) and completely inhibited by pyrimethamine. The renal clearance of endogenous NMN was 403 ± 61 in healthy male subjects, and it was significantly decreased to 119 ± 16 ml/min/kg by an oral dose of pyrimethamine (50 mg). These results support the utility of NMN as an endogenous in vivo probe for investigating MATE1 and MATE2-K in humans.

Villin and actin in the mouse kidney brush-border membrane bind to and are degraded by meprins, an interaction that contributes to injury in ischemia-reperfusion

Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin β gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu(744)-Val(745). Recombinant forms of rat meprin B and homomeric mouse meprin A had K(m) values for villin and actin of ∼1 μM (0.6-1.2 μM). The k(cat) values varied substantially (0.6-128 s(-1)), resulting in different efficiencies for cleavage, with meprin B having the highest k(cat)/K(m) values (128 M(-1)·s(-1) × 10(6)). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.

Molecular basis of aberrant expression of alkaline phosphatase in renal brush border membrane from renal cell carcinoma patients

Abstract The incidence of renal cell carcinoma (RCC) has been increasing worldwide. It continues to present a diagnostic and therapeutic challenge. It accounts for approximately 3% of adult malignancies and 90-95% of neoplasm arising from kidney. RCC originates from proximal tubular epithelium of adult kidney. Alkaline phosphatase is abundantly expressed on the BBM and serves as an excellent marker for its integrity. Earlier reports have demonstrated that the activities of brush border membrane (BBM) enzymes are altered in cancer cells. The present study was conducted to find out the molecular basis of altered expression/activity of ALP in BBM from RCC in comparison to normal renal BBM. Total 30 histopathologically confirmed cases of RCC were included in the present study. The specific activity of ALP was drastically reduced in homogenate as well as in BBM from RCC as compared to BBM from adjacent normal kidney parenchyma. SDS-PAGE study showed that the BBM proteins of higher molecular weights were poorly expressed in BBM from RCC as compare to BBM from adjacent normal kidney parenchyma. Incubation of gel with BCIP/NBT dye showed that the expression of ALP in BBM from RCC was reduced as compared to adjacent normal kidney BBM. Western blot analysis using anti ALP antibody also confirmed the reduced expression of ALP in RCC BBM. Further, to check whether this reduced expression of ALP occurs at mRNA level, a semi quantitative RT-PCR was done, which showed markedly reduced expression of ALP mRNA in RCC as compared to adjacent normal kidney.

Structure–activity studies on novel polymyxin derivatives that carry only three positive charges

Polymyxin B and colistin are pentacationic lipopeptides that possess a cyclic heptapeptide portion, a linear tripeptide portion, and a fatty acyl tail. They are used, in spite of nephrotoxicity, to treat infections caused by extremely multiresistant Gram-negative bacteria. We have recently developed novel derivatives, that carry three cationic charges only. Some of them, including NAB739, are directly antibacterial whereas others, including NAB7061, lack the direct activity but sensitize bacteria to other antibiotics. NAB739 and NAB7061 differ from the old polymyxins in their renal handling and have reduced affinity to kidney brush border membrane. To further study the structure–activity relationships, we here synthesized eight additional derivatives and tested their antibacterial activity. NAB751 carries methylheptanoyl as the fatty acyl instead of octanoyl in NAB739 and was as active as NAB739, whereas NAB750 with dodecanoyl was less active. NAB781 and NAB782 with the linear peptide portion Ser-DSer and Ser-Ser-DSer, respectively, were less active than NAB739 that carries Thr-DSer. NAB771 with Thr at position 8 in the cyclic portion (instead of Dab in NAB7061) and Thr-Dab as the linear peptide portion (instead of Thr-Abu in NAB7061), resembled NAB7061 in its activity. However, replacement of two Dab residues in the cyclic portion with Thr greatly decreased the activity, even though the loss of the cationic charges was compensated by introducing two Dab residues in the linear portion. These findings reveal that subtle structural modifications have a major effect on the antibacterial activity and that it is possible to design numerous tricationic polymyxin derivatives that are antibacterial.

Abstract B28: Molecular basis of aberrant expression of alkaline phosphatase in renal brush border membrane from renal cell carcinoma patients

Abstract The incidence of renal cell carcinoma (RCC) has been increasing worldwide. It continues to present a diagnostic and therapeutic challenge. It accounts for approximately 3% of adult malignancies and 90% to 95% of neoplasm arising from kidney. RCC originates from proximal tubular epithelium of adult kidney. Alkaline phosphatase is abundantly expressed on the BBM and serves as an excellent marker for its integrity. Earlier reports have demonstrated that the activities of brush border membrane (BBM) enzymes are altered in cancer cells. The present study was conducted to find out the molecular basis of altered expression/activity of ALP in BBM from RCC in comparison to normal renal BBM. Total 30 histopathologically confirmed cases of RCC were included in the present study. The specific activity of ALP was drastically reduced in homogenate as well as in BBM from RCC as compared to BBM from adjacent normal kidney parenchyma. SDS-PAGE study showed that the BBM proteins of higher molecular weights were poorly expressed in BBM from RCC as compare to BBM from adjacent normal kidney parenchyma. Incubation of gel with BCIP/NBT dye showed that the expression of ALP in BBM from RCC was reduced as compared to adjacent normal kidney BBM. Western blot analysis using anti-ALP antibody also confirmed the reduced expression of ALP in RCC BBM. Further, to check whether this reduced expression of ALP occurs at mRNA level, a semiquantitative RT-PCR was done, which showed markedly reduced expression of ALP mRNA in RCC as compared to adjacent normal kidney. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B28.

Novel polymyxin derivatives are effective in treating experimental Escherichia coli peritoneal infection in mice

Novel synthetic polymyxin derivatives including NAB737 and NAB739 are as effective as polymyxin B in vitro against the common opportunistic pathogen Escherichia coli. Another derivative, NAB7061, lacks direct antibacterial action but sensitizes E. coli to several other antibacterial agents including macrolides. The renal handling of NAB739 and NAB7061 in rats differs from that of polymyxin B. Furthermore, the affinities of NAB739 and NAB7061 for isolated rat kidney brush border membrane are significantly lower than that of polymyxin B. Here we investigate the in vivo antibacterial effect of these compounds. The polymyxin derivatives were evaluated in an experimental murine peritonitis model. Immunocompetent mice were infected intraperitoneally with E. coli IH3080 and were subcutaneously treated with NAB737, NAB739 or NAB7061. A >4.0 log(10) reduction in bacterial load compared with saline control was achieved 6 h after initiation of treatment with 1 mg/kg of NAB739 twice or 4 mg/kg of NAB737 twice. Combination therapy with NAB7061 (5 mg/kg) twice and erythromycin (10 mg/kg) resulted during the same time course in a >2.0 log(10) reduction in bacterial load compared with saline control. Neither NAB7061 nor erythromycin was effective as monotherapy. Together with the ability to reduce bacterial load, the NAB compounds also improved the clinical status of the mice. We found that the three novel synthetic polymyxin B derivatives had a potent in vivo bactericidal effect against E. coli.

Potent and Specific Inhibition of mMate1-Mediated Efflux of Type I Organic Cations in the Liver and Kidney by Pyrimethamine

This report describes a potent and selective inhibitor of multidrug and toxin extrusion (MATE) protein, pyrimethamine (PYR), and examines its effect on the urinary and biliary excretion of typical Mate1 substrates in mice. In vitro inhibition studies demonstrated that PYR is a potent inhibitor of mouse (m)Mate1 (K(i) = 145 nM) among renal organic cation transporters mOctn1 and mOctn2 (K(i) > 30 microM), mOct1 (K(i) = 3.6 microM), and mOct2 (K(i) = 6.0 microM). PYR inhibited the uptake of metformin by kidney brush-border membrane vesicles (BBMVs) (K(i) = 41 nM) and canalicular membrane vesicles in the presence of outward gradient of H+. PYR treatment significantly increased the kidney-to-plasma ratio of tetraethylammonium, and both the liver- and kidney-to-plasma ratios of metformin in mice, whereas it did not affect their plasma concentrations and urinary excretion rates. Furthermore, the plasma lactate concentration, a biomarker for inhibition of gluconeogenesis by metformin, was significantly higher in the PYR-treated group than in the control group. These results not only suggest the importance of mMate1 in the efflux of organic cations into the urine and bile in mice but also the importance of canalicular efflux mediated by MATE proteins for the therapeutic efficacy of metformin. PYR is a potent inhibitor of human (h)MATE1 and hMATE2-K (K(i) = 77 and 46 nM, respectively) and H+ and organic cation exchanger in human kidney BBMVs (K(i) = 31 nM) in the presence of outward gradient of H+. Taken together, PYR can be used as a potent probe inhibitor of human MATE transporters.

Inhibition of Neutral Endopeptidase 24.11 does not Potentiate the Improvement in Glycemic Control Obtained with Dipeptidyl Peptidase-4 Inhibition in Diabetic Goto–Kakizaki Rats

The incretin hormone glucagon-like peptide-1 (GLP-1) has a number of effects (reviewed in [1]), which has made it a very attractive basis for the development of new therapies for type 2 diabetes. The effects include actions on the pancreatic islets as well as, for example, beneficial actions on the cardiovascular system [2]. The native peptide is, however, not clinically useful due to its rapid cleavage in vivo by dipeptidyl peptidase-4 (DPP-4) [3], whereby the peptide loses its insulinotropic action. By promoting the beneficial effects of endogenous GLP-1, DPP-4 inhibitors have shown great therapeutic potential [4], and therefore, have been implemented in the treatment of type 2 diabetes. DPP-4 is, however, not the only enzyme involved in the degradation of GLP-1. In vitro studies have shown that several enzymes located in the kidney brush border membrane are able to cleave GLP-1 [5], and although they are probably not all relevant in the in vivo metabolism of GLP-1, studies have indicated that at least neutral endopeptidase 24.11 (NEP) seems to be of importance. NEP is widely distributed [6], and is able to cleave GLP-1 at several sites in the mid-region and the COOH-terminal part of the peptide [7]. Furthermore, a physiological role of NEP in the degradation of GLP-1 has been demonstrated in the anaesthetized pig [8].

Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.

BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects

Guanylin and uroguanylin are small cysteine-rich peptides involved in the regulation of fluid and electrolyte homeostasis through binding and activation of guanylyl cyclases signaling molecules expressed in intestine and kidney. Guanylin is less potent than uroguanylin as a natriuretic agent and is degraded in vitro by chymotrypsin due to unique structural features in the bioactive moiety of the peptide. Thus, the aim of this study was to verify whether or not guanylin is degraded by chymotrypsin-like proteases present in the kidney brush-border membranes. The isolated perfused rat kidney assay was used in this regard. Guanylin (0.2 microM) induced no changes in kidney function. However, when pretreated by the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI - 1.0 microM; guanylin - 0.2 microM) it promoted increases in urine flow (DeltaUF of 0.25 +/- 0.09 mL.g(-1)/min, P < 0.05) and Na+ excretion (% Delta ENa+ of 18.20 +/- 2.17, P < 0.05). BTCI (1.0 microM) also increased %ENa+ (from 22.8 +/- 1.30 to 34.4 +/- 3.48, P < 0.05, 90 minutes). Furthermore, BTCI (3.0 microM) induced increases in glomerular filtration rate (GFR; from 0.96 +/- 0.02 to 1.28 0.02 mL.g(-1)/min, P < 0.05, 60 minutes). The present paper strongly suggests that chymotrypsin-like proteases play a role in renal metabolism of guanylin and describes for the first time renal effects induced by a member of the Bowman-Birk family of protease inhibitors.

Crystal structure of aminopeptidase N from human pathogen Neisseria meningitidis

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and peptides for maturation, activation, or degradation, and therefore are involved in numerous biological processes.1,2 They are broadly distributed throughout all kingdoms of life and are found in subcellular organelles, cytoplasm, and in membrane-bound fractions.3 Many aminopeptidases use a set of conserved residues within a structural scaffold to form an active site capable of binding either one or two divalent metal ions that aid catalysis. Zn+2, Co+2, and Mn+2 are being the most common metals found in the active site.4–8 One of more extensively studied members of the aminopeptidase family is aminopeptidase N (APN) [alternative names: alanine aminopeptidase; aminopeptidase M; microsomal aminopeptidase; GP150; CD13; (EC 3.4.11.2)]. The APN sequence family is large and broadly distributed and includes members found in bacteria and eukaryotes, including plants and mammals. PsiBlast search identified over 1000 APN family members. Typically APNs are monomeric or homodimeric. In higher eukaryotes these enzymes are expressed in many tissues, with the highest level found in the intestinal and kidney brush border membranes, brain, lung, blood vessels, and primary cultures of fibroblasts. The sequence analysis indicates that aminopeptidase N is a member of the M1 family of the MA clan of peptidases, also termed gluzincins.5 The amino acid sequence fingerprints of the M1 family of zinc-metallopeptidases are the HEXXH(X18)E (a zinc binding motif) and GXMEN (an exopeptidase motif).9 Prominent members of this family include mammalian membrane-bound aminopeptidases [P-LAP, aminopeptidase A (APA), thyrotropin-releasing hormone degrading enzyme (TRHDE)], cytosolic proteins [puromycin-sensitive aminopeptidase (PSA) and leukotriene A4 hydrolase (LTA4H)], and secretory proteins such as [adipocyte-derived leucine aminopeptidase (A-LAP) and aminopeptidase B (APB)].5,9 The APNs catalyze liberation of N-terminal amino acids from a broad spectrum of substrates including small peptides, amide, or arylamide. The N-terminal residue is a preferably neutral or basic amino acid, although it has been reported that an intact XPro dipeptide was released when the terminal hydrophobic residue was followed by a prolyl residue.10 The diversity of function that APNs play depends on their location and source tissue. Some APNs have been used commercially, such as the APN from Lactococcus lactis, which has been used in the food industry.11 Aminopeptidases N are also present in many pathogenic bacteria and represent potential drug targets.9 In this article, we report the crystal structure of APN from N. meningitides at 2.05-A resolution.

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Study of bradykinin metabolism by rat lung tissue membranes and rat kidney brush border membranes by HPLC with inductively coupled plasma—mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry

The coupling of the techniques, high-performance liquid chromatography (HPLC), orthogonal acceleration time-of-flight mass spectrometry (OATOF-MS) and inductively coupled plasma mass spectrometry (ICP-MS) provides a very powerful method for identifying and quantifying the products of bradykinin metabolism. In this study, we were able to identify the major metabolites of bradykinin degradation reported in the literature. In addition, a new bradykinin metabolite corresponding to bradykinin 5,9 fragment (BK-(5,9)-fragment) was identified as a product of neutral endopeptidase (NEP) activity. This finding establishes that NEP cleaves bradykinin simultaneously at the positions 4-5 and 7-8. We also demonstrate the equivalent participation of NEP and angiotensin-converting enzyme (ACE) within the rat lung tissue membranes (RLTM) in bradykinin degradation, suggesting its suitability as a model for the assay of dual ACE/NEP inhibitors. On the contrary, in rat kidney brush border membranes (KBBM), ACE is not significantly involved in bradykinin metabolism, with NEP being the major enzyme.