Limited capacity of proximal tubular proteolysis in mice with proteinuria.

Albuminuria is associated with the additional loss in the urine of small molecular weight proteins normally degraded by the proximal convoluted tubule (PCT), and competition for binding to the megalin/cubilin reuptake system has been considered the likely cause. We have previously reported that deficiency of the intrinsic lysosomal protein Limp-2 causes tubular proteinuria due to reduced fusion of endosomes with lysosomes in the PCT leading to inadequate proteolysis. To determine whether this mechanism also contributes to the tubular proteinuria induced by albumin overload in normal mice, wild-type (WT) mice received daily BSA injections intraperitoneally for 10 days, using untreated Limp-2(-/-) mice as positive controls for inadequate proteolysis. BSA overload induced significant urinary loss of megalin and cubilin ligands in WT mice. Tubular uptake of Alexa-conjugated BSA, administered by intravenous injection, was not reduced in the PCT of mice receiving intraperitoneal BSA. Expression of the tubular protein receptor megalin was also unchanged. There was a delay in proteolysis of reabsorbed proteins in WT mice receiving BSA, evidenced by an increased quantity of retinol-binding protein (RBP) in the kidney cortex, increased basal distribution of endocytosed RBP in cells of the PCT, and persistence of exogenous Alexa-conjugated BSA and RBP after injection. Upregulation of cathepsin L and normal fusion of lysosomes with endosomes were apparently not sufficient to maintain normal clearance of endocytosed proteins. The data suggest that in the presence of competition from albumin overload, reabsorption of filtered proteins is limited by the capacity of lysosomal degradation rather than receptor-mediated endocytosis.

Expression of the transmembrane lysosomal protein SCARB2/Limp-2 in renin secretory granules controls renin release.

BACKGROUND/AIMS: Renin processing and storage is believed to occur in lysosome-like structures in the afferent arteriole. SCARB2/Limp-2 is a transmembrane lysosomal protein responsible for the intracellular trafficking of ß-glucocerebrosidase. This study aimed to confirm the expression of SCARB2/Limp-2 in renin secretory granules, and explore its role in renin processing and secretion. METHODS: Co-localisation studies of (pro)renin with lysosomal membrane proteins, SCARB2/Limp-2, LAMP-1 and LAMP-2, were performed in mouse and human kidney sections. Intrarenal expression and secretion of (pro)renin in wild-type (WT) and Limp-2(-/-) mice were compared with and without stimulation. RESULTS: SCARB2/Limp-2, LAMP-1 and LAMP-2 co-localised with (pro)- renin in mouse and human kidney. Plasma renin concentration was increased in Limp-2(-/-) mice when compared to WT littermates. No change in (pro)renin expression, however, was observed in Limp-2(-/-) mouse kidney cortex by immunofluorescence microscopy, Western blotting, quantitative RT-PCR or the ultrastructural appearance of renin secretory granules. Acute stimulation of renin release by isoprenaline or hydralazine was similar in WT and Limp-2(-/-) mice. Following chronic salt restriction, however, immunofluorescence microscopy showed less (pro)renin expressed in Limp-2(-/-) compared with WT mouse kidneys, and there was significantly less prorenin but not renin by Western blotting in Limp-2(-/-) mouse kidney cortex, despite no difference in circulating renin levels. CONCLUSION: Renin secretory granules possess integral lysosomal proteins, confirming that they are indeed modified lysosomes. Limp-2 deficiency leads to a minor increase in circulating renin. Limp-2, however, is not required for acute or chronic stimulation of renin release.

Tubular proteinuria in mice and humans lacking the intrinsic lysosomal protein SCARB2/Limp-2.

Deficiency of the intrinsic lysosomal protein human scavenger receptor class B, member 2 (SCARB2; Limp-2 in mice) causes collapsing focal and segmental glomerular sclerosis (FSGS) and myoclonic epilepsy in humans, but patients with no apparent kidney damage have recently been described. We now demonstrate that these patients can develop tubular proteinuria. To determine the mechanism, mice deficient in Limp-2, the murine homolog of SCARB2, were studied. Most low-molecular-weight proteins filtered by the glomerulus are removed in the proximal convoluted tubule (PCT) by megalin/cubilin-dependent receptor-mediated endocytosis. Expression of megalin and cubilin was unchanged in Limp-2(-/-) mice, however, and the initial uptake of injected Alexa Fluor 555-conjugated bovine serum albumin (Alexa-BSA) was similar to wild-type mice, indicating that megalin/cubilin-dependent, receptor-mediated endocytosis was unaffected. There was a defect in proteolysis of reabsorbed proteins in the Limp-2(-/-) mice, demonstrated by the persistence of Alexa-BSA in the PCT compared with controls. This was associated with the failure of the lysosomal protease cathepsin B to colocalize with Alexa-BSA and endogenous retinol-binding protein in kidneys from Limp-2(-/-) mice. The data suggest that tubular proteinuria in Limp-2(-/-) mice is due to failure of endosomes containing reabsorbed proteins to fuse with lysosomes in the proximal tubule of the kidney. Failure of proteolysis is a novel mechanism for tubular proteinuria.

A global multi-hazard risk analysis of road and railway infrastructure assets.

Transport infrastructure is exposed to natural hazards all around the world. Here we present the first global estimates of multi-hazard exposure and risk to road and rail infrastructure. Results reveal that ~27% of all global road and railway assets are exposed to at least one hazard and ~7.5% of all assets are exposed to a 1/100 year flood event. Global Expected Annual Damages (EAD) due to direct damage to road and railway assets range from 3.1 to 22 billion US dollars, of which ~73% is caused by surface and river flooding. Global EAD are small relative to global GDP (~0.02%). However, in some countries EAD reach 0.5 to 1% of GDP annually, which is the same order of magnitude as national transport infrastructure budgets. A cost-benefit analysis suggests that increasing flood protection would have positive returns on ~60% of roads exposed to a 1/100 year flood event.

Characterizing the learning curve for a basic laparoscopic drill.

BACKGROUND: The psychomotor challenges inherent in laparoscopic surgery are evident by the steep procedural "learning curves" documented throughout the literature. Few methods have been described to evaluate learning curves. The cumulative summation (CUSUM) method is a criterion-based evaluation of the learning process. The purpose of this study is to describe the CUSUM learning curves for a simple task for individuals and for a group of novice laparoscopists. METHODS: Sixteen medical students undertook four weekly sessions of 10 laparoscopic pegboard transfers in the MISTELS system. Their performance was scored and recorded for each trial. CUSUM learning curves were constructed based on the goal of achieving mean scores for senior, intermediate, or junior laparoscopists >or=95% of the time. RESULTS: Based on senior criteria, one student achieved the goal by the 40th peg transfer trial. Based on intermediate criteria, three students achieved the goal by their 40th trial (trials 21 and 36), and for junior criteria, 10 students achieved the acceptable success rate by their 40th trial (range, 26-40). CONCLUSION: CUSUM analysis suggests criterion-based practice is useful for novice laparoscopists. It allows educators to track the progress of an individual toward target criteria for each MISTELS task, to more logically allocate time for training and set attainable goals, to objectively evaluate trainee acquisition of basic laparoscopic skills, and to identify trainees who need remediation.

Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase.

Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)P(OPh)2 is the most potent inhibitor for granzyme A, and Z-LysP(OPh)2 is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)P(OPh)2 was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)P(OPh)2, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)P(OPh)2 (kobs/[I] = 2220 M-1 s-1) was quite specific with much lower inhibition rates for granzyme K and trypsin (kobs/[I] = 3 and 97 M-1 s-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO2-Gly-Pro-(4-AmPhGly)P(OPh)2 with a second-order rate constant of 3650 M-1 s-1. The most potent inhibitor for granzyme K was 3, 3-diphenylpropanoyl-Pro-(4-AmPhGly)P(OPh)2 with a kobs/[I] = 1830 M-1 s-1; all other phosphonates inhibited granzyme K weakly (kobs/[I] < 60 M-1 s-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz-LysP(OPh)2 as the best inhibitor (kobs/[I] = 89 M-1 s-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.

Evaluating laparoscopic skills: setting the pass/fail score for the MISTELS system.

BACKGROUND: The McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) was developed to assess laparoscopic skills and to score them objectively. This system has been described previously. The purpose of the current study was to determine a pass/fail threshold. METHODS: In this study, 165 individuals were tested and grouped according to their clinical competency in laparoscopic surgery. The noncompetent group consisted of medical students and surgical residents in their first 2 years of training (n = 83). The competent group consisted of chief general surgical residents in their last year of training, laparoscopy fellows, and practicing laparoscopic surgeons (n = 82). The Mann-Whitney U test was used to evaluate differences in task performance between the two groups. RESULTS: There was a significant difference in total scores and individual MISTELS task scores between the noncompetent and competent laparoscopic surgeons (189 vs 372.5; p <0.0001). By setting specific pass/fail total score thresholds (cutoff scores), competent surgeons can be discriminated from noncompetent surgeons. CONCLUSION: An objective pass/fail evaluation can be given to individuals tested with the MISTELS system.

The prevention of vitamin D deficiency in the elderly.

Vitamin D deficiency is common in the house-bound and institutionalised elderly population of Britain. A study of patients over 65 years discharged with a diagnosis of osteomalacia from Greater Glasgow Health Board hospitals between 1970 and 1981 inclusive showed a low incidence in the 65 to 74 years age group but a steeply rising incidence in older age groups. The majority (83%) of patients were female. The fortification of margarine, butter and milk with concentrations of vitamin D acceptable to the general population does not produce significant elevations in serum 25-hydroxyvitamin D (25-OHD) levels in vitamin D-deficient elderly patients. Low intensity background ultraviolet radiation (UVR) and intermittent high intensity UVR produce significant elevations in serum 25-OHD levels in elderly patients but both methods have disadvantages which limit their widespread use. Vitamin D supplements equivalent to 10 micrograms daily produce significant elevations in serum 25-OHD levels in vitamin D-deficient elderly patients. A vitamin D supplement policy for the housebound and institutionalised elderly population of Britain is required.

Perforin-enhancing protein, a low molecular weight protein of cytotoxic lymphocyte granules, enhances perforin lysis.

Perforin is a 68 kD protein found in the granules of cytotoxic lymphocytes and is used by lymphocytes to form lethal pores in the membranes of the cells they kill. We and others have found that when perforin is purified, its lytic activity is markedly reduced. ELISAs indicated that our final recovery of perforin protein was excellent. We decided to determine if depletion of other granule proteins contributed to the loss of lytic activity. We isolated perforin to the point where lytic activity was diminished and added back granule proteins that had no lytic activity or detectable (antigenic) perforin. Perforin was isolated by Cu2+-immobilized metal affinity chromatography (IMAC) followed by phenyl-Superose hydrophobic interaction chromatography (HIC). Its lytic activity was enhanced by a low molecular weight (<15 kD) protein, perforin enhancing protein (PEPr). We have isolated PEPr by two methods, HIC and MonoQ. Nonlytic PEPr restored perforin to close to its original lytic activity. A protein similar if not identical to PEPr was also detectable as an 125I-labeled protein associated with lytic perforin. We propose that PEPr acts in conjunction with perforin to form lethal pores and suggest that PEPr may be the rat equivalent of the human cytotoxic lymphocyte protein, granulysin.

Perforin lytic activity is controlled by calreticulin.

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.

Purification and characterization of lymphocyte chymase I, a granzyme implicated in perforin-mediated lysis.

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.

Interaction between a Ca2+-binding protein calreticulin and perforin, a component of the cytotoxic T-cell granules.

Calreticulin is a component of cytotoxic T-lymphocyte and NK lymphocyte granules. We report here that granule-associated calreticulin terminates with the KDEL endoplasmic reticulum retrieval amino acid sequence and somehow escapes the KDEL retrieval system. In perforin knock-out mice calreticulin is still targeted into the granules. Thus, calreticulin will traffic without perforin to cytotoxic granules. In the granules, calreticulin and perforin are associated as documented by (i) copurification of calreticulin with perforin but not with granzymes and (ii) immunoprecipitation of a calreticulin-perforin complex using specific antibodies. By using calreticulin affinity chromatography and protein ligand blotting we show that perforin binds to calreticulin in the absence of Ca2+ and the two proteins dissociate upon exposure to 0.1 mM or higher Ca2+ concentration. Perforin interacts strongly with the P-domain of calreticulin (the domain which has high Ca2+-binding affinity and chaperone function) as revealed by direct protein-protein interaction, ligand blotting, and the yeast two-hybrid techniques. Our results suggest that calreticulin may act as Ca2+-regulated chaperone for perforin. This action will serve to protect the CTL during biogenesis of granules and may also serve to regulate perforin lytic action after release.