Long-term monitoring of renal function in poly-traumatized intensive care patients.

INTRODUCTION: For the long-term monitoring of kidney function, polytraumatized patients were examined and routine as well as specialized parameters were compared. MATERIALS AND METHODS: 30 patients of the Surgical Intensive Care Unit (ICU) were examined daily over the entire period they stayed in the ICU. The patients were retrospectively classified as either survivors or deceased patients. Group 1 consisted of 20 patients who resided in the ICU for 11-15 (Median 14) days before they could be transferred to a normal hospital unit. Group 2 consisted of 10 patients who had passed away after 13-18 (Median 16) days in the ICU. In addition to the routine parameters diuresis, serum creatinine and serum urea, specialized parameters for kidney function including the excretion rates of alpha1-microglobulin (alpha1-MG), N-Acetyl-beta-D-glucosaminidase (NAG), angiotensinase A (ATA) and immunoglobulin G (IgG) were determined. RESULTS: Similar biometric data were shown by all patients at admission into the ICU, but differences did exist regarding the Revised Trauma Score, Injury Severity Score and the APACHE-II-Score. In the period between the 5th and 8th day of intensive treatment almost all patients showed pathological excretion rates of tubular and glomerular parameters whereby no increased frequency of unusual events could be determined at these time-points. CONCLUSION: During treatment in the ICU, all examined patients showed at times pathological excretion rates of specialized kidney function parameters. Such transient damage was only apparent in a few of the patients when the standard parameters serum creatinine and serum urea were employed. In 90% of the surviving patients the kidney parameters had normalized until the time they were transferred, indicating that such parameters reflected the general state of health of these patients.

The relation between the psychological functioning of children with Down syndrome and their urine peptide levels and levels of serum antibodies to food proteins.

AIMS: To investigate the relation between psychological functioning of subjects with Down syndrome, and their levels of urine peptide and serum antibodies to food proteins. METHODS: 55 children with Down syndrome in a cross-sectional study. Psychological functioning was measured by the Stanford-Binet Intelligence Scale: Fourth Edition, McCarthy Scales of Children's Abilities and Fagan's computer based test of novelty preference. RESULTS: The participants, and their siblings, were found to have significantly increased total urine peptide levels. There were no significant correlations between peptide levels and psychological functioning. Significantly increased levels of IgG activity to gliadin and gluten, and IgA activity to gliadin, gluten and casein were found. There were significant negative correlations (Spearman r = -0.13 to -0.51) between psychological functioning, and IgG and IgA activity to gliadin and gluten. CONCLUSIONS: A significant relation between antibodies to gluten and psychological functioning was documented. The mechanism and potential causal link are still unknown.

Tubular urinary enzymes in acute post-infectious glomerulonephritis.

Tubular function of 17 pediatric patients with a mild form of acute post-infectious glomerulonephritis was prospectively evaluated by assessment of the urinary activity of proximal and distal tubule enzymes. Neutral-like endopeptidase (NEP-like) and angiotensin-converting enzyme (ACE) were the proximal tubule enzymes assessed, while prolyl-endopeptidase (PE) and serine-endopeptidase H1 and H2 were the distal tubule enzymes analyzed. Urine was collected at diagnosis (T0) and after 2 (T2) and 6 (T6) months of follow-up. NEP-like enzyme activity (nmol/mg creatinine; median+/-quartile range) was increased at diagnosis, and this remained stable during the first 6 months (T0 18.30+/-83.26, T2 17.32+/-49.56, T6 23.38+/-107.18). Urinary activity of the other enzymes was as follows: ACE (mU/ml per mg creatinine) T0 0.08+/-0.16, T2 0.06+/-0.10, T6 0.18+/-0.29; PE (nmol/mg creatinine) T0 6.70+/-84.87, T2 9.55+/-69.00, T6 13.67+/-28.70; serine-endopeptidase H1 (nmol/mg creatinine) T0 7.86+/-26.95, T2 17.17+/-59.37, T6 18.19+/- 79.14; and serine-thiol-endopeptidase H2 (nmol/mg creatinine) T0 3.06+/-21.97, T2 12.06+/-32.42, T6 16.22+/- 44.06. Thirty other healthy children matched for age and gender were considered as a control group. This group was assessed once and the results were: NEP-like activity 6.05+/-10.54, ACE 0.11+/-0.22, PE 7.10+/-13.36, H1 5.00+/-17.30, and H2 6.00+/-20.16. In conclusion, we observed that NEP-like and H1 enzymes exhibited significant increased urinary activity 6 months after the diagnosis. This increase occurred in spite of the disappearance of clinical symptoms, which occurred 2 months after the diagnosis. We believe that the increase in urinary enzymatic activity could be a manifestation of a silent tubular dysfunction following an episode of acute post-infectious glomerulonephritis.

Circulating adhesion molecules and neutral endopeptidase enzymuria in patients with urolithiasis and hydronephrosis.

OBJECTIVES: To measure the concentrations of adhesion molecules (intercellular adhesion molecule-1 ¿ICAM-1, E-selectin, and L-selectin) in the serum of patients with renal stones and patients with hydronephrosis caused by obstructive ureteral stones. Renal tubular enzymes were examined from their urine samples to evaluate whether neutral endopeptidase (NEP) behaved as traditional tubular enzyme markers (N-acetyl-beta-glucosaminidase ¿NAG and beta-galactosidase ¿beta-GAL) in their disease state. METHODS: Three groups were studied. Group 1 included 15 normal volunteers, group 2 included 12 patients with ureteral stones and ipsilateral hydronephrosis, and group 3 included 17 patients with renal stones in one kidney without hydronephrosis or hydrocalycosis. A single, overnight fasting blood and urine sample was collected from each subject. Serum levels of ICAM-1, E-selectin, and L-selectin were measured by enzyme-linked immunosorbent assay. NEP, NAG, and beta-GAL were measured from urine samples, and the enzyme activities were expressed per gram of creatinine. RESULTS: Serum levels of ICAM-1 were higher in groups 2 and 3 (522 +/- 95 and 329 +/- 42 ng/mL, respectively), but the differences were only significant between group 2 and group 1 (263 +/- 32 ng/mL) and group 2 and group 3 (P <0.05). Serum levels of L-selectin were lower in group 3 and were significantly different when compared with groups 1 and 2 (P <0. 05). The serum levels of E-selectin were not significantly different among these three groups. Urinary levels of NEP were lower in group 2, although the levels of NAG and beta-GAL were more elevated than in group 1. CONCLUSIONS: Serum levels of ICAM-1 were elevated in patients with unilateral hydronephrosis caused by a ureteral stone, and E-selectin and L-selectin levels did not change significantly. These findings suggest that ICAM-1 may play a role in renal immune injury to ureteral obstruction. The urinary NEP values were lower in patients with hydronephrosis caused by ureteral stones, and the traditional lysosomal enzymes were increased. The lower urinary NEP values might suggest impairment of ipsilateral renal function. Renal stones per se resulted in no significant changes in serum adhesion molecule levels, although the levels of L-selectin were significantly decreased.

Characterization of a kinin inactivating serine endopeptidase H2 (kininase) from human urine using fluorogenic substrates.

We have previously described a kinin-inactivating endopeptidase (H2), which was purified 19-fold from human urine by DEAE-cellulose chromatography and gel filtration. The enzyme was inhibited 100% by PMSF, TPCK and pOHMB. In the present communication, we further characterized this enzyme using the fluorogenic substrates Abz-RPPGFSPFRQ-EDDnp (Abz-BKQ-EDDnp) and Abz-FRQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl] ethylenediamine). Also a rapid, sensitive and specific assay for the H2 was developed. The enzyme hydrolyzed bradykinin (BK = RPPGFSPFR) at the F-S peptide bond, differing from the cleavage site F-R, in the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FRQ-EDDnp. Other enzymes present in urine as the serine endopeptidase H1, prolyl endopeptidase and neutral endopeptidase-like were not able to hydrolyze the related substrate Abz-FRQ-EDDnp. The determined Km for Abz-BKQ-EDDnp and Abz-FRQ-EDDnp were 0.79 microM and 3.02 microM, respectively. Using the fluorogenic substrates, we observed that PMSF and p-hydroxymercuribenzoate irreversibly inhibited the enzyme H2. E-64 was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. The inhibition observed in the presence of pOHMB was partially reversed by 2 mM cysteine. These results suggest that the H2 enzyme belongs to the subfamily of SH-containing serine proteases. Based on the molecular weight of isolated H2 (60 kDa), we believe that this enzyme originated from the kidney and may cleave the kinins filtered through the glomerulus and also that produced in the kidney.

Activation of the kallikrein kinin system in interstitial cystitis.

PURPOSE: We investigated whether the kallikrein kinin system is activated in interstitial cystitis by measuring urinary excretion rates of kinin peptides, active and total kallikrein, and the kininase neutral endopeptidase in women with interstitial cystitis. We compared these excretion rates to a control group of women with stress incontinence and normal bladder function. MATERIALS AND METHODS: Catheter urine was collected from subjects during a water diuresis (approximately 10 ml. per minute) before and after distention of the bladder with 100 ml. water. The contribution of the bladder wall to urinary kinins was assessed by measuring the change in kinin levels after 2 minutes of bladder stasis before and after distention. RESULTS: Absolute bradykinin and kallidin excretion rates were similar in women with interstitial cystitis and control subjects. Two minutes of bladder stasis after bladder distention increased urinary bradykinin (p = 0.02) but not kallidin excretion rates. Active and total kallikrein excretion rates were similar in patients with interstitial cystitis and control subjects. Neutral endopeptidase excretion rates were reduced in the initial urine collection from subjects with interstitial cystitis but were similar in both groups during later collection periods. CONCLUSIONS: These data provide evidence for increased bradykinin levels in the bladder wall of subjects with interstitial cystitis, which may be due in part to reduced neutral endopeptidase levels. These increased bradykinin levels may participate in the pathogenesis and symptomatology of interstitial cystitis.

Alteration in insulin-like growth factor-binding proteins (IGFBPs) and IGFBP-3 protease activity in serum and urine from acute and chronic renal failure.

The insulin-like growth factors, IGF-I and IGF-II, are proteins that promote cellular growth and differentiation of various organs, including the kidney. These peptides interact with high affinity cell surface receptors and bind to a family of IGF-binding proteins (IGFBPs). Altered serum and urinary IGFBP patterns in children with chronic renal failure have been previously described. In this study, we evaluated serum and urinary IGFBP profiles in acute renal failure patients (ARF; n = 10) and chronic renal failure patients (n = 10), using Western ligand blots. Most patients with acute or chronic renal failure showed decreased intact serum IGFBP-3 and increased serum IGFBP-2. Both groups displayed marked urinary IGFBP alterations, including increased urinary IGFBP-1 and totally absent urinary IGFBP-3, as detected by Western ligand blot. To evaluate altered IGFBP profiles, we performed IGFBP-3 protease assays with sera and urine from renal failure patients and normal controls. Although control urine had only minor protease activity (defined by the ability to degrade [125I]IGFBP-3), significant protease activity was found in urine from renal failure patients. The proteolytic pattern and susceptibility to protease inhibitors in most renal failure urine samples were the same as those seen in normal urine and with plasmin. Protease activity was completely inhibited by serine protease inhibitors. We speculate that urinary protease activity is mediated primarily by a serine protease(s), which may be involved in the modulation of renal IGF activity in health and disease.

Insulin-like growth factor binding protein-3 protease activity in the urine of children with chronic renal failure.

The insulin-like growth factors, IGF-1 and IGF-II, are polypeptides that potentiate cellular growth. In addition to binding to specific cell surface receptors, the IGFs bind with high affinity to a family of proteins, the insulin-like growth factor binding proteins (IGFBPs). Serum and urine IGFBP patterns are altered in individuals with chronic renal failure (CRF). We recently reported that the urinary IGFBP pattern of CRF patients is unique for increased insulin-like growth factor binding protein-1 (U-IGFBP-1) levels. In this study, we used western ligand blotting (WLB), western immunoblotting (WIB), and radioimmunoassay (RIA) to further evaluate serum and urine IGFBP profiles of children with CRF (n = 14). Five patients with CRF displayed decreased serum IGFBP-3 profiles by WLB. Serum IGFBP-3 WIB profiles were remarkable for 30- and 20-kDa fragments of IGFBP-3 not seen in control serum. Serum IGFBP-3 levels, as determined by RIA, were slightly elevated. Serum levels of IGFBP-2 also were increased, although not at a level reaching statistical significance. WLB of CRF urine revealed a large increase in U-IGFBP-1 and a complete absence of urinary IGFBP-3. Recent studies of serum from pregnant women and seminal plasma have demonstrated a similar absence of intact IGFBP-3, due to the presence of a specific IGFBP-3 protease. To evaluate whether an IGFBP-3 protease accounts for the absence of intact U-IGFBP-3 in children with CRF, urine and serum samples from individuals with CRF and controls were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Human seminal plasma proteinase inhibitor: action toward some trypsin-like arginine amidases from humans.

Human seminal plasma trypsin-like proteinase inhibitor (HSTPI) was separated and examined by trypsin Cellulofine affinity adsorption and Cellulofine GCL-300 gel filtration and its inhibitory action toward some arginine amidases obtained from the urine, semen, and blood of humans. HSTPI showed strong inhibitory action toward two types of human seminal plasma basic arginine amidases (BHSAA-L and -A), human seminal plasma acidic arginine amidase with affinity to lima bean trypsin inhibitor (LBTI) column (AHSAA-L), and human acrosin and thrombin. Conversely, no or little inhibition was observed toward human urinary arginine amidase-2, human high molecular weight urokinase, or human seminal plasma acidic arginine amidase with affinity to aprotinin column (AHSAA-A, tissue kallikrein). Measurement of Ki values of BHSAA-L with affinity to LBTI column toward HSTPI and LBTI revealed that the arginine amidase had a stronger affinity for LBTI than that for HSTPI. This indicates that it is the difference in Ki values that allows BHSAA-L to be separated by the LBTI affinity adsorption method from human seminal plasma containing a large amount of HSTPI.

Atrial natriuretic factor and liver disease.

A working formulation for the role of ANF in the sodium retention of cirrhosis is summarized in Figure 4. Sodium retention is initiated early in cirrhosis, either as a result of hepatic venous outflow block or of primary vasodilation. The consequent intravascular volume expansion causes increases in ANF levels. At this stage of disease, the rise in ANF level is sufficient to counterbalance the antinatriuretic influences. However, this occurs at the expense of an expanded intravascular volume with the potential for overflow ascites. With progression of disease, disruption of intrasinusoidal Starling forces and loss of volume from the vascular compartment into the peritoneal compartment occur. This underfilling of the circulation may attenuate further increases in plasma ANF and promotes the activation of antinatriuretic factors. At this later stage of disease, elevated levels of ANF are insufficient to counterbalance antinatriuretic influences. Thus the role of ANF in cirrhosis is primarily beneficial in that it successfully attenuates the antinatriuretic forces in the compensated stage. Raised ANF levels have two potential deleterious effects. First, ANF may exacerbate arterial vasodilation, leading to further sodium retention. The primacy of vasodilatation has been proposed as an alternate formulation to the overflow and underfill hypotheses. Second, Epstein et al. found higher basal ANF levels in cirrhotic patients with edema than in those patients without edema. ANF is known to reduce plasma volume in anephric animals and to increase the ultrafiltration coefficients of isolated capillaries. Therefore it is conceivable that in the clinical setting in which antinatriuretic factors limit the renal responsiveness to ANF but in which ANF levels are elevated (i.e., cirrhosis, congestive heart failure, primary kidney disease), ANF itself may contribute to edema formation at the level of the peripheral microcirculation. In general, ANF likely has no primary role in the sodium retention in cirrhosis. In early compensated cirrhosis, ANF may maintain sodium homeostasis despite the presence of mild antinatriuretic factors. In late ascitic cirrhosis renal resistance to ANF develops, rendering it ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients.

The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The other Proteus proteinases had similar patterns but slightly different mobilities. In each case all proteinase activity in culture supernatants was demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be associated with only the triple-band complex; all three bands were proteolytically active. The P. mirabilis proteinase was resistant to inhibitors of both serine and thiol proteinases but strongly inhibited by metal chelators, although it was not affected by phosphoramidon, an inhibitor of the thermolysin group of bacterial metalloproteinases. Active proteinase was detected in urine samples from P. mirabilis-infected patients; this is consistent with our detection of immunoglobulin A fragments of a size suggestive of P. mirabilis proteinase activity.

Endopeptidase and carboxypeptidase activities in human urine which hydrolyze bradykinin.

1. We have fractionated the bradykinin inactivating activity of human urine by stepwise elution chromatography on DEAE-cellulose and recovered 95% of the inactivating activity and 29% of the protein (absorbance at A280 nm). 2. Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin converting activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides. 3. Cleavage sites demonstrated for Fractions A through G were: Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pro7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G). 4. The relative molecular weight of the bradykininase activity present in each fraction, determined by gel filtration, was: 16 kDa (A), 70 kDa (B), 60 kDa (C), 88 kDa (D), 230 kDa (E), 45 kDa (F) and 49 kDa (G). 5. Bradykinin inactivating activity was inhibited 50-100% by 3 mMEDTA (A, B, D, E and G), 1 mMM 2-mercaptoethanol (A, B, C and G), 0.1 microM Hg2+ (A, C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Zn2+ (C), 60 microM BPP5a and 40 microM BPP9a (D), 0.1 microM phosphoramidon (E) and 3 mM sodium p-hydroxymercuribenzoate (G). 6. The properties of some of these bradykinin inactivating activities correspond to enzymes previously described in urine and tissues: carboxypeptidases (Fractions A and B), angiotensin I converting enzyme (Fraction D), neutral endopeptidase (Fraction E). However, the chymotrypsin-like activity of Fractions C and F and the prolylendopeptidase activity of Fraction G have not been described before in urine and they are being purified in order to obtain a more accurate characterization.

Further characterization of endopeptidase H2 a serine proteinase from human urine.

A human urine serine proteinase chymotrypsin like hydrolyzes the peptide bonds: Phe-Ser (kinin); Gly-Gly, Leu-Arg, Phe-Lys (neuropeptides) and Gln-Gln (substance P). Endopeptidase H2 hydrolyzes better oligopeptides with 4 to 18 aminoacid residues than larger peptides, it does not hydrolyzes kininogen or proenkephalin. The enzyme behaves as an oligoendopeptidase.

Characterization of dipeptidyl and tripeptidyl aminopeptidases in human kidney soluble fraction.

In an attempt to identify improved biochemical markers for the diagnosis of early kidney damage via urinary analysis of kidney derived enzymes, we have undertaken the systematic identification, quantification and characterization (following purification via anion exchange and gel filtration chromatography, and preparative electrophoresis) of the dipeptidyl and tripeptidyl aminopeptidases in normal human kidney soluble extract (with which the majority of the cellular activity of these enzymes is associated). Four chromatographically separable enzyme types were identified as follows (% relative activity in parentheses): dipeptidyl aminopeptidase I (EC 3.4.14.1; 24%); dipeptidyl aminopeptidase II (EC 3.4.14.2; 8%); dipeptidyl aminopeptidase IV (EC 3.4.14.5; 61%); tripeptidyl aminopeptidase (unclassified; 7%). Comparison of the levels of activity for the above enzyme types in normal and pathological urine may lead to an improvement upon existing procedures for the early detection of renal damage.

Proteolytic activity in mouse urine: relationship to the kidney metallo-endopeptidase, meprin.

Meprin, a brush border kidney metallo-endopeptidase is present as the major endopeptidase in mouse urine. The enzyme is freely soluble and can be detected enzymically or immunologically. Mice can be partitioned into two phenotypes that differ by 10-20-fold in the amount of meprin in kidney membranes; this phenotypic variation is reflected in urinary activities. We propose a role for meprin in the degradation of other urinary proteins.

Nutritional status of men under hypokinesia.

Hypokinesia (diminished muscular activity) elicits substantial changes in energy and nutritional requirements in humans. The objective of this investigation was to determine the nutritional status of six physically healthy men aged 19 to 21 years under 95 days of hypokinesia without the use of any preventive measures. For the simulation of the hypokinetic effect the men were placed under 95 days of an ad libitum bed rest regimen. During the background period, that is, prior to exposure to hypokinesia, the caloric value of the diet was 3124 kcal per day; under hypokinesia, the caloric value was 2745 kcal per day. In calculating the nutritional requirements of men under hypokinesia, several biochemical parameters were measured. Effects of hypokinesia demonstrated included certain changes in the enzymatic activity of glands; increased excretion of nitrogenous compounds in the urine; increased blood cholesterol content; changes in the amount of blood sugar; changes in acid-base balance; increased elimination of fluid, calcium, and phosphorus; impaired supply of vitamins to the organism; and increased energy expenditure. It was concluded that the nutritional status of men underwent substantial changes under conditions of diminished muscular activity.

Urinary excretion of neutral proteinases in nephrotic rats with a glomerular disease.

A proliferative glomerulonephritis was induced in rats pre-immunized with rabbit IgG by injecting intravenously a sub-nephrotoxic dose of rabbit anti-glomerular basement membrane (GBM) IgG (A rats). Most rats (80%) developed a severe proteinuria (greater than 100 mg/24 hr) within two to five days after the injection of anti-GBM IgG. At the same time, microscopic examination of the kidneys revealed a glomerular infiltration by mononuclear phagocytes and a prominent decrease in the intensity of the colloidal iron reaction in glomeruli. A non-proliferative glomerular disease was induced in another group of rats (B rats) by intraperitoneal administration of aminonucleoside of puromycin. A marked proteinuria (greater than 100 mg/24 hr) occurred after six days in 90% of animals. Histochemical studies then revealed a decrease in staining intensity of glomeruli for polyanion. No glomerular hypercellularity was noted. In normal rats and in non-proteinuric A or B rats, the 24 hour urinary excretion of neutral proteinases ranged from 1.4 to 7.8 units (mean value +/- SEM, 4.69 +/- 0.60, N = 11), that of laminin ranged from 100 to 3,900 ng (mean value +/- SEM, 1,154 +/- 325, N = 10), and that of type IV collagen ranged from 160 to 420 ng (mean value +/- SEM, 306 +/- 26.5 ng, N = 8). In proteinuric rats from groups A (N = 11) and B (N = 9), the 24 hour urinary excretion of neutral proteinases significantly increased (mean values +/- SEM, 38.55 +/- 8.66 U for A rats and 42.17 +/- 7.92 U for B rats) and ran parallely with that of proteins, laminin and type IV collagen.(ABSTRACT TRUNCATED AT 250 WORDS)