Developmental loss, but not pharmacological suppression, of renal carbonic anhydrase 2 results in pyelonephritis susceptibility.

Carbonic anhydrase II knockout (Car2-/-) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2-/- and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2-/- versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2-/- kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.

Mitochondria-Associated Matrix Metalloproteinases 2 and 9 in Acute Renal Pathologies.

Activities of MMP-2 and MMP-9 in the cytoplasm and mitochondria of kidney cells were evaluated on the models of acute renal pathologies: pyelonephritis, rhabdomyolysis, and ischemia/reperfusion of the kidney. In acute pyelonephritis, a significant increase in the level of MMP-2 and MMP-9 in kidney cells and the appearance of mitochondrial MMP-2 isoform with a lower molecular weight, but still exhibiting proteolytic activity were observed. A direct correlation between the level of MMP-2 and MMP-9 in the kidney and the severity of inflammation in pyelonephritis was revealed. Obviously, the appearance of active protease in the mitochondria of the kidney cells could have an impact on their functioning and, generally, on the fate of renal cells in this pathology.

An endogenous ribonuclease inhibitor regulates the antimicrobial activity of ribonuclease 7 in the human urinary tract.

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has a broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7, but regulation of its antimicrobial activity has not been well defined. Here, we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on the antimicrobial activity of RNase 7. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time polymerase chain reaction showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.

The antibacterial shield of the human urinary tract.

Antibacterial peptides and proteins maintain the sterility of the human urinary tract. A broad-spectrum antimicrobial protein, ribonuclease 7 (RNase 7), previously discovered to play a role in controlling the growth of bacteria on human skin, has now been shown to have an important antibacterial function in the human urinary tract.

Ribonuclease 7, an antimicrobial peptide upregulated during infection, contributes to microbial defense of the human urinary tract.

The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and enzyme-linked immunosorbant assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection and has antibacterial activity against uropathogens at micromolar concentrations.

[Antilysozyme activity of microorganisms causing acute and chronic pyelonephritis].

Etiological structure of urine microflora was studied in patients with acute and chronic pyelonephritis. Gram-negative microorganisms dominated. Antilysozyme activity of 175 bacterial strains was studied. Detectability and potency of persisting potential were assessed regarding infectious-inflammatory process course. High antilysozyme activity of bacteria was revealed. This indicates potential ability of the bacteria to persist in the patients.

Urinary N-acetyl-beta-glucosaminidase and the selection of children for radiologic evaluation after urinary tract infection.

Urinary levels of N-acetyl-beta-glucosaminidase (NAG) were measured in 147 consecutively enrolled children younger than 13 years of age with urinary tract infection to determine whether elevated levels were a predictor of urologic abnormalities. The children were classified as having cystitis if results of 0 or 1 of the following tests were positive and as having pyelonephritis if results of greater than or equal to 2 tests were positive: (1) temperature greater than 38 degrees C, (2) serum C-reactive protein greater than 1 mg/dL, (3) erythrocyte sedimentation rate greater than 25 mm/h, and (4) 1-deamino-8-D-arginine vasopressin-renal concentrating protein less than 810 mOsm/kg. Urinary NAG to creatinine ratios did not distinguish cases of cystitis from those of pyelonephritis. Urinary NAG was useful in identifying children with cystitis who had vesicoureteral reflux of grades II through V. Of 6 children with cystitis and vesicoureteral reflux, 5 had levels of NAG more than 1 SD above the mean, whereas of 75 children without vesicoureteral reflux, only 15 had such an elevation (P = .003). Of those children with a normal NAG level, 60 (98.4%) had normal radiologic evaluation results, and only 1 child (1.6%) had vesicoureteral reflux. Levels of NAG did not identify children with pyelonephritis who had vesicoureteral reflux. It is concluded that (1) urinary NAG is of no value in localizing the site of urinary tract infection, and (2) an NAG level within 1 SD of the mean in a child with cystitis indicates a low risk of urologic abnormalities, and radiologic evaluation may be omitted unless infection recurs.

Oxygen-dependent and -independent mechanisms of renal injury in experimental ascending pyelonephritis.

Pyelonephritis is the most common urinary tract infection affecting females of all age groups. Despite concerted efforts the mechanism of renal injury in pyelonephritis is not clearly understood. In the present study we have made an attempt to characterise the mediators of inflammatory insult in an experimental model of ascending pyelonephritis. Mice infected with Escherichia coli O6:K13:H1 were sacrificed at 2, 7 and 14 days post-infection. Luminol-dependent chemiluminescence response, NADPH oxidase, acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase activities were monitored in circulating as well as renal phagocytic cells in order to determine the role of reactive oxygen species and lysosomal enzymes in genesis of renal injury. We have demonstrated that reactive oxygen species are generated at the initiation of infection and the levels increase progressively during the course of infection. While intracellular release of lysosomal enzymes was seen in all groups, extracellular release was primarily observed at 7 and 14 days post-infection only. The results indicate that while reactive oxygen species play a significant role in tissue injury during all stages of infection, lysosomal enzyme release in extracellular milieu augments tissue destruction at later stages only.

Stabilities of N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes in urine: advantage of NAG isoenzyme B measurement in clinical applications.

N-Acetyl-beta-D-glucosaminidase (NAG) is a widely used urinary enzyme for the assessment of renal diseases. We studied the stabilities of NAG isoenzymes in urine at 37 degrees C by enzyme assay and ELISA using a model simulating in vivo conditions. The stabilities were found to be affected by the pH. Under mild acidic condition (about pH 6), there was no significant loss of enzymatic activity of NAG isoenzyme A, enzymatic activity of NAG isoenzyme B and immunological activity of NAG isoenzyme B even after 8 h incubation. In contrast, under alkaline condition (about pH 8), the enzymatic activity of NAG isoenzyme A was rapidly lost, whereas both enzymatic and immunological activities of NAG isoenzyme B were maintained at more than 80% of their initial values. Also, we found that the ratios of endogenous NAG isoenzyme B to total NAG were elevated in alkaline urine samples. These results indicate that NAG isoenzyme A, which is a major isoenzyme in normal urine (pH 5-7), seems to be inactivated in alkaline urine. Our results suggest that for alkaline urine, NAG isoenzyme B should be measured to avoid misinterpretation of total NAG enzymatic activities.

Diagnostic significance of different urinary enzymes in patients suffering from chronic renal diseases.

The urinary enzymes alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), and ribonuclease (EC 3.1.4.22) were measured in 66 healthy persons and 52 patients suffering from chronic renal diseases (pyelonephritis, glomerulonephritis). The residual renal function of patients characterized by 99mTc-diethylenetriaminopentaacetate isotope clearance was only moderately reduced. Except for gamma-glutamyltransferase, patients generally showed increased urinary enzyme excretions. N-Acetyl-beta-D-glucosaminidase was more sensitive to detect renal dysfunction than the other enzymes and the conventional parameters serum creatinine, total protein excretion, and the measurement of glomerular filtration rate. The determination of this enzyme can be recommended as a suitable diagnostic parameter in nephrology.

New sensitive markers for the detection of experimental ascending pyelonephritis.

Kinetic parameters (Km and Vmax) of renal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, leucine-aminopeptidase and gamma-glutamyltranspeptidase were used as markers for the early detection of pyelonephritis. Km of all the enzymes studied remained unaltered. The Vmax of all the enzymes studied were found to be significantly decreased (p less than 0.05) 3 or 4 days postinfection and onwards in the left obstructed kidney. The Vmax of alkaline phosphatase and leucine-aminopeptidase was found to be significantly increased (p less than 0.05) in early stages and decreased (p less than 0.05) in later stages of infection in the right unobstructed kidney. No histopathological lesions confirming pyelonephritis could be seen 7 days postinfection in the left kidney and right kidney remained histopathologically unaltered. This demonstrated that BBM enzymes are much earlier disturbed as compared to histopathological changes.

Prevention of tissue injury in an ascending mouse model of chronic pyelonephritis--role of free radical scavengers.

The role of free radical scavengers in preventing the tissue injury using a non obstructive, ascending mouse model for chronic pyelonephritis was assessed. The parameters taken into consideration are Luminol Dependent Chemiluminescence (LDCL), histopathology and some biochemical investigations. We have observed that both catalase and Dimethyl-Sulfoxide (DMSO, free radical scavengers) were able to prevent the free radical mediated tissue injury and ultimate renal scarring, irrespective of the bacterial strain studied.

Urinary lactate dehydrogenase isoenzyme analysis in adult population.

This investigation was a systemic study on an adult population of urinary lactate dehydrogenase (LDH) isoenzyme analysis for the distinction between upper and lower urinary tract infections. The study included 160 urine samples from patients and healthy individuals. On the basis of clinical symptoms, urinary bacterial colony counts, renal function tests and radiologic findings, the adults were divided into pyelonephritis group, cystitis group, pelvic lesion group, and control group. This technique correctly identified 23 of 26 patients with pyelonephritis by the presence of elevated LDH-V (over 10 percent) and all of 12 patients with cystitis by the presence of elevated LDH-I (over 60 relative units) but low LDH-V (below 10 percent or lower than LDH-I). In the pelvic group, the results of eight patients were consistent with cystitis and four with pyelonephritis. Our study confirms the sensitivity and specificity of the LDH isoenzyme technique for the differential diagnosis of urinary tract infection on adult patients and is consistent with previous studies on pediatric patients. However, one should be cautious to interpret the results of LDH isoenzymogram before extra-urinary tract lesions are excluded.

Presence of immunoreactive tissue kallikrein in human polymorphonuclear (PMN) leucocytes.

The presence of tissue kallikrein in polymorphonuclear (PMN) leucocytes from normal human blood and from patients with rheumatoid arthritis and pyelonephritis was investigated. Immunoreactive tissue kallikrein was specifically detected in neutrophil leucocytes. PMN leucocytes displayed a granular staining. In the synovial membrane and kidney, the cells were aggregated into pockets as part of an inflammatory infiltrate. The presence of immunoreactive tissue kallikrein in human PMN leucocytes may provide a new insight into the importance of this enzyme in inflammation.

[Behavior of certain forms of enzymuria in subjects treated with pipemidic acid]. / Considerazioni sul comportamento di alcune enzimurie in soggetti trattati con acido pipemidico.

Three groups of subjects (I with infections of the lower urinary ways; II with chronic pyelonephritis; III healthy controls) were treated with pipemidic acid for 10 consecutive days and the behaviour of their urinary NAG and AAP was studied. It was discovered that the healthy group and the patients with infections of the lower urinary ways showed no significant variations in the urinary activity of the two enzymes following the administration of pipemidic acid. However there was a distinct reduction in both enzymes in patients with chronic pyelonephritis who presented demonstrable bacterial sensitivity to the drug. Apart from the fact that it produces no nephrotoxic effects at least in the doses used and for the treatment period adopted, the drug is therefore considered to reduce renal inflammation that is beneficial to individuals with chronic interstitial nephropathy.

[Effect of antibiotic chemotherapy on urinary N-acetyl-B-D-glucosaminidase activity in patients with chronic pyelonephritis]. / Effetti della terapia chemio-antibiotica sull'attività NAG-urinaria in pazienti affetti da pielonefrite cronica.

The behaviour of N-acetyl-B-D-Glucosaminidase (NAG) was studied in the urine of patients with chronic pyelonephritis under pharmacological treatment for renewed attacks of acute renal inflammation. It was noted that by the end of target chemo-antibiotic treatment all patients presented a reduction in the inflammation and hence the tubular distress. Once treatment was suspended several patients showed an increase tendency in the activity of the enzyme followed by the reappearance of significant bacteriuria and/or leukocyturia. The acute attack of renal inflammation would not have been recognised if the enzymuria technique had been used to monitor the patients.

[Behaviour of serum and urinary lysozyme after renal transplantation (author's transl)]. / Verhalten des Lysozyms in Serum und Harn nach Nierentrasplantation

The behaviour of serum and urinary lysozyme was investigated before and after renal transplantation in 20 patients. The mean postoperative observation time was 67.8 (10 to 212) days. In 11 patients with reversible olig-anuria due to prolonged preoperative ischaemia, lysozymuria lasted for a period of 17 days after surgery, whereas in 8 patients with immediate transplant function lysozymuria disappeared 7 days after transplantation. Serum lysozyme concentrations were markedly elevated before transplantation in all patients. In patients with transplant failure due to ischaemia, normalization of serum lysozyme levels was achieved 28 days after surgery; patients with immediate function showed normal serum lysozyme levels already 7 days after transplantation. Prolonged lysozymuria was also noticed in 2 cases with irreversible ischaemic transplant failure, in 1 case with recurrence of glomerulonephritis and in 1 further case with acute pyelonephritis in the transplant. In 7 cases with an acute renal rejection crisis, lysozymuria was evident 0.7 days before clinical diagnosis of rejection. Serum lysozyme levels showed a strong correlation with serum correlation with serum creatinine concentrations. Therefore, lysozymuria in renal transplant patients indicates tubular transplant damage of varied aetiology. Elevated serum lysozyme levels, on the other hand, seem to reflect a reduced glomerular filtration rate.

[NAG (N-acetyl-beta-D-glucosaminidase)--a sensitive marker for disorders of kidney function]. / NAG--Ein sensitiver Marker für Nierenfunktionsstörungen.

In a clinical study we tested the use of the lysosomal enzyme NAG as a parameter of kidney function. Following prospective randomization, we examined NAG excretion during cisplatin treatment with/without nephroprotection, after intravenous urography with ionic/non-ionic contrast media, during lower/upper urinary tract infections and before/after extracorporeal shockwave lithotripsy for intrarenal calculi (first-generation equipment used). Measurements were performed in 3-h urine specimens and in urine collected over 24 h, using a simple method of analysis. A correlation between NAG leakage and functional disorder of the renal tubular cells seemed likely on the basis of additional clinical and experimental data. Increases, in some cases dramatic, in NAG excretion were observed after the administration of cisplatin and ionic contrast media, in acute pyelonephritis, and after extracorporeal shockwave lithotripsy. However, the increase in NAG excretion was less impressive during cisplatin therapy when nephroprotective amino acids were infused, and in the urography group when non-ionic contrast media were used. Infections of the lower urinary tract did not increase NAG excretion. The results indicate that NAG is a sensitive marker of occult renal dysfunction, which can be checked by non-invasive techniques and can be used in a clinic setting to detect functional disorders of the kidney.

[Assessment of fructose-1,6-biphosphatase in urine of children with acute pyelonephritis]. / Ocena aktywnosci fruktozo-1,6 dwufosfatazy w moczu dzieci chorych na ostre odmiedniczkowe zapalenie nerek.

UNLABELLED: We assessed the excretion of fructose-1,6-bisphosphatase (FBP) and N-acetyl-beta-D-glucosaminidase (NAG) in 52 children (aged 4.1 +/- 2.3): group I--26 children with acute pyelonephritis (APN), in whom the examination were carried out twice: A--before treatment, B--after 14-21 days of antibacterial treatment, group II--21 healthy children. Activity of FBP in urine was found in 80% children from group I and II, and activity of NAG was found in all children from both groups. In examination A mean excretion of FBP and NAG was higher than in healthy children (p < 0.05). After antibacterial treatment excretion of both enzymes decreased to values, which did not differ from control group (p > 0.05). High correlation between FBP and NAG (r = 0.9355; p = 0.00001) was shown only in 14 children, in whom the course of acute pyelonephritis was serious (CRP > 20 mg%, leucocytosis > 10 x 10(9), and renal swelling in ultrasonography). CONCLUSION: Increased excretion of FBP in urine is found mainly in children with severe course of acute pyelonephritis, in whom the correlation between NAG and FBP is observed.

[Effect of galascorbin on activity of lactate and malate dehydrogenases and their isoenzymic spectrum in experimental pyelonephritis]. / Vlianie galaskorbina na aktivnost' laktat-, malatdegidrogenazy i ikh izofermentnyi spektr pri éksperimental'nom pielonefrite.

It is shown that per os administration of galascorbin to animals with acute experimental pyelonephritis in a dose of 100 mg/1 kg of mass per day normalizes the total activity of lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37) and their isoenzymic spectrum in kidney tissue and blood serum. The content of pyruvic and lactic acids in blood serum also normalizes, that promotes a favourable course of the disease.