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.

Bacterial control of host gene expression through RNA polymerase II.

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coli develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II­dependent (Pol II­dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coli 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II­dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur.

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.

Urinary N-acetyl-beta-D-glucosaminidase as a diagnostic marker of acute pyelonephritis in children.

INTRODUCTION: Prompt diagnosis and localization of pyelonephritis are of great importance in children. The urinary excretion of enzymes, and in particular N-acetyl-beta-D-glucosaminidase (NAG), is considered a simple noninvasive marker for detection of renal tubular dysfunction due to pyelonephritis. This study was performed to determine the diagnostic value of urinary NAG in acute pyelonephritis. MATERIALS AND METHODS: In a quasi-experimental study conducted on 72 children with confirmed pyelonephritis, we measured urinary NAG, creatinine, and NAG-creatinine ratio before and after the treatment. Diagnostic values of these parameters were evaluated by considering the patients before and after the treatment as disease-positive and disease-negative groups, respectively. RESULTS: The patients were 18 boys (25.0%) and 54 girls (75.0%) with a mean age of 43.0 +/- 39.0 months. The mean levels of urinary NAG were 12.20 +/- 6.14 U/L and 5.46 +/- 7.98 U/L before and after the treatment, respectively (P < .001). The sensitivity and specificity of urinary NAG-creatinine ratio for diagnosis of pyelonephritis were 73.6% and 77.3%, respectively, with a cutoff point of 10.16 U/g (area under the curve = 0.76, 95% confidence interval, 0.67 to 0.76). Significantly higher levels of urinary NAG were found in those who had a negative urine culture at diagnosis (8.8 +/- 10.4 U/L) compared to those with a positive urine culture (4.5 +/- 8.7 U/L). CONCLUSIONS: We concluded that urinary NAG is elevated in children with pyelonephritis and it can be considered as a further criterion in the diagnosis of upper urinary tract infection.

Expression of COX-2 in normal and pyelonephritic kidney, renal intraepithelial neoplasia, and renal cell carcinoma.

OBJECTIVES: The role of inflammation in carcinogenesis is unknown. To determine the relationship between cyclooxygenase 2 (COX-2) expression, inflammation, and carcinogenesis in human renal cell carcinoma (RCC), we looked for COX-2 expression in normal and pyelonephritic kidney, renal intratubular neoplasia (RIN), and RCC tissues. METHODS: COX-2 expression was assessed immunohistochemically in tissues obtained from 20 pyelonephritic kidneys, 16 normal kidneys, 19 RIN, and 75 RCC cases. RESULTS: COX-2 expression was found to be positive in 64% of RCCs. It was positive in 13 chronic pyelonephritic (65%), 9 normal (56%), and 15 RIN (79%) cases. COX-2 expression was significantly higher in RCC and RIN than the normal and pyelonephritic cases (p<0.001 and p<0.001, respectively). No statistically significant difference was noted between RCC and RIN cases. CONCLUSIONS: Although the function of COX-2 in tumor development has not been exactly elucidated, the increased expression of COX-2 in RIN and RCC might be a factor that may play a role in the development of RIN or progression to RCC, which warrants further research.

[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.

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.

[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.

Uropathogenic Escherichia coli toxins induce caspase-independent apoptosis in renal proximal tubular cells via ERK signaling.

BACKGROUND: Pyelonephritis is a risk factor for renal tubular epithelial cell damage. Recent studies have shown that Escherichia coli and/or its toxins may stimulate apoptotic cell death in renal tubular cells, but the underlying molecular mechanisms remain to be elucidated. METHODS: Confluent LLC-PK(1) cells were exposed to E. coli toxins from overnight cultures of the uropathogenic O6K13H1 (O6) and the nonpathogenic W3110. The cell death was studied with morphological and biological assay. RESULTS: E. coli soluble toxins from uropathogenic O6:K13:H1(O6) strain were found to induce apoptosis in a dose- and time-dependent manner in LLC-PK1 cells. The expression of FasR and the phosphorylation of ERK1/2 were significantly upregulated by O6 soluble toxins in a time-dependent manner. Cell death was completely inhibited by two specific ERK1/2 inhibitors, but not by a broad caspase inhibitor, zVAD-fmk, implicating a caspase-independent pathway via ERK. Moreover, we found that lysophosphatidic acid could trigger a survival signal through G-proteins and PI3K. CONCLUSION: We demonstrate that apoptosis induced by uropathogenic E. coli toxins is dependent on ERK1/2. Caspases, although being activated, are not necessary for cell death, and they act after the ERK signaling at which point cells become committed to cell death or can be rescued.

Polymorphonuclear elastase as a diagnostic marker of acute pyelonephritis in children.

OBJECTIVE: Experimental evidence suggests that neutrophils and their metabolites play an important role in the pathogenesis of pyelonephritis. The aim of this study was to investigate the diagnostic value of polymorphonuclear elastase-a(1)-antitrypsin complex (E-a(1)-Pi) for the detection of acute pyelonephritis in children. METHODS: Eighty-three patients, 29 boys and 54 girls, 25 days to 14 years of age, with first-time symptomatic urinary tract infection were prospectively studied. Fifty-seven healthy children served as controls. Dimercaptosuccinic acid (DMSA) scan and voiding cystourethrography were performed in all patients. Plasma and urinary E-a(1)-Pi, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), neutrophil count, urinary N-acetyl-beta-glucosaminidase (NAG), N-acetyl-beta-glucosaminidase b (NAG b), and creatinine levels were measured in all patients on admission and 3 days after the introduction of antibiotics. The same markers were also measured in the control subjects. RESULTS: Planar DMSA scintigraphy demonstrated changes of acute pyelonephritis in 30 of 83 children (group A). It was normal in the remaining 53 children (group B). The sex and age distributions were not significantly different between the 2 groups, as well as between the patients and the control subjects (group C). Nineteen of the 53 children with a normal DMSA had body temperature >/=38 degrees C, whereas all but 4 children with abnormal DMSA had temperature >/=38 degrees C. Therefore, the temperature was significantly different between these 2 groups. The sensitivity and specificity of fever (>/=38 degrees C) as an indicator of renal involvement based on isotopic findings were 86% and 64%, respectively. Given the significant number of the febrile children with normal DMSA scintiscans, group B was subdivided into B(1) with 19 febrile children (14 boys and 5 girls) and B(2) with 34 children whose body temperature was below 38 degrees C (8 boys and 26 girls). The sex and age distribution was significantly different between groups B(1) and B(2). The mean age of group B(1) was.78 years (range: 28 days to 9 years; median:.25 years; standard deviation: 2.1). All but 1 child in this group were younger than 1 year of age. In contrast, in group B(2), there were only 4 infants, the remaining 30 children were older than 2.5 years (mean age: 6 years; median: 7 years; standard deviation: 3.5; range: 34 days to 12 years). The mean duration of fever before hospital admission was 2.8 days for group A and 1.8 days for group B(1). This difference was not statistically significant. Similarly, body temperature was not significantly different between these 2 groups. The distribution of plasma E-a(1)-Pi values was normal in the control subjects. The sensitivity and specificity of plasma E-a(1)-Pi, as an indicator of renal involvement, were 96% and 50%, respectively, taking the 95th percentile of the reference range as a cutoff value. However, considering as a cutoff value the level of 72 microg/dL (95th percentile of group B(2)), its sensitivity and specificity were 74% and 86%, respectively. Plasma E-a(1)-Pi levels were significantly elevated in group A compared with group B and in both groups, the plasma E-a(1)-Pi values were significantly higher than in the control subjects. A significant difference also was noticed between group A and each of the subgroups B(1) and B(2) and also between the subgroups themselves. Plasma E-a(1)-Pi concentrations correlated significantly with neutrophil count in groups A (r =.3), B (r =.4), and B(2) (r =.46), but the correlation was not significant in group B(1.) ESR levels showed, among the different groups, similar differences with those of E-a(1)-Pi values. Unlike E-a(1)-Pi, CRP levels were comparable between groups A and B(1), which both consisted of febrile children. Neutrophil count was not significantly different between subgroups B(1) and B(2). (ABSTRACT TRUNCATED)

[Homeostatic and antioxidant defense enzymes activity in chronic pyelonephritis]. / Pokazateli gomeostaza i funktsional'noe sostoianie fermentov antioksidantnoi zashchity pri khronicheskom pielonefrite.

As shown by the analysis of chronic pyelonephritis, manifestations in patients with high and low activity of antioxidant system enzymes, patients with high catalase activity have more effective defense of red cells, glomerular epithelium. This was concluded from marked differences in urinary excretion of metabolites, hemoglobin levels compared to those in patients with low activity of catalase. In low activity of glutathione peroxidase, inflammation gains activity, nonspecific resistance weakens, excretion of some metabolites inhibits vs patients with high activity of this enzyme. The data provide criteria for diagnosis of antioxidant system defects and show necessity of antioxidant drugs inclusion into the therapeutic scheme (unitiol, antioxidant complex of vitamins A, E, C, P).

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.

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.

The possible role of granulocyte elastase in renal damage from acute pyelonephritis.

During acute inflammatory processes, extracellular release of granulocyte elastase can contribute to subsequent tissue damage. To test our hypothesis that extracellular elastase release during acute pyelonephritis may contribute to subsequent renal parenchymal damage, we compared the intracellular and extracellular activities of the lysozyme elastase of human polymorphonuclear cells (PMN) when incubated in vitro with bacterial strains causing renal infection that led to either renal damage or no damage. Urine bacterial cultures were obtained from patients with acute pyelonephritis (flank pain, costovertebral angle tenderness, fever > 38 degrees C, bacteriuria, pyuria, and leukocytosis). Renal damage was demonstrated by cortical scarring on followup intravenous pyelography and/or diminished function on 131iodine hippuran renal scan. Mean extracellular elastase activity (mu units/PMN) was 0.15 for unstimulated PMN, 0.07 for PMN stimulated by bacteria not associated with renal damage, and 1.20 for the PMN stimulated by strains associated with renal damage. Mean intracellular elastase activity (mu units/PMN) was 3.73 for unstimulated PMN, 3.48 for PMN stimulated by bacteria not associated with renal damage, and 3.31 for the PMN stimulated by strains associated with renal damage. Extracellular granulocyte elastase activity was thus significantly higher (P = 0.0001) in PMN stimulated by bacterial strains associated with renal damage. Extracellular release of elastase may contribute to the pathogenesis of renal damage in pyelonephritis.

[The metabolic aspects of the sequelae of the accident at the Chernobyl Atomic Electric Power Station in the course of kidney diseases]. / Metabolichni aspekty naslidkiv avariï na ChAES v perebihu zakhvoriuvan' nyrok.

As many as 159 patients with chronic pyelonephritis and 100 those with glomerulonephritis residing in ecologically favourable, and on contaminated territories for 6-8 years after Chernobyl accident, were examined for indices of lipid peroxidation, antioxidant system and energy exchange. The above renal disease were found to be accompanied by a rise in the activity of lipid peroxidation and accumulation of excess of their products in cellular membranes and urine. In the antioxidant system, the activity of NAD-H2-dependent factors and an inhibitor of free radical oxidation of superoxiddismutase tends to decrease. There occur changes in isoenzyme spectrum of lactate and malatedehydrogenases. The above shifts appear to be manifest in those persons having been exposed to low radiation doses. Antioxidants and stabilizers of the biological membranes were shown to be useful in correcting the above metabolic processes.

Pyelonephritis: renal urokinase and sialidase (neuraminidase) activity in rats fed a standard laboratory diet.

Renal stone formation can be caused by many different and varied disturbances, some of which are poorly understood. The relationship between urinary infection and renal stone formation has not been completely clarified. It is argued that renal stones form primarily as a consequence of the hydrolysis of urea by the bacterial enzyme urease. However, no explanation is given for microorganisms that produce urease only occasionally or not at all. The question arises as to whether the infection-induced microorganisms might not be playing a double role in renal stone formation by not only producing urease, but also by affecting in vivo urokinase (UK) and sialidase (SA) activity. With this in mind, the effect of Escherichia coli on renal UK and SA activity has been studied in male rats with a normal diet. The renal UK (P = 0.208) and SA (P = 0.2135) activities did not differ significantly between the two kidneys of the same rat. In contrast, when drainage from one kidney of a rat was externally obstructed, the UK and SA activities differed significantly between kidneys (P < 0.015). An increase in UK (r = 0.6456, P < 0.0001) and SA (r = 0.7507, P < 0.0001) activity was observed over time in the obstructed kidney. Subcutaneous injections with E coli reduced the UK activity of the obstructed kidney significantly (p = 0.017). However, the SA activity remained the same (P = 0.3929).(ABSTRACT TRUNCATED AT 250 WORDS)