Shuta Kubo

Suppression of renal scarring by prednisolone combined with ciprofloxacin in ascending pyelonephritis in rats

To prevent renal scarring, which occurs at the end stage of chronic pyelonephritis due to vesicoureteral reflux of infected urine, immediate antimicrobial treatment is reported to be essential. When treatment is delayed, the antimicrobial agent is believed to be effective only in eliminating bacteria, not in preventing scar formation. Using the ascending pyelonephritis model in rats, we investigated the effect of immediate or delayed treatment with ciprofloxacin and that of delayed treatment with a combination of ciprofloxacin and prednisolone in preventing renal scarring following infection. An inoculum of 1 x 10(9) colony forming units (cfu)/0.1 ml. of the HM32 strain of Escherichia coli, which was isolated from a patient with a urinary tract infection, was injected directly into the rat bladder, and the urethra was clamped for 4 hours in each rat. Treatment by ciprofloxacin (15 mg./kg., twice a day for 5 days) alone or in combination with prednisolone (2 mg./kg., once a day for 4 days) was initiated 6 or 72 hours after bacterial inoculation. The kidneys of each rat were examined 6 weeks later. Immediate treatment by ciprofloxacin significantly inhibited renal scarring (no scarring was seen in any of the 8 rats), but delayed treatment had no effect on scarring (4 of 8 rats showed scarring) when compared with the untreated controls (7 of 8 rats showed scarring). However, the addition of prednisolone to the delayed treatment with ciprofloxacin significantly inhibited renal scarring (only 1 of 10 rats showed scarring) when compared with the untreated controls (7 of 8 rats showed scarring). These data suggest that prednisolone is effective in preventing renal scarring which occurs due to vesicoureteral reflux when the initiation of antimicrobial treatment is delayed.

Enhanced chemiluminescence response of polymorphonuclear leukocytes by new quinolone antimicrobials.

Some of the many antimicrobial agents (beta-lactams, macrolides, aminoglycosides, tetracyclines, new quinolones; NQs) were reported to have a bactericidal or bacteriostatic effect cooperating with host defense mechanisms including polymorphonuclear neutrophils (PMNs). We investigated the effect of new quinolone antimicrobials on chemiluminescence (CL) response of human PMNs. Among many NQs, we chose ofloxacin, lomefloxacin, fleroxacin, sparfloxacin, AM-1155, NM-394, Q-35, Y-26611 and T-3761. Twenty-five or 100 micrograms/ml of fleroxacin and ofloxacin enhanced luminol-dependent CL response of PMNs up to 1.5-2.0 times compared to the drug free condition. Other antimicrobial agents, however, inhibited CL response. This suggested that fleroxacin and ofloxacin were more efficient in the treatment of bacterial infections with respect to the interaction between antimicrobials and PMNs.

Fleroxacin Enhancement of Superoxide Production by Polymorphonuclear Leukocytes: The Role of Protein Kinases

New quinolone (NQ) antimicrobials may influence the functions of polymorphonuclear leukocyes (PMNs). Fleroxacin (FLRX), one of the newer NQs which has a long half-life in blood and a strong bactericidal effect, was examined for its influence on superoxide production by PMNs. Augmentation of superoxide production by PMNs when stimulated with phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (fMLP) was observed following the addition of 25, 50, 100 and 200 micrograms/ml of FLRX. In addition, the effects of staurosporine and H-7, inhibitors of protein kinase C (PKC), and of genistein, a tyrosine kinase (TK) inhibitor, on FLRX-enhanced superoxide production were examined. Superoxide production augmented by FLRX was diminished by the addition of staurosporine and H-7, when PMNs were stimulated with PMA, and by the addition of genistein, when PMNs were stimulated with fMLP. These results suggest that FLRX augments superoxide production by PMNs through enhancing the activities of phosphorylation by PKC or TK within the signal transduction pathway in PMNs.

Preventive Effect of Ulinastatin on Renal Scarring in Rat Model of Pyelonephritis Induced by Direct or Ascending Infection with Serratia marcescens or Escherichia coli

Renal scarring is considered to be a characteristic of reflux nephropathy. The effects of ulinastatin, a strong inhibitor of polymorphonuclear leukocyte elastase, on renal scarring following direct parenchymal or intravesical ascending infection by Serratia marcescens or Escherichia coli were determined. Four days of treatment with ulinastatin initiated 2 or 5 days after infection prevented renal scarring. Doses of 1,000-4,000 units/kg inhibited renal scar formation, but 8,000 units/kg did not. These results suggest that it may be possible to limit renal scar formation in pyelonephritis by the use of an appropriate pharmacologic agent.

Effect of Ebselen on Renal Scarring in Rats following Renal Infection

Renal scarring, which occurs following refluxing pyelonephritis, is considered to be involved in the development of reflux nephropathy. Prevention of renal scar formation requires immediate initiation of antimicrobial treatment; treatment delay results in renal scarring. We demonstrate that Ebselen, an antioxidant agent, given at a dose of 15 mg/kg twice a day prevents renal scarring in rats following direct renal parenchymal bacterial inoculation. In addition, using an ascending pyelonephritis model, which clinically resembles refluxing pyelonephritis in humans, we show that when initiation of antimicrobial treatment was delayed, coadministration of Ebselen prevents renal scar formation. These results show that Ebselen is effective in preventing renal scarring and suggest that the clinical use of this drug may prevent renal scar formation following pyelonephritis and progression to reflux nephropathy.

Renal scarring by mannose-sensitive adhesin of Escherichia coli type 1 pili.

Most Escherichia coli isolates from patients with pyelonephritis possess both pap (mannose-resistant) pili and type 1 (mannose-sensitive) pili. In the experimental pyelonephritis model of rats, the mannose-sensitive-piliated strain caused severe renal scarring, whereas the mannose-resistant or nonpiliated strain did not. Type 1 pili consist of several subunits; one major subunit and other minor subunits. One of the minor subunits, adhesin, is responsible for mannose-sensitive adhesion to eukaryotic cells. The role of adhesin was examined in scar formation after infection with a newly constructed adhesin-deficient mutant which has pilus structure but cannot agglutinate guinea pig erythrocytes. A mutant plasmid, pYMZ84, containing a deletion in the adhesin gene of type 1 pili, failed to agglutinate guinea pig erythrocytes even though the bacteria expressed pili morphologically indistinguishable from those produced by plasmid pSH2, carrying the intact genes for the type 1 pili. E. coli harboring pYMZ84 caused negligible or minimal renal scarring, whereas E. coli harboring pSH2 caused severe renal scarring in rats. These data suggest that the mannose-sensitive adhesin of type 1 pili stimulates renal scarring.

Influence of Hyperosmotic Environment Comparable to the Renal Medulla Upon Membrane NADPH Oxidase of Human Polymorphonuclear Leukocytes

Hyperosmotic environment in the renal medulla seems important for bacterial pyelonephritis because it exerts inhibitory influences upon the function of polymorphonuclear leukocytes (PMN). Urea and NaCl primarily contribute to high osmolarity in the renal medulla. We previously reported that PMN function was actually suppressed in phagocytosis, intracellular bacterial killing and superoxide generation in the hyperosmotic solution of urea and NaCl. In the present report, to verify the mechanism of this inhibitory effect, a kinetic study for NADPH oxidase in the cell membrane, the key enzyme complex of superoxide generation, was carried out in the cell membrane-solubilizing system under the hyperosmotic condition caused by urea or NaCl. Urea directly denaturated NADPH oxidase, and its inhibitory mechanism was reversible and uncompetitive with a decrease in Vmax and Km, while NaCl had no effect upon it, maintaining Lineweaver-Burk plots in the same position as those of the control. This result suggests that urea at least produces an inhibitory effect upon PMN through the direct inactivation of NADPH oxidase, although NaCl was unable to do so.

Effect of Prednisolone on Renal Scarring in Rats Following Infection with Serratia marcescens

Renal scarring is considered a criterion of reflux nephropathy and the end stage of pyelonephritis. Prednisolone, a strong anti-inflammatory drug, at doses of 1 or 2 mg/kg prevented renal scarring in rats following infection with Serratia marcescens. Four or 8 mg/kg of prednisolone, however, did not inhibit renal scar formation. In a time course experiment, renal scarring was prevented when 4-day treatment with prednisolone was initiated 2, 5, or 13 days after infection. These results show that prednisolone is effective in preventing such scarring and suggest the clinical use of this drug for preventing renal scar formation after pyelonephritis and reflux nephropathy.

Effect of pili of Serratia marcescens on superoxide production and phagocytosis of human polymorphonuclear leukocytes

PURPOSE To determine the role played by superoxide in renal scar formation following renal infection. MATERIALS AND METHODS The piliation of bacteria was assessed for its capacity to interact with human polymorphonuclear leukocytes (PMNs). Two recombinant strains having either MS or MR pili of Serratia marcescens were constructed. RESULTS The MS-piliated strain stimulated superoxide production of PMNs twice as much as the MR- or nonpiliated strains did. The MS-piliated strain was more susceptible to phagocytosis than was the MR- or nonpiliated strain. CONCLUSION These data suggest that the MS-piliated strain stimulates superoxide production of PMNs associated with phagocytosis, which leads to tissue damage in infected organs.

Effect of prednisolone on ascending renal infection due to biofilm disease and lower urinary tract obstruction in rats

A model of renal infection due to lower urinary tract obstruction and biofilm disease was constructed for the study of renal scarring by inserting glass beads coated with bacterial biofilm into the bladder of rats and then clamping the urethra. We previously reported the effect of antimicrobial therapy used in combination with the anti-inflammatory agent prednisolone to prevent renal scarring. In this study we investigated the effect of prednisolone on renal scar formation using our new model. Renal scarring could not be prevented in the group in which prednisolone was administered in the period during which the urethra was regularly being clamped. In contrast, scarring was prevented in the group that began to receive prednisolone after the period of clamping had ended. Therefore, in cases of lower urinary tract obstruction prednisolone should only be administered for the prevention of renal scarrring after the obstruction has been resolved.