Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of alpha Gal(1-4) beta Gal binding in virulence of a wild-type strain.

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.

Recruitment of renin gene-expressing cells in adult rat kidneys.

To define whether angiotensin I-converting enzyme (ACE) inhibition affects the distribution of renin gene-expressing cells within the kidney, a control group of adult male Wistar-Kyoto rats (C, n = 7) was compared with a group of rats treated with enalapril (E, n = 6) for 5 days. Renin mRNA distribution was assessed using in situ hybridization to a 35S-labeled 28 mer oligonucleotide complementary to rat renin mRNA. Whereas in control rats renin mRNA was confined to a juxtaglomerular location, in enalapril-treated rats, renin mRNA extended proximally along the length of the afferent arteriole. The percent of visible afferent arteriolar length containing renin mRNA was higher in enalapril-treated (71.7 +/- 2.8%) than in control (49.6 +/- 2.1%) rats (P less than 0.0001). These findings were accompanied by an increase in the percent of juxtaglomerular apparatuses (JGAs) containing renin mRNA (71 +/- 2.2 vs. 49 +/- 2.9%; E vs. C, P less than 0.0001). Also, the intensity of the JGA hybridization signals was higher in enalapril-treated (757 +/- 59 grains/JGA) than in control (167 +/- 11 grains/JGA) rats (P less than 0.00001). We conclude that the increased kidney renin gene expression elicited by ACE inhibition is the result of an increase in renin mRNA content per JGA, an increase in the number of JGAs expressing the renin gene, and a recruitment of renin gene-expressing cells along the afferent arteriole.

Distribution of renin mRNA and its protein in the developing kidney.

The intrarenal distribution of renin changes markedly during maturation. To determine whether renin gene expression changes along the developing renal vasculature, renin mRNA distribution was assessed using in situ hybridization histochemistry. Fetal, newborn, and adult kidney tissue sections from Wistar-Kyoto rats were hybridized with an oligonucleotide complementary to rat renin mRNA. In fetal kidneys, renin mRNA was found in the vascular pole of juxtamedullary glomeruli and along afferent, interlobular, and arcuate arteries. In kidneys from newborn rats, renin mRNA localized throughout the whole length of afferent arterioles, but was not detected in interlobular or arcuate arteries. In adult kidneys, hybridization signals were less intense and confined to the juxtaglomerular apparatus. Immunolocalization of renin with a polyclonal anti-rat renin antibody paralleled closely the mRNA distribution. Northern blot analyses demonstrated that renin mRNA levels were higher in fetal and newborn (20- and 10-fold, respectively) than in adult kidneys. We conclude the following. 1) The fetal kidney expresses the renin gene. 2) Expression of the renin gene is subjected to developmental changes. 3) As maturation progresses, localization of renin synthesis and storage shifts from large intrarenal arteries to a restricted, classical juxtaglomerular site in the afferent arteriole.