Urinary production of HD5 in infected urine samples.

<p>HD5 levels in sterile urine samples and urine samples infected with uropathogenic <i>E.coli</i>. The square boxes depict urinary proHD5 standardized to urine creatinine detected by ELISA assay using monoclonal antibody 8C8. The open circles depict urinary proHD5 and mature HD5 standardized to urine creatinine by immunoblot analysis. ProHD5 and mature HD5 were not detected in sterile urine samples using polyclonal HD5 antisera.</p

HD5 is expressed throughout the urothelium of the ureter and bladder.

<p>Immunohistochemistry demonstrates HD5 expression (brown/arrows) throughout the urothelium of the human bladder (<b>A</b>) and ureter (<b>C</b>). Immunostaining was most prominent in the luminal surfaces (brown/arrows). Immunostaining was not detected in the smooth muscle layers of the bladder or ureter (+). Negative control bladder (<b>B</b>) and ureter (<b>D</b>) showed no immunostaining. Magnification 20×.</p

HD5 expression increases with pyelonephritis.

<p>(<b>A</b>) <i>DEFA5</i> mRNA transcript levels were quantified by real-time PCR in non-infected kidney tissue and in kidney tissue with pyelonephritis. Shown are the results for three independent samples. In the table below, the mean transcript levels are shown with the SEM. <i>DEFA5</i> expression was significantly greater with pyelonephritis (<i>p</i> = 0.019). (<b>B</b>) To confirm the increase in message is accompanied by an increase in HD5 protein production, cationic peptides from the same non-infected kidney tissues (NL) and kidney tissue with pyelonephritis (P) were subjected to SDS PAGE followed by Western immunoblot analysis. Each lane contained the equivalent of 800 µg of cationic protein. Silver stained PAGE gels (top panel) confirmed equal protein loading into each lane. Immunoblot analysis for GAPDH and HD5 (middle panel). Serial dilutions of proHD5 (200 ng–70 ng) were subjected to SDS PAGE followed by Western immunoblot analysis (bottom panel).</p

Expression of <i>DEFA5</i> in human kidney, ureter, and bladder.

<p><i>DEFA5</i> mRNA transcript levels were quantified by real-time PCR in non-infected kidney, ureter, bladder. Shown are the results of three independent samples. In the table below, the mean transcript levels are shown with the SEM. <i>DEFA5</i> expression was significantly greater in the lower urinary tract (<i>p</i> = 0.014).</p

HD5 production in non-infected human kidney and kidney with pyelonephritis.

<p>Immunohistochemistry demonstrates HD5 production in isolated renal tubules (brown/arrows) in non-infected renal cortex (<b>A</b>) and medulla (<b>C</b>). With pyelonephritis, HD5 production increased in the renal tubules of the cortex (<b>B</b>) and medulla (<b>D</b>). The glomeruli (+) show no immunostaining in non-infected kidney samples and with pyelonephritis. Negative controls showed no immunostaining (not shown). Magnification 20×.</p

Tubular HD5 expression in states of sterility and infection.

<p>Human kidney labeled for HD5 (green), nuclei (blue) and nephron specific markers (red). Segment markers consisted of AQP-2 for collecting tubules (CT), THP for the loop of Henle (LOH), and AQP-1 for proximal tubules (PT). <b>A/B</b>: HD5 (green) was produced in the collecting tubules (red apical AQP-2 staining) of non-infected kidney tissue (A) and with pyelonephritis (B). Arrows indicate HD5 is produced in other nephron segments. <b>C/D</b>: HD5 (green) was expressed in the loops of Henle (red THP staining) in non-infected kidney tissue (C) and with pyelonephritis (D). <b>E/F</b>: HD5 (green) shows minimal production in the proximal tubules (red AQP-1 staining) of non-infected kidney tissue (E) and with pyelonephritis (F). Magnification 40×.</p

HD5 is present in infected urine samples.

<p>HD5 levels in sterile urine samples (n = 15) and urine samples infected with uropathogenic <i>E.coli</i> (<i>n</i> = 15). Cationic peptides from non-infected urine (NL) and infected urine (IN) were subjected to SDS PAGE followed by Western immunoblot analysis. Each lane contained the equivalent of 300 µg of urinary cationic protein. (<b>A</b>) Silver stained PAGE gels with 150 ng ProHD5 as standard to confirm equal protein loading into each lane. (<b>B</b>) Western blot analysis with 200 ng ProHD5 as standard.</p

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

<div><p>Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified <i>Regenerating islet-derived 3 gamma</i> (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic <i>Escherichia coli</i> (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC <i>in vitro</i>, but does kill <i>Staphylococcus saprophyticus</i> in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.</p></div

The human RegIIIγ orthologue, HIP/PAP, is secreted in infected human urine.

<p>(<b>A</b>) HIP/PAP levels were quantified by ELISA and adjusted for urine creatinine to control for differences in urine concentration. The resulting PAP/Cr ratio is significantly elevated in urine from patients with positive urine cultures, compared to sterile urine (n = 26 urines/group; * <i>P</i> < 0.0001, Mann-Whitney U test). The horizontal line indicates the mean. (<b>B</b>) Western blotting demonstrates absent of HIP/PAP in sterile (S) urine, whereas infected (I) urine from two children with UTI exhibits immunoreactivity.</p

Recombinant RegIIIγ protein exhibits bactericidal activity toward <i>S</i>. <i>saprophyticus</i> but not UPEC, and <i>RegIIIγ</i> deletion does not affect susceptibility to experimental UTI.

<p>(<b>A</b>) Recombinant RegIIIγ kills <i>S</i>. <i>saprophyticus</i> not UPEC in a dose-dependent manner. 10⁵ CFU bacteria were incubated with indicated concentrations of recombinant C-terminally cleaved RegIIIγ for 2 hours at 37°C in 10 mM MES, pH 5.5 with 25 mM NaCl, and colonies were enumerated. (<b>B-C</b>) Experimental UTI. Comparable bacterial burden in wildtype (WT) and <i>RegIIIγ</i>^<i>-/-</i> urinary tracts following inoculation with 10⁸ CFU of UPEC strain UTI89 (<b>B</b>) or <i>S</i>. <i>saprophyticus</i> (<b>C</b>). There was no significant difference between genotypes by Mann-Whitney U test (<i>p</i> > 0.05). Horizontal lines indicate geometric means.</p