Human alpha defensin 5 expression in the human kidney and urinary tract

Abstract

Background: The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.

Methodology/principal findings: Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.

Conclusions/significance: DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Expression of DEFA5 in human kidney, ureter, and bladder.

DEFA5 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. DEFA5 expression was significantly greater in the lower urinary tract (p = 0.014).

Figure 2. HD5 expression increases with pyelonephritis.

(A) DEFA5 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. DEFA5 expression was significantly greater with pyelonephritis (p = 0.019). (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).

Figure 3. HD5 is expressed throughout the urothelium of the ureter and bladder.

Immunohistochemistry demonstrates HD5 expression (brown/arrows) throughout the urothelium of the human bladder (A) and ureter (C). 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) and ureter (D) showed no immunostaining. Magnification 20×.

Figure 4. HD5 production in non-infected human kidney and kidney with pyelonephritis.

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

Figure 5. Tubular HD5 expression in states of sterility and infection.

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). A/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. C/D: HD5 (green) was expressed in the loops of Henle (red THP staining) in non-infected kidney tissue (C) and with pyelonephritis (D). E/F: 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×.

Figure 6. HD5 is present in infected urine samples.

HD5 levels in sterile urine samples (n = 15) and urine samples infected with uropathogenic E.coli (n = 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. (A) Silver stained PAGE gels with 150 ng ProHD5 as standard to confirm equal protein loading into each lane. (B) Western blot analysis with 200 ng ProHD5 as standard.

Figure 7. Urinary production of HD5 in infected urine samples.

HD5 levels in sterile urine samples and urine samples infected with uropathogenic E.coli. 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.