Role of hypoxia inducible factor-1α (HIF-1α) in innate defense against uropathogenic Escherichia coli infection

Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infections (UTI) affecting approximately 150 million people worldwide. Here, we revealed the importance of transcriptional regulator hypoxia-inducible factor-1 α subunit (HIF-1α) in innate defense against UPEC-mediated UTI. The effects of AKB-4924, a HIF-1α stabilizing agent, were studied using human uroepithelial cells (5637) and a murine UTI model. UPEC adherence and invasion were significantly reduced in 5637 cells when HIF-1α protein was allowed to accumulate. Uroepithelial cells treated with AKB-4924 also experienced reduced cell death and exfoliation upon UPEC challenge. In vivo, fewer UPEC were recovered from the urine, bladders and kidneys of mice treated transurethrally with AKB-4924, whereas increased bacteria were recovered from bladders of mice with a HIF-1α deletion. Bladders and kidneys of AKB-4924 treated mice developed less inflammation as evidenced by decreased pro-inflammatory cytokine release and neutrophil activity. AKB-4924 impairs infection in uroepithelial cells and bladders, and could be correlated with enhanced production of nitric oxide and antimicrobial peptides cathelicidin and β-defensin-2. We conclude that HIF-1α transcriptional regulation plays a key role in defense of the urinary tract against UPEC infection, and that pharmacological HIF-1α boosting could be explored further as an adjunctive therapy strategy for serious or recurrent UTI

The effect of puberty on the development of early diabetic microvascular disease in insulin-dependent diabetes

We studied the prevalence of early diabetic retinopathy and nephropathy in 21 prepubertal and 55 late-pubertal subjects with insulin-dependent diabetes (IDD). All subjects had IDD of 5–7 years duration at the time of evaluation. The prevalence of early diabetic retinopathy was significantly greater in the late-pubertal subjects than prepubertal subjects (33% vs. 9.5%, P = 0.05), despite similar glycosylated hemoglobin values between the two groups (11.7 ± 2.7% vs. 10.1 ± 1.6%) at the time of evaluation. Nephropathy was infrequent in late-pubertal subjects (9%), and absent in the prepubertal subjects. We hypothesize that puberty plays an important role in the development of microvascular complications of IDD, and that increases in growth factors, sex hormones and deterioration in glycemic control at the time of puberty may each enhance the development of diabetic microvascular disease

A randomized, double-blind, placebo-controlled trial on intravenous ibuprofen L-lysine for the early closure of nonsymptomatic patent ductus arteriosus within 72 hours of birth in extremely low-birth-weight infants

ABSTRACT A multicenter, double-blind, randomized, placebo-controlled trial was conducted to evaluate the efficacy and safety of intravenous (IV) ibuprofen (L-lysine) for the early closure of nonsymptomatic patent ductus arteriosus (PDA) within 72 hours of birth in extremely low-birth-weight (ELBW) infants with evidence of ductal shunting by echocardiogram. Eleven sites enrolled 136 infants with nonsymptomatic early PDA (gestational age < 30 weeks; body weight 500 to 1000 g) to receive a 3-day course (10 mg/kg, 5 mg/kg, and 5 mg/kg) of IV ibuprofen (N = 68) or placebo (N = 68). Cardiac echocardiogram was performed on study days 1 and 14, and with rescue. Infants were followed to 36 weeks postconceptional age. Patient demographics, mean (standard deviation), were similar between ibuprofen and placebo: birth weight: 798.5 g (128.7) versus 797.3 g (132.8); gestational age: 26.1 weeks (1.3) versus 26.2 weeks (1.4); and age at first dose: 1.5 days (0.7). The intent-to-treat analysis of the primary endpoint, subjects rescued, died, or dropped through study day 14, was 21/68 (30.9%) with ibuprofen and 36/68 (52.9%) for placebo (P = 0.005). Death, intraventricular hemorrhage, necrotizing enterocolitis, daily fluid intake/output, liver function, bronchopulmonary dysplasia, and retinopathy of prematurity did not differ. A trend toward decreased periventricular leukomalacia by ibuprofen was noted. IV ibuprofen was effective and safe in the early closure of PDA in preterm neonates

Reduced UPEC-mediated inflammatory damage to bladder epithelium with AKB-4924 pretreatment.

<p>(<b>A, B</b>) ELISA analysis shows pro-inflammatory cytokines IL-6, IL-1β or KC in the bladders or kidneys are reduced in AKB-4924 treated mice 18–24 h post UPEC CFT073 infection. Results are compiled from more than two independent experiments (n > 3 per group). (<b>C</b>) Myeloperoxidase (MPO) assay of bladder supernatants (n = 9–11) indicates significant decrease in MPO activity in AKB-4924 pre-treated bladders 18h post UPEC CFT073 infection; Error bar = S.E.M *<i>P</i> < 0.05, Student’s two-tailed unpaired t-test. (<b>D</b>) Representative immunolocalization images of bladder MPO (red) and nuclei (DAPI, blue) of 18 h UTI89-infected mice with or without 4924 pre-treatment (n = 3–4 per group). Scale bar = 50 μm.</p

AKB-4924 reduces UPEC-mediated cytotoxicity and inflammation in human uroepithelial cells.

<p>5637 Cells were infected with UPEC CFT073 for 2h at MOI ~20. (<b>A</b>) Live/dead cell staining illustrates viable (green) or dead (red) cells. Scale bar = 100 μm. (<b>B</b>) Percentage death in uninfected (UI), DMSO and AKB-4924 pre-treated 5637 cells (n > 7). (<b>C</b>) Total attached cells per field of view. Counts were made from multiple random fields of view (10x objective, n > 7) from independent samples (n = 3). Error bar = S.E.M, ***<i>P</i> < 0.001, **<i>P</i> < 0.01, *<i>P</i> < 0.05 by student’s two-tailed unpaired t-test. (<b>D</b>) Western blots of paxillin, phospho-p38 and phospho-p65 expression of uninfected (UI), UPEC-infected 5637 cells with prior exposure of DMSO or AKB-4924 (n = 3 per group). Data represent one of two independent experiments. (<b>E</b>) ELISA detection of pro-inflammatory cytokine proteins IL-6, IL-1β and IL-8 released by DMSO or AKB-4924 pre-treated 5637 cells 2 h post- infection (n = 4 per group). Results are pooled from 3 independent experiments.</p

Pretreatment with AKB-4924 impairs UPEC urinary tract colonization in C57BL/6 mice.

<p>(<b>A</b>) Bladders from female C57BL/6 mice treated with 1 h vehicle, or 0.2 mg/mL AKB-4924, were recovered after 18–24 h UPEC CFT073 infection. Real-time qPCR shows increased Hif-1 mRNA in AKB-4924 treated animals. (<b>B</b>) Real-time qPCR and ELISA show increased VEGF mRNA and protein expression in AKB-4924 treated group. Data are generated from two independent repeats (n > 5 per experiment). (<b>C</b>) UPEC recoveries from urine (n = 15), bladder (n = 25) and both kidneys (n = 18) of mice that received 1 h of 4924 pre-treatment were significantly decreased compared to vehicle treated mice. Data is presented as mean +/- SEM, generated from 3–4 independent experiments, **<i>P</i> < 0.01. <i>Ex vivo</i> gentamicin protection assay revealed intracellular bacterial CFU from mice 18 h post-infection (n = 10,12)</p

Mice lacking HIF-1 in their bladder epithelium are more susceptible to UPEC urinary tract colonization.

<p>(<b>A</b>) Level of Hif-1 transcripts in bladders of HIF-1^-/- (<i>Hif1αflox/flox</i>/K14-Cre⁺) mice compared to wild-type mice (n = 3). (<b>B</b>) WT or HIF-1^-/- mice were infected with UPEC CFT073 and bladders were harvested 24 h post-infection for CFU enumeration (n = 16). (<b>C</b>) WT or HIF-1^-/- mice were pre-treated with 1 h of 0.2mg of AKB-4924 and infected with UPEC. Bladders were harvested 18 h post-infection for CFU enumeration (n = 7). (<b>D</b>) Images of representative UPEC infected bladders from littermates (n = 3–4 per group) with or without prior AKB-4924 treatment. Error bar = S.E.M ***<i>P</i> < 0.001, **<i>P</i> < 0.01, *<i>P</i> < 0.05, Student’s two-tailed unpaired t-test. Results are mean values from 2 or more independent experiments.</p

AKB-4924 pretreatment enhances production of nitric oxide and host defense peptides during UPEC infection.

<p>(<b>A</b>) Nitrite production from AKB-4924 treated 5637 cells was significantly higher than from DMSO treated cells 2 h post-infection. (Uninfected, n = 6; UPEC CFT073 infected, n = 12). Left panel: Absolute nitrite production (μM). Right panel: Relative nitrite productions per log CFU per mL (n = 6). (<b>B</b>) Nitrite released from WT (iNOS +/+) and heterozygous (iNOS +/-) per log CFU per bladder (gram) (n = 4–5) (<b>C</b>) WT (iNOS +/+), iNOS+/- and iNOS-/- C57BL/6 mice were infected with UPEC CFT073 for 18–24h before bladders were harvested for CFU enumeration (n = 7, 6). (<b>D</b>) WT and iNOS +/- C57BL/6 mice were pre-treated with 0.2 mg AKB-4924 or ωehicle for 1 h followed by infection with UPEC CFT073 (n = 4–6). (<b>E</b>) Human AMP β-defensin 2 (hBD2) (n = 8) and cathelicidin LL-37 (n = 7–10) mRNA level in UPEC CFT073 infected 5637 cells. Results were normalized to β-actin. (<b>F</b>) Real-time qPCR shows murine AMP βdefensin 2 (mβD2) and CRAMP mRNA level in bladders (n = 10–12). Error bar = S.E.M **<i>P</i> < 0.01, *<i>P</i> < 0.05, Student’s two-tailed unpaired t-test. (<b>G</b>) Representative images illustrating the distribution of murine cathelicidin (red) and nuclei (DAPI, blue) in the mucosa of UTI89 infected bladders with vehicle or AKB-4924 pre-treatment (n = 6). Upper panel scale bar = 50 μm. Bottom panel: higher magnitude focused on the uroepithelial cell layer of bladder. Scale bar = 25 μm. (<b>H</b>) Representative western blot of mouse bladder left uninfected (n = 2) or infected overnight with UPEC CFT073 (n = 3). Infected animals were pre-treated with vehicle or AKB-4924 for 2 h. Relative level of cathelicidin level was measured using Image J and normalized to β-actin from 2 independent western blot experiments (2–3 different animals in each western blot).</p

AKB-4924 stabilizes HIF-1α protein and reduces UPEC-mediated infection of cultured human uroepithelial cells.

<p>(<b>A</b>) Western blot illustrating expression of HIF-1α (120 kDa) in the nuclear fraction of uninfected human uroepithelial cell line 5637 left untreated (UT), or treated with DMSO (negative control), DFO (desferrioxamine mesylate; positive control) or AKB-4924 for 2 h. Nuclear matrix protein P84 was used as loading control. Bar graph shows relative intensity of protein bands from repeated experiments (n = 2). (<b>B</b>) Real-time qPCR showing relative HIF-1α and VEGF mRNA expression in uninfected (UI) or UPEC CFT073 infected (2 h) 5637 cells without treatment, or (<b>C</b>) DMSO (mock treatment and 4924 pre-treated cells followed by UPEC infection (n = 10) (<b>D</b>) Bacterial counts from 2 h DMSO or AKB-4924 treated 5637 cells followed by 2 h infection with UPEC CFT073 to measure total bacteria, or 2 h infection with additional 2 h gentamicin (100 μg/mL) treatment to measure intracellular bacteria (n = 3 per group). Shown as mean +/- S.E.M., *<i>P</i> < 0.05, Student’s unpaired t-test. Results are pooled from three independent experiments.</p

AKB-4924 attenuates urinary tract infection.

<p>(<b>A</b>) Bladders isolated from female C57BL/6 mice infected with UPEC CFT073 for 6 h received localized treatment with 0.2 mg of AKB-4924 via transurethral injection for 16 h. Less bacteria were recovered from infected bladders treated with AKB-4924 compared to vehicle. Real-time qPCR shows elevated level of (<b>B</b>) murine AMP CRAMP and (<b>C</b>) mBD2 mRNA (n >11) in AKB-4924 treated animals. Error bar = S.E.M, **<i>P</i> < 0.01, *<i>P</i> < 0.05, Student’s unpaired two-tailed t-test. Results are compiled from 2 independent experiments.</p