Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Plant Phenolics Inhibit Focal Adhesion Kinase and Suppress Host Cell Invasion by Uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic and polyphenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here we tested a panel of four well-studied phenolic compounds - caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate - for effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses, and likely contribute to the development of chronic and recurrent infections. Using cell culture-based assays, we found that only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK, or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model, and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.

Predicted Mutation Strength of Nontruncating PKD1 Mutations Aids Genotype-Phenotype Correlations in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) often results in ESRD but with a highly variable course. Mutations to PKD1 or PKD2 cause ADPKD; both loci have high levels of allelic heterogeneity. We evaluated genotype-phenotype correlations in 1119 patients (945 families) from the HALT Progression of PKD Study and the Consortium of Radiologic Imaging Study of PKD Study. The population was defined as: 77.7% PKD1, 14.7% PKD2, and 7.6% with no mutation detected (NMD). Phenotypic end points were sex, eGFR, height-adjusted total kidney volume (htTKV), and liver cyst volume. Analysis of the eGFR and htTKV measures showed that the PKD1 group had more severe disease than the PKD2 group, whereas the NMD group had a PKD2-like phenotype. In both the PKD1 and PKD2 populations, men had more severe renal disease, but women had larger liver cyst volumes. Compared with nontruncating PKD1 mutations, truncating PKD1 mutations associated with lower eGFR, but the mutation groups were not differentiated by htTKV. PKD1 nontruncating mutations were evaluated for conservation and chemical change and subdivided into strong (mutation strength group 2 [MSG2]) and weak (MSG3) mutation groups. Analysis of eGFR and htTKV measures showed that patients with MSG3 but not MSG2 mutations had significantly milder disease than patients with truncating cases (MSG1), an association especially evident in extreme decile populations. Overall, we have quantified the contribution of genic and PKD1 allelic effects and sex to the ADPKD phenotype. Intrafamilial correlation analysis showed that other factors shared by families influence htTKV, with these additional genetic/environmental factors significantly affecting the ADPKD phenotype.

Imaging-based diagnosis of autosomal dominant polycystic kidney disease.

The clinical use of conventional ultrasonography (US) in autosomal dominant polycystic kidney disease (ADPKD) is currently limited by reduced diagnostic sensitivity, especially in at-risk subjects younger than 30 years of age. In this single-center prospective study, we compared the diagnostic performance of MRI with that of high-resolution (HR) US in 126 subjects ages 16-40 years born with a 50% risk of ADPKD who underwent both these renal imaging studies and comprehensive PKD1 and PKD2 mutation screening. Concurrently, 45 healthy control subjects without a family history of ADPKD completed the same imaging protocol. We analyzed 110 at-risk subjects whose disease status was unequivocally defined by molecular testing and 45 unaffected healthy control subjects. Using a total of >10 cysts as a test criterion in subjects younger than 30 years of age, we found that MRI provided both a sensitivity and specificity of 100%. Comparison of our results from HR US with those from a previous study of conventional US using the test criterion of a total of three or more cysts found a higher diagnostic sensitivity (approximately 97% versus approximately 82%) with a slightly decreased specificity (approximately 98% versus 100%) in this study. Similar results were obtained in test subjects between the ages of 30 and 40 years old. These results suggest that MRI is highly sensitive and specific for diagnosis of ADPKD. HR US has the potential to rival the diagnostic performance of MRI but is both center- and operator-dependent.

Imaging classification of autosomal dominant polycystic kidney disease: a simple model for selecting patients for clinical trials.

The rate of renal disease progression varies widely among patients with autosomal dominant polycystic kidney disease (ADPKD), necessitating optimal patient selection for enrollment into clinical trials. Patients from the Mayo Clinic Translational PKD Center with ADPKD (n=590) with computed tomography/magnetic resonance images and three or more eGFR measurements over ≥6 months were classified radiologically as typical (n=538) or atypical (n=52). Total kidney volume (TKV) was measured using stereology (TKVs) and ellipsoid equation (TKVe). Typical patients were randomly partitioned into development and internal validation sets and subclassified according to height-adjusted TKV (HtTKV) ranges for age (1A-1E, in increasing order). Consortium for Radiologic Imaging Study of PKD (CRISP) participants (n=173) were used for external validation. TKVe correlated strongly with TKVs, without systematic underestimation or overestimation. A longitudinal mixed regression model to predict eGFR decline showed that log2HtTKV and age significantly interacted with time in typical patients, but not in atypical patients. When 1A-1E classifications were used instead of log2HtTKV, eGFR slopes were significantly different among subclasses and, except for 1A, different from those in healthy kidney donors. The equation derived from the development set predicted eGFR in both validation sets. The frequency of ESRD at 10 years increased from subclass 1A (2.4%) to 1E (66.9%) in the Mayo cohort and from 1C (2.2%) to 1E (22.3%) in the younger CRISP cohort. Class and subclass designations were stable. An easily applied classification of ADPKD based on HtTKV and age should optimize patient selection for enrollment into clinical trials and for treatment when one becomes available.

Evidence of a third ADPKD locus is not supported by re-analysis of designated PKD3 families.

Mutations to PKD1 and PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The absence of apparent PKD1/PKD2 linkage in five published European or North American families with ADPKD suggested a third locus, designated PKD3. Here we re-evaluated these families by updating clinical information, re-sampling where possible, and mutation screening for PKD1/PKD2. In the French-Canadian family, we identified PKD1: p.D3782_V3783insD, with misdiagnoses in two individuals and sample contamination explaining the lack of linkage. In the Portuguese family, PKD1: p.G3818A segregated with the disease in 10 individuals in three generations with likely misdiagnosis in one individual, sample contamination, and use of distant microsatellite markers explaining the linkage discrepancy. The mutation PKD2: c.213delC was found in the Bulgarian family, with linkage failure attributed to false positive diagnoses in two individuals. An affected son, but not the mother, in the Italian family had the nonsense mutation PKD1: p.R4228X, which appeared de novo in the son, with simple cysts probably explaining the mother's phenotype. No likely mutation was found in the Spanish family, but the phenotype was atypical with kidney atrophy in one case. Thus, re-analysis does not support the existence of a PKD3 in ADPKD. False positive diagnoses by ultrasound in all resolved families shows the value of mutation screening, but not linkage, to understand families with discrepant data.

Somatotroph pituitary adenoma with acromegaly and autosomal dominant polycystic kidney disease: SSTR5 polymorphism and PKD1 mutation.

A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6 years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106 ng/mL (normal 0-5) and insulin-like growth factor (IGF-1) 811 ng/mL (normal 48-255) blood levels. A magnetic resonance imaging scan disclosed a 3.0 cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C>A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.

Successful disease-specific induced pluripotent stem cell generation from patients with kidney transplantation.

INTRODUCTION: End-stage renal disease (ESRD) is a major public health problem. Although kidney transplantation is a viable therapeutic option, this therapy is associated with significant limitations, including a shortage of donor organs. Induced pluripotent stem (iPS) cell technology, which allows derivation of patient-specific pluripotent stem cells, could provide a possible alternative modality for kidney replacement therapy for patients with ESRD. METHODS: The feasibility of iPS cell generation from patients with a history of ESRD was investigated using lentiviral vectors expressing pluripotency-associated factors. RESULTS: In the present article we report, for the first time, generation of iPS cells from kidney transplant recipients with a history of autosomal-dominant polycystic kidney disease (ADPKD), systemic lupus erythematosus, or Wilms tumor and ESRD. Lentiviral transduction of OCT4, SOX2, KLF4 and c-MYC, under feeder-free conditions, resulted in reprogramming of skin-derived keratinocytes. Keratinocyte-derived iPS cells exhibited properties of human embryonic stem cells, including morphology, growth properties, expression of pluripotency genes and surface markers, spontaneous differentiation and teratoma formation. All iPS cell clones from the ADPKD patient retained the conserved W3842X mutation in exon 41 of the PKD1 gene. CONCLUSIONS: Our results demonstrate successful iPS cell generation from patients with a history of ESRD, PKD1 gene mutation, or chronic immunosuppression. iPS cells from autosomal kidney diseases, such as ADPKD, would provide unique opportunities to study patient-specific disease pathogenesis in vitro.