Pkd1 Haploinsufficiency Increases Renal Damage and Induces Microcyst Formation following Ischemia/Reperfusion

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1^+/− and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploinsufficient mice, a process that apparently depends on a relative deficiency of p21 activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic renal disease, with a prevalence of 1:400 to 1:1000. Mutations of the PKD1 gene are responsible for approximately 85% of the disease cases, whereas approximately 15% are caused by mutations in PKD2.¹ Only half of patients reach the age of 58 yr without ESRD.² ADPKD is a systemic disorder, however, including extrarenal manifestations typically represented by liver cysts, intracranial aneurysms, and heart valve alterations.The PKD1 gene encodes polycystin 1, a large glycoprotein with an approximately 3000–amino acid extracellular portion that comprises domains that seem to be involved in protein–protein and protein–carbohydrate interactions. A number of studies support the involvement of the primary apical cilium in the pathogenesis of PKD by modulating signal transduction via intracellular Ca²⁺ transients.³ Polycystins 1 and 2 (PC1 and PC2) are thought to participate actively in this process.^3–6 Ciliary mechanosensation has also been associated with STAT6-dependent changes in gene expression.⁷ In addition, the cellular effects of polycystins seem to rely on interaction with the cytoskeleton and mediation of cell–cell adhesion.^8,9PC1 and PC2 activate a number of other pathways. PC1 may function as a G protein–coupled receptor.¹⁰ Its activation, following a process dependent on PC2, may also activate JAK2, leading to phosphorylation and activation of STAT1 and generation of STAT1 homodimers.¹¹ These dimers bind to the p21 promoter in the nucleus, promoting its upregulation, reduction of Cdk2 activity, and cell arrest in G0/G1. It has also been shown that PC1 induces phosphatidylinositol 3 kinase–dependent Akt activation,¹² whereas its C-terminus may interact with tuberin, regulating mammalian target of rapamycin activity.¹³ Moreover, PC1 is subjected to an autoproteolytic process in the G protein-coupled receptor proteolytic site domain, generating an extracellular N-terminal fragment,¹⁴ whereas its C-terminus seems to be cleaved, to be translocated to the nucleus, and to activate AP-1.¹⁵Ischemia/reperfusion (IR) injury is a common cause of acute kidney injury (AKI), including patients with ADPKD. The cellular damage is secondary to a chain of biochemical and biologic abnormalities.^16,17 An abnormal proliferative response of ADPKD cells to cAMP has been reported, apparently associated with defective intracellular Ca²⁺ homeostasis,¹⁸ and animal models of PKD have been associated with dysregulated cell-cycle activity.¹⁹ Piontek et al.,²⁰ however, have shown that the effects of Pkd1 inactivation in mice are determined by a developmental switch on postnatal day 13. Interestingly, in this model, cellular proliferation was not significantly increased.In this scenario, we hypothesized that a lower PC1 biologic activity might amplify the IR injury degree. Although the focal cyst formation in ADPKD is likely dependent on a two-hit mechanism,²¹ the functional effects of PC1 seem to rely on activity thresholds.²² PKD1-haploinsufficient kidney cells, therefore, might be unable to achieve the required PC1 activity level when exposed to IR. Although studies on the ischemia/PC2 relation have brought some potential contributions to this question,^23,24 the relationship between PC1 and IR is basically unknown. By coordinating cell planar polarity in renal tubules, PC1 might exert a protective effect after an ischemic insult. In this study, this potential mechanism was investigated in Pkd1^+/− mice obtained from an inbred mouse line with a Pkd1 null mutation. Our findings of a more severe renal lesion in Pkd1-null heterozygotes suggest an increased risk for renal IR injury in Pkd1-haploinsufficient mice. Development of tubular dilation (TD) and microcysts (MCs) and increased renal fibrosis, in turn, suggest that the IR aggression has a higher long-term negative impact on Pkd1^+/− kidneys.     

Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high on antiretroviral therapy

OBJECTIVE: To examine the relationships between blood CD4 natural regulatory T (Treg) cells, plasma HIV RNA level, CD4 T-cell count and immune activation in untreated HIV-infected patients and immunodeficient patients beginning antiretroviral therapy (ART), using a novel phenotype to define Treg cells (CD25CD127CD4). Data were compared with established Treg cell markers (FoxP3, CTLA-4 and GITR). METHODS: Twenty-nine untreated HIV-infected patients with CD4 T-cell counts of < 300 or > 400/microl were compared in a cross-sectional study and 12 patients beginning combination ART with < 100 CD4 T cells/mul were followed for 1 year on therapy. Three- and four-colour flow cytometry was used to quantitate proportions of Treg cells. RESULTS: In control donors and patients with high CD4 T-cell counts, 28-89% (median 60%) of CD25CD127CD4 cells were FoxP3, but < 10% expressed GITR or CTLA-4. Immunodeficient patients also had CD4-negative lymphocytes with the phenotype FoxP3CD127. Proportions of CD25CD127 cells and activated (HLA-DR) cells in the CD4 T-cell population were increased in patients with low CD4 T cell counts. The proportion of CD25CD127CD4 T cells correlated positively with plasma HIV RNA level and CD4 T-cell activation, but inversely with CD4 T-cell count. Longitudinal studies of 12 patients receiving ART in two distinct cohorts (Western Australia and Malaysia) showed that the proportion of CD25CD127CD4 cells decreased slightly over time, but remained above levels seen in non-HIV controls. CONCLUSIONS: Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high after 1 year on ART.     

Optimal nutritional status assessment parameters in gastroenterological patients on hospital admission

BACKGROUND/AIM: There are no recommendations for the optimal nutritional status assessment parameters (NSAPs) in the current literature. The aim of this study was to define the optimal NSAPs for nutritional status assessing in gastroenterological patients on hospital admission. METHODS: Nutritional status of 612 gastroenterological patients was evaluated at the admission using 6 NSAPs: unintentional weight loss (WL), body mass index (BMI), triceps skinfold thickness (TSF), mid-upper arm muscle circumference (MAMC), serum albumin concentration (ALB), and lymphocyte counts (LYM). According to their nutritive status, the patients were classified as well nourished (normally nourished and obese), moderately undernourished and severely undernourished. Based on the similarities and differences in the assessment results, obtained according to each of 6 parameters, the optimal nutritional assessment parameters were defined, separately for the well-nourished/undernourished patients and for moderately/severely undernourished patients. RESULTS: The incidence of malnutrition was in the range 5.9-29.7%. The results based on MAMC, ALB, and LYM were similar (25.2-29.7%; p > 0.05), while the results based on WL, BMI, and TSF differed significantly (5.9-19.9%; p = 0.001-0.015). The assessment based on BMI was the most severe criterion, while the assessment according to MAMC was the mildest criterion in selection of malnourished patients. The assessment according to MAMC was the mildest criterion for the selection of severe malnourished patients (severely malnouorished patients accounted for 70.1%), while BMI and LYM were the most severe criteria (severely malnouorished patients accounted for 22.2% and 27.3%, respectively). The results based on BMI and LYM were similar (Wilcoxon test; p > 0.05). CONCLUSION: Combining BMI with MAMC is sufficient for the assessment of the nutritional status of gastroenterological patients on admission, as well as for differentiation between severely malnourished and moderately malnourished patients.     

Percutaneous Transhepatic Portography for the Treatment of Early Portal Vein Thrombosis After Surgery

We treated three cases of early portal vein thrombosis (PVT) by minimally invasive percutaneous transhepatic portography. All patients developed PVT within 30 days of major hepatic surgery (one case each of orthotopic liver transplantation, splenectomy in a previous liver transplant recipient, and right extended hepatectomy with resection and reconstruction of the left branch of the portal vein for tumor infiltration). In all cases minimally invasive percutaneous transhepatic portography was adopted to treat this complication by mechanical fragmentation and pharmacological lysis of the thrombus. A vascular stent was also positioned in the two cases in which the thrombosis was related to a surgical technical problem. Mechanical fragmentation of the thrombus with contemporaneous local urokinase administration resulted in complete removal of the clot and allowed restoration of normal blood flow to the liver after a median follow-up of 37 months. PVT is an uncommon but severe complication after major surgery or liver transplantation. Surgical thrombectomy, with or without reconstruction of the portal vein, and retransplantation are characterized by important surgical morbidity and mortality. Based on our experience, minimally invasive percutaneous transhepatic portography should be considered an option toward successful recanalization of early PVT after major liver surgery including transplantation. Balloon dilatation and placement of a vascular stent could help to decrease the risk of recurrent thrombosis when a defective surgical technique is the reason for the thrombosis.     

Dynamic self-assembly and control of microfluidic particle crystals

Engineered two-phase microfluidic systems have recently shown promise for computation, encryption, and biological processing. For many of these systems, complex control of dispersed-phase frequency and switching is enabled by nonlinearities associated with interfacial stresses. Introducing nonlinearity associated with fluid inertia has recently been identified as an easy to implement strategy to control two-phase (solid-liquid) microscale flows. By taking advantage of inertial effects we demonstrate controllable self-assembling particle systems, uncover dynamics suggesting a unique mechanism of dynamic self-assembly, and establish a framework for engineering microfluidic structures with the possibility of spatial frequency filtering. Focusing on the dynamics of the particle-particle interactions reveals a mechanism for the dynamic self-assembly process; inertial lift forces and a parabolic flow field act together to stabilize interparticle spacings that otherwise would diverge to infinity due to viscous disturbance flows. The interplay of the repulsive viscous interaction and inertial lift also allow us to design and implement microfluidic structures that irreversibly change interparticle spacing, similar to a low-pass filter. Although often not considered at the microscale, nonlinearity due to inertia can provide a platform for high-throughput passive control of particle positions in all directions, which will be useful for applications in flow cytometry, tissue engineering, and metamaterial synthesis.     

Towards high-throughput identification of endocrine disrupting compounds with mass spectrometry

High-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking has the ability to monitor the ligand-dependent dimerization of the human estrogen receptor α ligand binding domain (hERα LBD) in solution. Because only ER ligands enhance the homodimer abundance, we evaluated the ability of this label-free approach for identifying endocrine disrupting compounds (EDCs) in a high-throughput manner. This was achieved by combining an automated liquid handler with an automated MS acquisition procedure, which allowed a five-fold gain in operator time compared to a fully manual approach. To detect ligand binding with enough confidence, the receptor has to be incubated with at least a 10 μM concentration of the test compound. Based on the increase of the measured homodimer intensity, eight compounds with a relative binding affinity (RBA, relative to the natural hormone estradiol) >7% were identified as ER ligands among the 28 chemicals tested. Two other compounds, quercetin and 4-tert-amylphenol, were also identified as ER ligands, although their RBAs have been reported to be only 0.01% and 0.000055%, respectively. This suggests that these two ligands have a higher affinity for hERα LBD than reported in the literature. The high-mass MALDI approach thus allows identifying high affinity EDCs in an efficient way.     

Laparoscopic reconstruction after esophageal resection for perforation: a new surgical approach

The authors present 2 cases of esophageal perforation treated using a new 2-step approach, consisting of esophageal resection and delayed reconstruction of the digestive tract after laparoscopic preparation and transposition of the stomach. The method is characterized by the minimally invasive insertion of a gastric tube through the precardial esophageal stump for postoperative enteral nutrition, and by the use of a laparoscopic method in the reconstruction step for gastrolysis and transposition of the stomach. The benefits lie in the opportunity for enteral feeding preparatory to the reconstruction, with no need for any gastrostomy or jejunostomy, and with fewer complications and a better recovery after reconstruction surgery thanks to the use of a laparoscopic method instead of a laparotomy.