Glucocorticoid-Induced Leucine Zipper Promotes Neutrophil and T-Cell Polarization with Protective Effects in Acute Kidney Injury

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Fluorescent Microangiography Is a Novel and Widely Applicable Technique for Delineating the Renal Microvasculature

Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections

Pharmacological Blockade of Soluble Epoxide Hydrolase Attenuates the Progression of Congestive Heart Failure Combined With Chronic Kidney Disease: Insights From Studies With Fawn-Hooded Hypertensive Rats

An association between congestive heart failure (CHF) and chronic kidney disease (CKD) results in extremely poor patient survival rates. Previous studies have shown that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, improves the survival rate in CHF induced by aorto-caval fistula (ACF) and attenuates CKD progression. This prompted us to examine if sEH inhibitor treatment would improve the outcome if both experimental conditions are combined. Fawn-hooded hypertensive (FHH) rats, a genetic model showing early CKD development was employed, and CHF was induced by ACF. Treatment with an sEH inhibitor was initiated 4 weeks after ACF creation, in FHH and in fawn-hooded low-pressure (FHL) rats, a control strain without renal damage. The follow-up period was 20 weeks. We found that ACF FHH rats exhibited substantially lower survival rates (all the animals died by week 14) as compared with the 64% survival rate observed in ACF FHL rats. The former group showed pronounced albuminuria (almost 30-fold higher than in FHL) and reduced intrarenal EET concentrations. The sEH inhibitor treatment improved survival rate and distinctly reduced increases in albuminuria in ACF FHH and in ACF FHL rats, however, all the beneficial actions were more pronounced in the hypertensive strain. These data indicate that pharmacological blockade of sEH could be a novel therapeutic approach for the treatment of CHF, particularly under conditions when it is associated with CKD

Effects of Hepatocyte CD14 Upregulation during Cholestasis on Endotoxin Sensitivity

Cholestasis is frequently related to endotoxemia and inflammatory response. Our previous investigation revealed a significant increase in plasma endotoxin and CD14 levels during biliary atresia. We therefore propose that lipopolysacharides (LPS) may stimulate CD14 production in liver cells and promote the removal of endotoxins. The aims of this study are to test the hypothesis that CD14 is upregulated by LPS and investigate the pathophysiological role of CD14 production during cholestasis. Using Western blotting, qRT-PCR, and promoter activity assay, we demonstrated that LPS was associated with a significant increase in CD14 and MD2 protein and mRNA expression and CD14 promoter activity in C9 rat hepatocytes but not in the HSC-T6 hepatic stellate cell line in vitro. To correlate CD14 expression and endotoxin sensitivity, in vivo biliary LPS administration was performed on rats two weeks after they were subjected to bile duct ligation (BDL) or a sham operation. CD14 expression and endotoxin levels were found to significantly increase after LPS administration in BDL rats. These returned to basal levels after 24 h. In contrast, although endotoxin levels were increased in sham-operated rats given LPS, no increase in CD14 expression was observed. However, mortality within 24 h was more frequent in the BDL animals than in the sham-operated group. In conclusion, cholestasis and LPS stimulation were here found to upregulate hepatic CD14 expression, which may have led to increased endotoxin sensitivity and host proinflammatory reactions, causing organ failure and death in BDL rats

Activation of Rac-1 and RhoA contributes to podocyte injury in chronic kidney disease

Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD

Phenotypically Heterogeneous Podoplanin-expressing Cell Populations Are Associated with the Lymphatic Vessel Growth and Fibrogenic Responses in the Acutely and Chronically Infarcted Myocardium

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRalpha, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRbeta or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development

Effects of gender and gonadectomy on growth and plasma cholesterol levels in pigs

We conducted two studies to determine the effect of gender, gonadectomy (GDX) on growth and plasma cholesterol levels in pigs. In experiment 1, five sham-operated and five GDX female Landrace pigs (26 kg) were allowed to have free access to water and feed up to market weight (approximately 100 kg). Body weight and feed consumption were recorded biweekly, and daily body weight gain, daily feed intake and feed efficiency (gain/feed) were calculated during the feeding period. In experiment 2, 10 male (26 kg) and 10 female (26 kg) Landrace pigs were used; five male and five female pigs were assigned to sham-operated or GDX. Pigs were allowed to have free access to water and a diet without added cholesterol (Table 1) until they were 6 months old (male 104 and female 98 kg) and thereafter they were fed a hypercholesterolemic diet (Table 1) containing 0.5% cholesterol and 0.1% cholate for 10 days. GDX of female pigs increased average daily gain (P<0.05), compared with their sham-operated counterparts during the growing-finishing period, but had no effect (P>0.05) on feed efficiency. Plasma cholesterol levels in pigs fed a hypercholesterolemic diet for 10 days were much higher (P<0.05) in females than in males (161 vs 104 mg/100 mL plasma), and were increased by GDX only in male pigs. HDL-cholesterol/LDL+VLDL-cholesterol ratio appeared to be higher in males than in females, and was not influenced by GDX in either sex. Results suggested that the lower growth rate of female pigs than their male counterparts is attributable to the ovarian activity, and the lower plasma cholesterol level in male than in female pigs fed a hypercholesterolemic diet is due to the testicular activity

Phenotypically Heterogeneous Podoplanin-expressing Cell Populations Are Associated with the Lymphatic Vessel Growth and Fibrogenic Responses in the Acutely and Chronically Infarcted Myocardium

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRalpha, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRbeta or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development