Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes.

The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.

Metformin protects rat hepatocytes against bile acid-induced apoptosis.

BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

Functional TRPV6 channels are crucial for transepithelial Ca2+ absorption.

TRPV6 is considered the primary protein responsible for transcellular Ca2+ absorption. In vitro studies demonstrate that a negatively charged amino acid (D) within the putative pore region of mouse TRPV6 (position 541) is critical for Ca2+ permeation of the channel. To elucidate the role of TRPV6 in transepithelial Ca2+ transport in vivo, we functionally analyzed a TRPV6D541A/D541A knockin mouse model. After weaning, mice were fed a regular (1% wt/wt) or Ca2+-deficient (0.02% wt/wt) diet and housed in metabolic cages. Blood was sampled for Ca2+ measurements, and the expression of Ca2+ transport proteins was analyzed in kidney and duodenum. Intestinal 45Ca2+ uptake was measured in vivo by an absorption assay. Challenging the mice with the Ca2+-deficient diet resulted in hypocalcemia in wild-type and TRPV6D541A/D541A mice. On a low-Ca2+ diet both mouse strains displayed increased expression of intestinal TRPV6, calbindin-D(9K), and renal TRPV5. TRPV6D541A/D541A mice showed significantly impaired intestinal Ca2+ uptake compared with wild-type mice, and duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice. On a normal diet, serum Ca2+ concentrations normalized in both mouse strains. Under these conditions, intestinal Ca2+ uptake was similar, and the expression levels of renal and intestinal Ca2+ transport proteins were not affected. We demonstrate that TRPV6D541A/D541A mice exhibit impaired transcellular Ca2+ absorption. Duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice, albeit insufficient to correct for the diminished Ca2+ absorption. Under normal conditions, when passive Ca2+ transport is predominant, no differences between wild-type and TRPV6D541A/D541A mice were observed. Our results demonstrate a specific role for TRPV6 in transepithelial Ca2+ absorption.

Characterization of vitamin D-deficient klotho(-/-) mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho(-/-) mice?

BACKGROUND: Klotho(-/-) mice display disturbed Ca(2+) and vitamin D homeostasis. Renal cytochrome p450 27b1 (Cyp27b1), the enzyme that catalyzes the hydrolysis to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is increased in klotho(-/-) mice, and a 1,25(OH)(2)D(3)-deficient diet partially normalized Ca(2+) homeostasis in these klotho(-/-) mice. The aim of the present study was to further delineate the interplay between 1,25(OH)(2)D(3) and klotho and their relative contribution to the Ca(2+) homeostasis of klotho(-/-) mice. METHODS: Double-klotho(-/-)/Cyp27b1(-/-) mice were generated and mice aged 8-12 weeks were housed in metabolic cages to collect 24-h urine. Blood samples were taken and the animals were sacrificed, and the kidney and duodenum tissues were sampled for RNA extraction. The bone was fixed in 10% v/v formalin and analysed by microcomputed tomography (µCT) scans. RESULTS: Klotho(-/-)/Cyp27b1(-/-) mice, like Cyp27b1(-/-) mice, displayed significantly decreased serum total calcium concentrations compared with wild-type mice (1.44 ± 0.03 and 2.25 ± 0.02 mM) along with normal urinary total calcium excretion. Hyperphosphataemia of klotho(-/-) mice normalized to wild-type levels in klotho(-/-)/Cyp27b1(-/-) mice. The mRNA levels of duodenal transient receptor potential vanilloid subtype 6 (TRPV6) and calcium-binding protein-D(9K), and renal calbindin-D(28K) and NCX1 were significantly reduced in the double knockouts compared with wild-type or klotho(-/-) mice. Elevated TRPV5 protein levels in klotho(-/-) mice normalized to wild type in klotho(-/-)/Cyp27b1(-/-) mice, but were decreased in Cyp27b1(-/-) mice. µCT scans showed that klotho(-/-)/Cyp27b1(-/-) mice, as Cyp27b1(-/-) mice, display significant bone hypomineralization and severely decreased bone mass. Klotho(-/-) mice show a reduced bone mass and increased trabecular numbers. CONCLUSIONS: Klotho(-/-)/Cyp27b1(-/-) mice resemble Cyp27b1(-/-) mice. Since 1,25(OH)(2)D(3) is absent in these mice, our results imply that Ca(2+) homeostasis in klotho(-/-) mice is affected by their excessive 1,25(OH)(2)D(3) levels.

Transient receptor potential melastatin 6 knockout mice are lethal whereas heterozygous deletion results in mild hypomagnesemia.

BACKGROUND: Hypomagnesemia with secondary hypocalcemia is due to disturbed renal and intestinal magnesium (Mg(2+)) (re)absorption. The underlying defect is a mutation in the transient receptor potential melastatin type 6 (TRPM6), a Mg(2+)-permeable ion channel expressed in the kidney and intestine. Our aim was to characterize homozygous (-/-) and heterozygous (+/-) TRPM6 knockout mice with respect to Mg(2+) homeostasis. METHODS: TRPM6(+/-) mice were bred on a normal (0.19% wt/wt Mg(2+)) and high (0.48% wt/wt Mg(2+)) Mg(2+) diet. In the offspring, 24-hour urinary Mg(2+) and calcium excretion as well as serum concentrations of both were determined. TRPM6 mRNA expression in the kidney and colon was measured. RESULTS: On the regular diet, 30% of the offspring were TRPM6 wild-type ((+/+)), 70% were TRPM6(+/-), and none were TRPM6(-/-). The genotypic distribution of the litters remained the same on the 0.48% Mg(2+) diet. In TRPM6(+/-) mice on both diets, serum Mg(2+) levels were significantly lower, and renal and intestinal TRPM6 mRNA expression was reduced. Urinary Mg(2+) excretion was unaffected. CONCLUSIONS: Homozygous TRPM6 deletion is embryonic lethal in mice. Heterozygous deletion of TRPM6 results in a mild hypomagnesemia. The Mg(2+)-enriched diet could not compensate for either embryonic lethality or hypomagnesemia caused by TRPM6 deficiency.

Unconjugated bile salts shuttle through hepatocyte peroxisomes for taurine conjugation.

UNLABELLED: Bile acid-CoA:amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis. In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation. Recently, we showed that BAAT is a peroxisomal protein, implying shuttling of bile salts through peroxisomes for reconjugation. However, the subcellular location of BAAT remains a topic of debate. The aim of this study was to obtain direct proof for reconjugation of bile salts in peroxisomes. Primary rat hepatocytes were incubated with deuterium-labeled cholic acid (D(4)CA). Over time, media and cells were collected and the levels of D(4)CA, D(4)-tauro-CA (D(4)TCA), and D(4)-glyco-CA (D(4)GCA) were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Subcellular accumulation of D(4)-labeled bile salts was analyzed by digitonin permeabilization assays and subcellular fractionation experiments. Within 24 hours, cultured rat hepatocytes efficiently (>90%) converted and secreted 100 µM D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 µg/mL digitonin led to the complete release of D(4)CA, D(4)GCA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cytosolic marker). Full release of D(4)TCA, catalase, and BAAT was only observed at 500 µg/mL digitonin, indicating the presence of D(4)TCA in membrane-enclosed organelles. D(4)TCA was detected in fractions of purified peroxisomes, which did not contain D(4)CA and D(4)GCA. CONCLUSION: We established a novel assay to study conjugation and intra- and transcellular transport of bile salts. Using this assay, we show that cholic acid shuttles through peroxisomes for taurine-conjugation.

Anti-oxidants do not prevent bile acid-induced cell death in rat hepatocytes.

BACKGROUND: Bile acids, reactive oxygen species (ROS) and inflammatory cytokines are crucial regulators of cell death in acute and chronic liver diseases. The contribution of each factor to hepatocyte death, either apoptosis or necrosis, has not been clarified as yet. It has been suggested that the generation of oxidative stress by bile acids contributes to hepatocyte death during cholestasis and bile acid toxicity, although the beneficial role of ROS prevention in bile acid-mediated cell death is not fully understood. AIM: Study the effects of anti-oxidants in bile acid-induced cell death in vitro. METHODS: Primary rat hepatocytes were exposed to the bile acids glycochenodeoxycholic acid (GCDCA) or taurolithocholic acid-3 sulphate in the absence or presence of ROS scavengers or anti-oxidants. Haeme oxygenase (HO)-1 mRNA levels were analysed by quantitative polymerase chain reaction. Apoptosis was quantified by acridine orange staining and caspase-3 activity assay. Necrosis was detected by Sytox green staining. RESULTS: Anti-oxidants do not attenuate bile acid-induced cell death. Furthermore, bile acid exposure does not enhance the mRNA expression of the oxidative stress-responsive gene HO-1. The Src-kinase inhibitor, SU6656, does reduce GCDCA-induced apoptosis and necrosis. CONCLUSIONS: In hepatocytes, bile acid-induced toxicity is not prevented by scavengers of oxidative stress. The beneficial effects observed in patients might be because of the contribution of ROS and cytokines rather than bile acid-mediated oxidative stress. However, the use of specific Src kinase inhibitors might be a useful tool to prevent bile acid-induced injury in liver diseases.

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes.

UNLABELLED: Caveolae are a subtype of cholesterol-enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin-1 is an essential component of caveolae. Hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin-1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin-1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin-1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin-1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno-electron microscopy. Green fluorescent protein (GFP)-tagged caveolin-1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X-100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid-dependent regulation of caveolin-1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference-mediated silencing of caveolin-1 and caveolin-1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin-1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid-coenzyme A:amino acid N-acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP-caveolin-1 accumulated in catalase-positive organelles. Peroxisomal caveolin-1 is associated with detergent-resistant microdomains. Caveolin-1 expression is strongly repressed by the peroxisome proliferator-activated receptor-alpha agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%-80% reduced caveolin-1 levels and in livers of caveolin-1 knockout mice. CONCLUSION: Caveolin-1 is enriched in peroxisomes of hepatocytes. Caveolin-1 is not required for peroxisome biogenesis, but this unique subcellular location may determine its important role in hepatocyte proliferation and lipid metabolism.

Klotho prevents renal calcium loss.

Disturbed calcium (Ca(2+)) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca(2+) absorption is primary, which causes increased urinary Ca(2+) excretion, leading to elevated 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its sequelae. Here, we assessed intestinal Ca(2+) absorption, bone densitometry, renal Ca(2+) excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho(-/-) mice. We observed elevated serum Ca(2+) and fractional excretion of Ca(2+) (FE(Ca)) in klotho(-/-) mice. Klotho(-/-) mice also showed intestinal Ca(2+) hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D(9K) increased. In the kidney, klotho(-/-) mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D(28K), implying a failure to absorb Ca(2+) through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-alpha-hydroxylase (1alphaOHase), and calbindin-D(9K) excluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca(2+) wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca(2+) handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca(2+) loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis.

The role of transient receptor potential channels in kidney disease.

The transient receptor potential (TRP) superfamily consists, in mammals, of six protein subfamilies, TRPC, TRPM, TRPV, TRPA, TRPML and TRPP. TRPs are cation channels involved in many physiological processes and in the pathogenesis of various disorders. In the kidney, TRP channels are expressed along the nephron, and a role for some of these channels in renal function has been proposed. TRPC3 is thought to facilitate calcium ion influx into the principal cells of the collecting duct in response to vasopressin. TRPM3 and TRPV4 might be osmosensors, whereas the TRPP1/TRPP2 complex could function as a mechanosensor in the cilia of renal epithelial cells. A number of kidney diseases have also been linked to dysfunctional activity of TRPs. TRPC6 dysfunction has been associated with the onset of focal segmental glomerosclerosis; TRPP2 dysfunction is linked to autosomal-dominant polycystic kidney disease, TRPM6 mutations underlie hypomagnesemia with secondary hypocalcemia, and TRPV1 dysfunction is implicated in renal hypertension. A link between TRPC1 dysfunction and diabetic nephropathy has also been suggested in an animal model. Animal studies have implicated a role for TRPV5 in idiopathic hypercalciuria and vitamin D-dependent rickets, although these observations have not been confirmed in patients. This Review focuses on the role of renal TRP channels in health and disease.