Renal medullary Na-K-ATPase and hypoxic injury in perfused rat kidneys

Renal medullary Na-K-ATPase and hypoxic injury in perfused rat kidneys. We wished to see if chronic alterations in Na-K-ATPase activity in the medullary thick ascending limb would modify the susceptibility of its cells to the hypoxic injury produced by perfusion of the isolated kidney. Rats were fed a diet high (64%) or low (8%) in protein for three weeks. Renal medullary Na-K-ATPase was 75 ± 12 U/mg protein/hr (mean ± SE) in the high protein group and 44 ± 3 in rats given low protein. After 90 minutes of perfusion, the kidneys of rats fed a high protein diet showed almost all mTAL cells near the inner medulla with severe damage (93 ± 4.8%), whereas the same zone in perfused kidneys of rats on a low protein diet showed only 47 ± 7.7% injury. In a similar fashion, damage to mTAL cells seen in perfused kidneys was greatly augmented by compensatory renal hypertrophy produced by removal of the contralateral kidney two weeks earlier, and by a diet high in potassium given for two weeks, procedures which also increased the activity of medullary Na-K-ATPase. The results suggest that the level of transport work of medullary cells mediated by Na-K-ATPase is a determinant of the vulnerability of mTAL cells to hypoxic injury

Protective action of glycine in cisplatin nephrotoxicity

Protective action of glycine in cisplatin nephrotoxicity. Because glycine is cytoprotective for kidney cells in vitro, we investigated its possible action in vivo to protect rats against cisplatin nephrotoxicity, a well-established experimental model of renal tubular injury. Glycine was infused at a dose of 1mmol per 100g body weight per hour for 75 minutes, starting 15 minutes before cisplatin, 5mg per kg, was injected intravenously. Plasma concentration of glycine rose to 3.5mmol per liter at the time cisplatin was injected. These rats were compared with cisplatin-treated animals treated with L-alanine or with isotonic saline. After five days plasma creatinine of saline-treated rats given cisplatin had risen threefold to 2.6 ± 1.5mg per 100ml (mean ± SD), as creatinine clearance fell to 25% of baseline (0.14 ± 0.05ml/min/100g). Morphological evaluation disclosed extensive damage involving all S3 segments in the outer medulla as well as the medullary rays of the cortex. In contrast, in rats treated with glycine, plasma creatinine rose only to 1.2 ± 0.2mg/100ml and creatinine clearance was maintained at 75% of baseline (0.35 ± 0.05ml/min/100g). Glycine also attenuated the weight loss, polyuria, increased fractional excretion of sodium and potassium, decreased urinary osmolality, and renal glycosuria observed in control, saline-treated rats after cisplatin, while substantially decreasing the percentage of S3 tubules with evident morphological injury. Renal platinum content was unaffected by glycine. The administration of L-alanine or the delayed infusion of glycine, starting one hour after cisplatin was given, did not prevent cisplatin toxicity. Thus, high plasma concentrations of glycine achieved during a brief period of time when cisplatin is administered, markedly attenuate cisplatin nephrotoxicity

Vascular bed-specific regulation of the von Willebrand factor promoter in the heart and skeletal muscle

A region of the human von Willebrand factor (VWF) gene between -2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between -843 and -620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at -56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed-specific expression of VWF. (Blood. 2011;117(1):342-351

RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG

ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knockdown on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knockdown of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver. (Blood. 2011; 118(4):1145-1153)</p

PGC-1&amp;agr; Induces SPP1 to Activate Macrophages and Orchestrate Functional Angiogenesis in Skeletal Muscle

RationaleMechanisms of angiogenesis in skeletal muscle remain poorly understood. Efforts to induce physiological angiogenesis hold promise for the treatment of diabetic microvascular disease and peripheral artery disease but are hindered by the complexity of physiological angiogenesis and by the poor angiogenic response of aged and patients with diabetes mellitus. To date, the best therapy for diabetic vascular disease remains exercise, often a challenging option for patients with leg pain. Peroxisome proliferation activator receptor-γ coactivator-1α (PGC-1α), a powerful regulator of metabolism, mediates exercise-induced angiogenesis in skeletal muscle.ObjectiveTo test whether, and how, PGC-1α can induce functional angiogenesis in adult skeletal muscle.Methods and resultsHere, we show that muscle PGC-1α robustly induces functional angiogenesis in adult, aged, and diabetic mice. The process involves the orchestration of numerous cell types and leads to patent, nonleaky, properly organized, and functional nascent vessels. These findings contrast sharply with the disorganized vasculature elicited by induction of vascular endothelial growth factor alone. Bioinformatic analyses revealed that PGC-1α induces the secretion of secreted phosphoprotein 1 and the recruitment of macrophages. Secreted phosphoprotein 1 stimulates macrophages to secrete monocyte chemoattractant protein-1, which then activates adjacent endothelial cells, pericytes, and smooth muscle cells. In contrast, induction of PGC-1α in secreted phosphoprotein 1(-/-) mice leads to immature capillarization and blunted arteriolarization. Finally, adenoviral delivery of PGC-1α into skeletal muscle of either young or old and diabetic mice improved the recovery of blood flow in the murine hindlimb ischemia model of peripheral artery disease.ConclusionsPGC-1α drives functional angiogenesis in skeletal muscle and likely recapitulates the complex physiological angiogenesis elicited by exercise