The results highlight the need to provide access to appropriate in-language advance care planning resources and promotion of advance care planning across the Chinese community.

Vascular endothelial growth factor (VEGF) acts primarily as an endothelial cell mitogen via the "endothelial cell-specific" receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR). Only a few nonendothelial cells have been shown to possess functional VEGF receptors. We therefore examined the rat renal tubular epithelial cell line NRK52-E. NRK52-E expressed VEGFR-1 and VEGFR-2 mRNA and protein by RT-PCR, Northern blotting, Western blotting, immunofluorescence, and ligand binding. Serum-starved NRK52-E incubated with VEGF showed a significant increase in [(3)H]thymidine incorporation compared with control (2.3-fold at 1-10 ng/ml, P < 0. 05; 3.3-fold at 50-100 ng/ml, P < 0.01). VEGF also protected NRK52-E from hydrogen peroxide-induced apoptosis and necrosis compared with control (annexin-V-FITC-positive cells, 39 vs. 54%; viable cells, 50. 5 vs. 39.7%). Immunohistochemical staining using a variety of antibodies showed expression of both VEGF receptors in normal rat renal tubules in vivo. Because VEGF induced a proliferative and an antiapoptotic response in renal tubular epithelial cells, these data suggest that VEGF may act as a survival factor for renal tubular epithelium in vivo.

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.

The AMP-activated protein kinase (AMPK) is a key controller of cellular energy metabolism. We studied its expression and regulation by salt handling in the kidney. Immunoprecipitation and Western blots of protein lysates from whole rat kidney using subunit-specific antibodies showed that the alpha1-catalytic subunit is expressed in the kidney, associated with the beta2- and either gamma1- or gamma2-subunits. Activated AMPK, detected by immunohistochemical staining for phospho-Thr172 AMPK (pThr172), was expressed on the apical surface of the cortical thick ascending limb of the loop of Henle, including the macula densa, and some parts of the distal convoluted tubule. Activated AMPK was also expressed on the basolateral surface of the cortical and medullary collecting ducts as well as some portions of the distal convoluted tubules. AMPK activity was increased by 25% in animals receiving a high-salt diet, and this was confirmed by Western blotting for pThr172. Low-salt diets were associated with reduced levels of the alpha-subunit of AMPK, which was highly phosphorylated on Thr172. Surprisingly, both low- and high-salt media transiently activated AMPK in the macula densa cell line MMDD1, an effect due to changes in osmolality, rather than Na+ or Cl- concentration. This study, therefore, demonstrates regulation of AMPK by both a high- and a low-salt intake in vivo and suggests a role for the kinase in the response to changes in osmolality within the kidney.