The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK coimmunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca 2+ -calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca 2+ -calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaMbinding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.z 1999 Federation of European Biochemical Societies.

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.

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Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis and Goodpasture’s disease, is associated with particular Human Leukocyte Antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigated the molecular mechanism of Goodpasture’s disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T cell self-epitope derived from the α3 chain of Type IV collagen (α3135-145)1–4. While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR152. We show that autoreactive α3135-145-specific T cells expand in patients with Goodpasture’s disease and, in α3135-145-immunized HLA-DR15 transgenic mice, α3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture’s disease. HLA-DR15 and HLA-DR1 exhibited distinct peptide repertoires and binding preferences and presented the α3135-145 epitope in different binding registers. HLA-DR15-α3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. HLA-DR1-induced Tregs confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered α3135-145-specific TCR usage, HLA-DR15-α3135-145 tetramer+ Foxp3− Tconv and HLA-DR1-α3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes, and patients with Goodpasture’s disease display a clonally expanded α3135-145-specific CD4+ T cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Tregs that leads to protection or causation of autoimmunity.

In the kidney nitric oxide (NO) has numerous important functions including the regulation of renal haemodynamics, maintenance of medullary perfusion, mediation of pressure-natriuresis, blunting of tubuloglomerular feedback, inhibition of tubular sodium reabsorption and modulation of renal sympathetic neural activity. The net effect of NO in the kidney is to promote natriuresis and diuresis. Significantly, deficient renal NO synthesis has been implicated in the pathogenesis of hypertension. All three isoforms of nitric oxide synthase (NOS), namely neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3) are reported to contribute to NO synthesis in the kidney. The regulation of NO synthesis in the kidney by NOSs is complex and incompletely understood. Historically, many studies of NOS regulation in the kidney have emphasized the role of variations in gene transcription and translation. It is increasingly appreciated, however, that the constitutive NOS isoforms (nNOS and eNOS) are also subject to rapid regulation by post-translational mechanisms such as Ca 2+ flux, serine/threonine phosphorylation and protein-protein interactions. Recent studies have emphasized the role of post-translational regulation of nNOS and eNOS in the regulation of NO synthesis in the kidney. In particular, a role for phosphorylation of nNOS and eNOS at both activating and inhibitory sites is emerging in the regulation of NO synthesis in the kidney. This review summarizes the roles of NO in renal physiology and discusses recent advances in the regulation of eNOS and nNOS in the kidney by post-translational mechanisms such as serine/threonine phosphorylation.

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.

Aims/hypothesis: We compared the predictive performance of a GFR based on serum cystatin C levels with commonly used creatinine-based methods in subjects with diabetes.

The ultrasensitive energy sensor AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and energy-consuming pathways. AMPK is highly expressed in the kidney where it is reported to be involved in a variety of physiological and pathological processes including ion transport, podocyte function, and diabetic renal hypertrophy. Sodium transport is the major energy-consuming process in the kidney, and AMPK has been proposed to contribute to the coupling of ion transport with cellular energy metabolism. Specifically, AMPK has been identified as a regulator of several ion transporters of significance in renal physiology, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), the Na(+)-K(+)-2Cl(-) cotransporter (NKCC), and the vacuolar H(+)-ATPase (V-ATPase). Identified regulators of AMPK in the kidney include dietary salt, diabetes, adiponectin, and ischemia. Activation of AMPK in response to adiponectin is described in podocytes, where it reduces albuminuria, and in tubular cells, where it reduces glycogen accumulation. Reduced AMPK activity in the diabetic kidney is associated with renal accumulation of triglyceride and glycogen and the pathogenesis of diabetic renal hypertrophy. Acute renal ischemia causes a rapid and powerful activation of AMPK, but the functional significance of this observation remains unclear. Despite the recent advances, there remain significant gaps in the present understanding of both the upstream regulating pathways and the downstream substrates for AMPK in the kidney. A more complete understanding of the AMPK pathway in the kidney offers potential for improved therapies for several renal diseases including diabetic nephropathy, polycystic kidney disease, and ischemia-reperfusion injury.