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.

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.

These data indicate that reduced phosphorylation of ACC after renal injury contributes to the development of TIF, and that phosphorylation of ACC is required for metformin's antifibrotic action in the kidney.

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.

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

Although synthesis of VEGF mRNA and protein is not increased during ischemia reperfusion injury, pre-existing VEGF in the tubular cell cytoplasm redistributes to the basolateral aspect of the cells. These data suggest that the kidney may have evolved unique patterns of VEGF regulation to cope with acute hypoxia.

Activation of nuclear factor‐kappa B (NF‐κB) is one of the most important pro‐inflammatory mechanisms in disease. In this study, we show that 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), an intermediate in nucleoside metabolism, inhibits signalling by NF‐κB in three cell types, including bovine aortic endothelial cells (BAEC). The block in the NF‐κB signalling pathway occurred beyond degradation of IκB‐α and movement of p65 into the nucleus of BAEC. There was, however, reduced binding of NF‐κB from AICAR‐treated cells to a κB‐consensus oligonucleotide, suggesting that part of the mechanism was a reduction in NF‐κB DNA‐binding activity. Although AICAR is metabolized to ZMP and then adenosine, adenosine had no effect on activation of an NF‐κB reporter. ZMP, however, activates the metabolic stress‐sensing AMP‐activated protein kinase (AMPK). Transfection of active AMPK into BAEC reduced NF‐κB reporter activity compared with a kinase‐dead mutant, suggesting that part of the ability of AICAR to inhibit NF‐κB signalling is due to activation of AMPK. Inhibition of NF‐κB signalling may be important in the anti‐inflammatory action of drugs such as sulfasalazine and methotrexate, which led to the accumulation of AICAR within target cells.

SUMMARYConflicting reports exist regarding the effects of interleukin-10 (IL-10) on mesangial cells. There have been reports of both proliferative and antiproliferative effects, and both proinflammatory and antiinflammatory effects of IL-10 on mesangial cells. However, the potential for IL-10 to affect glomerulonephritis characterized by mesangial proliferation is not known. To test the hypothesis that IL-10 would limit experimental mesangial proliferative glomerulonephritis, IL-10 was administered to rats in which mesangial proliferative glomerulonephritis was induced by administration of anti-Thy 1 antibody. Compared to control treated rats, IL-10 treated rats showed less proliferation, with fewer cells in glomeruli. Glomerular cellular proliferation was reduced, assessed by the numbers of cells within glomeruli expressing either proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine. Glomerular macrophage influx (but not the proportion of glomerular macrophages that were PCNA positive) was reduced by IL-10 administration. There was no significant reduction in glomerular asmooth muscle actin staining. IL-10 treatment resulted in reduced renal IL-1b mRNA expression and reduced glomerular ICAM-1 expression, but renal expression of MCP-1 and osteopontin mRNA was unaltered. This study demonstrates that in experimental mesangial proliferative glomerulonephritis IL-10 diminishes inflammatory cell recruitment and mesangial cell proliferation. The effects of IL-10 in inhibiting mesangial cell proliferation are likely to be due to a combination of direct effects of IL-10 on mesangial cells and effects mediated by macrophages.