Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD+) when compared with diabetic patients with normoalbuminuria (DKD−) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.

The best-known function of the serine protease tissue-type plasminogen activator (tPA) is as a thrombolytic enzyme. However, it is also found in structures of the brain that are highly vulnerable to hypoxia-induced cell death, where its association with neuronal survival is poorly understood. Here, we have demonstrated that hippocampal areas of the mouse brain lacking tPA activity are more vulnerable to neuronal death following an ischemic insult. We found that sublethal hypoxia, which elicits tolerance to subsequent lethal hypoxic/ischemic injury in a natural process known as ischemic preconditioning (IPC), induced a rapid release of neuronal tPA. Treatment of hippocampal neurons with tPA induced tolerance against a lethal hypoxic insult applied either immediately following insult (early IPC) or 24 hours later (delayed IPC). tPA-induced early IPC was independent of the proteolytic activity of tPA and required the engagement of a member of the LDL receptor family. In contrast, tPA-induced delayed IPC required the proteolytic activity of tPA and was mediated by plasmin, the NMDA receptor, and PKB phosphorylation. We also found that IPC in vivo increased tPA activity in the cornu ammonis area 1 (CA1) layer and Akt phosphorylation in the hippocampus, as well as ischemic tolerance in wild-type but not tPA-or plasminogen-deficient mice. These data show that tPA can act as an endogenous neuroprotectant in the murine hippocampus.

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte aVb 3 integrin-dependent migration in vitro. Furthermore, aVb 3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinaselike phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent aVb 3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD seratreated human podocytes, where it prevented aVb 3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.

The serine proteinase tissue-type plasminogen activator (tPA) and the serine proteinase inhibitor neuroserpin are both expressed in areas of the brain with the highest vulnerability to hypoxia/ischemia. In vitro studies show that neuroserpin inhibits tPA and, to a lesser extent, urokinase-type plasminogen activator and plasmin. Experimental middle cerebral artery occlusion (MCAO) increases tPA activity and neuroserpin expression in ischemic tissue, and genetic deficiency of tPA or either treatment with or overexpression of neuroserpin decreases the volume of the ischemic lesion following MCAO. These findings have led to the hypothesis that neuroserpin's neuroprotection is mediated by inhibition of tPA's alleged neurotoxic effect. Ischemic preconditioning is a natural adaptive process whereby exposure to a sublethal insult induces tolerance against a subsequent lethal ischemic injury. Here we demonstrate that exposure to sublethal hypoxia/ischemia increases the neuroserpin expression in the hippocampal CA1 layer and cerebral cortex, and that neuroserpin induces ischemic tolerance and decreases the volume of the ischemic lesion following MCAO in wild-type and tPAdeficient (tPA ؊/؊ ) neurons and mice. Plasmin induces neuronal death, and this effect is abrogated by either neuroserpin or the NMDA receptor antagonist MK-801. Neuroserpin also attenuated kainic acid-induced neuronal death. Our data indicate that the neuroprotective effect of neuroserpin is due to inhibition of plasmin-mediated excitotoxin-induced cell death and is independent of neuroserpin's ability to inhibit tPA activity.

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.

Studies suggest that altered renal lipid metabolism plays a role in the pathogenesis of diabetic kidney disease and that genetic or pharmacological induction of cholesterol efflux protects from the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we tested whether altered lipid metabolism contributes to renal failure in the Col4a3 knockout mouse model for Alport Syndrome. There was an eight fold increase in the cholesterol content in renal cortexes of mice with Alport Syndrome. This was associated with increased glomerular lipid droplets and cholesterol crystals. Treatment of mice with Alport Syndrome with hydroxypropyl-β-cyclodextrin (HPβCD) reduced cholesterol content in the kidneys of mice with Alport Syndrome and protected from the development of albuminuria, renal failure, inflammation and tubulointerstitial fibrosis. Cholesterol efflux and trafficking related genes were primarily affected in mice with Alport Syndrome and were differentially regulated in the kidney cortex and isolated glomeruli. HPβCD also protected from proteinuria and mesangial expansion in a second model of non-metabolic kidney disease; adriamycin-induced nephropathy. Consistent with our experimental findings, microarray analysis confirmed dysregulation of several lipid related genes in glomeruli isolated from kidney biopsies of patients with primary FSGS enrolled in the NEPTUNE study. Thus, lipid dysmetabolism occurs in non-metabolic glomerular disorders such as Alport Syndrome and FSGS, and HPβCD improves renal function in experimental Alport Syndrome and FSGS.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Fn14) are expressed in neurons. Here we demonstrate that TWEAK induces a dose-dependent increase in neuronal death and that this effect is independent of TNF-α and mediated by NF-κB pathway activation. Incubation with TWEAK induces apoptotic cell death in wild-type (Wt) but not in Fn14 deficient (Fn14 −/− ) neurons. Intracerebral injection of TWEAK induces accumulation of poly(ADP-ribose) polymers (PAR) in Wt but not in Fn14 −/− mice. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in Wt neurons, and decreases cell survival in Wt but not in Fn14 −/− or TWEAK deficient (TWEAK −/− ) neurons. Experimental middle cerebral artery occlusion (MCAO) increases the expression of TWEAK and Fn14 mRNA and active caspase-3, and the cleavage of poly(ADPribose)polymerase-1 with accumulation of PAR in the ischemic area in Wt but not Fn14 −/− mice. Together, these results suggest a model where in response to hypoxia/ischemia the interaction between TWEAK and Fn14 in neurons induces PARP-1 activation with accumulation of PAR polymers and cell death via NF-κB pathway activation. This is a novel pathway for hypoxia/ ischemia-induced TWEAK-mediated cell death and a potential therapeutic target for ischemic stroke. KeywordsCerebral ischemia; Tumor necrosis factor-like weak inducer of apoptosis (TWEAK); Fibroblast growth factor-inducible 14 (Fn14); Poly(ADP-ribose)polymerase-1 (PARP-1); Poly(ADP-ribose) polymers (PAR) Corresponding author: Manuel Yepes, Department of Neurology and Center for Neurodegenerative Disease, Whitehead Biomedical Research Building, 615 Michael Street, Suite 505J, Atlanta, Georgia 30322. Telephone: (404) 712 8358. Fax: (404) 727 3728. myepes@emory.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 December 29. JA, (Winkles, 2008)], which has been linked to cell death during cerebral ischemia (Schneider et al., 1999a). TWEAK has been reported to stimulate cell proliferation (Lynch et al., 1999;Harada et al., 2002;Donohue et al., 2003), migration (Harada et al., 2002;Tran et al., 2003;Donohue et al., 2003) and differentiation (Polek et al., 2003), as well as the expression of pro-inflammatory molecules (Chicheportiche et al., 1997;Saas et al., 2000;Harada et al., 2002;Chicheportiche et al., 2002;Xu et al., 2004;Kim et al., 2004;Jin et al., 2004).TWEAK was initially described as a poor cytotoxic agent that in...

Background: Nephrin is an immunoglobulin-like protein that facilitates insulin release by pancreatic beta cells.Results: Nephrin phosphorylation at tyrosine residues responsible for SH2 domain binding is a Dynamin-dependent phenomenon, and it is necessary for glucose-stimulated insulin release in insulinoma cells and human islets.Conclusion: Dynamin-dependent Nephrin phosphorylation is necessary for glucose-stimulated insulin secretion.Significance: Pharmacological modulation of Nephrin phosphorylation may facilitate pancreatic beta cell function.