Nucleotide phosphohydrolysis by the ecto-59-nucleotidase (CD73) is the main source for extracellular generation of adenosine. Extracellular adenosine subsequently signals through four distinct adenosine A receptors (Adora1, Adora2a, Adora2b, or Adora3). Here, we hypothesized a functional role for CD73-dependent generation and concomitant signaling of extracellular adenosine during diabetic nephropathy. CD73 transcript and protein levels were elevated in the kidneys of diabetic mice. Genetic deletion of CD73 was associated with more severe diabetic nephropathy, whereas treatment with soluble nucleotidase was therapeutic. Transcript levels of renal adenosine receptors showed a selective induction of Adora2b during diabetic nephropathy. In a transgenic reporter mouse, Adora2b expression localized to the vasculature and increased after treatment with streptozotocin. Adora2b 2/2 mice experienced more severe diabetic nephropathy, and studies in mice with tissue-specific deletion of Adora2b in tubular epithelia or vascular endothelia implicated endothelial Adora2b signaling in protection from diabetic nephropathy. Finally, treatment with a selective Adora2b agonist (BAY 60-6583) conveyed potent protection from diabetes-associated kidney disease. Taken together, these findings implicate CD73-dependent production of extracellular adenosine and endothelial Adora2b signaling in kidney protection during diabetic nephropathy.
Recent studies implicate neuronal guidance molecules in the orchestration of inflammatory events. For example, previous studies demonstrate a functional role for netrin-1 in attenuating acute kidney injury. Here, we hypothesized a kidney-protective role for netrin-1 during chronic kidney disease, such as occurs during diabetic nephropathy. To study the role of netrin-1 during diabetic nephropathy, we induced diabetes in mice at age of 8 weeks by streptocotozin (STZ) treatment. Sixteen weeks after STZ treatment, we examined the kidneys. Initial studies in wild-type mice demonstrated robust induction of renal, urinary and plasma netrin-1 protein levels during diabetic nephropathy. Subsequent genetic studies in mice with partial netrin-1 deficiency (Ntrn1+/− mice) revealed a more severe degree of diabetic nephropathy, including more severe loss of kidney function (albuminuria, glomerular filtration rate, histology). We subsequently performed pharmacologic studies with recombinant netrin-1 treatment given continuously via osmotic pump. Indeed, netrin-1 treatment was associated with attenuated albuminuria and improved histologic scores for diabetic nephropathy compared to controls. Consistent with previous studies implicating purinergic signaling in netrin-1-elicited tissue-protection, mice deficient in the Adora2b adenosine receptor were not protected. Taken together, these studies demonstrate a functional role for endogenous netrin-1 in attenuating diabetic kidney disease.
Background: Myelomeningocele (MMC) results in lifelong neurologic and functional deficits. Currently, prenatal repair of MMC closes the defect, resulting in a 50% reduction in postnatal ventriculoperitoneal shunting. However, this invasive fetal surgery is associated with significant morbidities to mother and baby. We have pioneered a novel reverse thermal gel (RTG) to cover MMC defects in a minimally invasive manner. Here, we test in-vitro RTG long-term stability in amniotic fluid and in vivo application in the Grainy head-like 3 (Grhl3) mouse MMC model. Materials and methods: RTG stability in amniotic fluid (in-vitro) was monitored for 6 mo and measured using gel permeation chromatography and solutionegel transition temperature (lower critical solution temperature). E16.5 Grhl3 mouse fetuses were injected with the RTG or saline and harvested on E19.5. Tissue was assessed for RTG coverage of the gross defect and inflammatory response by immunohistochemistry for macrophages. Results: Polymer backbone molecular weight and lower critical solution temperature remain stable in amniotic fluid after 6 mo. Needle injection over the MMC of Grhl3 fetuses successfully forms a stable gel that covers the entire defect. On harvest, some animals demonstrate >50% RTG coverage. RTG injection is not associated with inflammation. Conclusions: Our results demonstrate that the RTG is a promising candidate for a minimally invasive approach to patch MMC. We are now poised to test our RTG patch in the large preclinical ovine model used to evaluate prenatal repair of MMC.
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