Inflammation plays a vital role in the development of diabetic nephropathy, but the underlying regulatory mechanisms are only partially understood. Our previous studies demonstrated that, during acute inflammation, endothelial heparan sulfate (HS) contributes to the adhesion and transendothelial migration of leukocytes into perivascular tissues by direct interaction with l-selectin and the presentation of bound chemokines. In the current study, we aimed to assess the role of endothelial HS on chronic renal inflammation and fibrosis in a diabetic nephropathy mouse model. To reduce sulfation of HS specifically in the endothelium, we generated Ndst1f/fTie2Cre+ mice in which N-deacetylase/N-sulfotransferase-1 (Ndst1), the gene that initiates HS sulfation modifications in HS biosynthesis, was expressly ablated in endothelium. To induce diabetes, age-matched male Ndst1f/fTie2Cre− (wild type) and Ndst1f/fTie2Cre+ mice on a C57Bl/6J background were injected intraperitoneally with streptozotocin (STZ) (50 mg/kg) on five consecutive days (N = 10–11/group). Urine and plasma were collected. Four weeks after diabetes induction the animals were sacrificed and kidneys were analyzed by immunohistochemistry and qRT-PCR. Compared to healthy controls, diabetic Ndst1f/fTie2Cre− mice showed increased glomerular macrophage infiltration, mannose binding lectin complement deposition and glomerulosclerosis, whereas these pathological reactions were prevented significantly in the diabetic Ndst1f/fTie2Cre+ animals (all three p < 0.01). In addition, the expression of the podocyte damage marker desmin was significantly higher in the Ndst1f/fTie2Cre− group compared to the Ndst1f/fTie2Cre+ animals (p < 0.001), although both groups had comparable numbers of podocytes. In the cortical tubulo-interstitium, similar analyses show decreased interstitial macrophage accumulation in the diabetic Ndst1f/fTie2Cre+ animals compared to the diabetic Ndst1f/fTie2Cre− mice (p < 0.05). Diabetic Ndst1f/fTie2Cre+ animals also showed reduced interstitial fibrosis as evidenced by reduced density of αSMA-positive myofibroblasts (p < 0.01), diminished collagen III deposition (p < 0.001) and reduced mRNA expression of collagen I (p < 0.001) and fibronectin (p < 0.001). Our studies indicate a pivotal role of endothelial HS in the development of renal inflammation and fibrosis in diabetic nephropathy in mice. These results suggest that HS is a possible target for therapy in diabetic nephropathy.
Cerebrospinal fluid is a biofluid contiguous with brain and spinal cord and a plausible tissue for measuring concentrations of central nervous system drugs and biomarkers. Lumbar puncture enables single timepoint sampling while lumbar catheterization enables repeated collection over a period of time. We report here our experience with the procedural safety of both techniques across two phase 1 studies of ESB1609, a brain penetrant sphingosine-1-phosphate receptor agonist, alongside the technical considerations, opportunities, and challenges of implementation. Across both studies, good quality samples were obtained through lumbar puncture and lumbar catheterization. Mild adverse events of post dural puncture headaches were common among participants undergoing lumbar catheterization and resolved with simple analgesics. Surprisingly we found that blood contamination was absent for single lumbar punctures but was detectable throughout the entire lumbar catheter sampling periods, although levels of hemoglobin were very low. We determined that for certain neurological therapeutics, serial cerebrospinal fluid sampling via lumber catheterization in healthy volunteers can provide informative pharmacokinetic and biomarker data but has a relatively high risk of post dural puncture headache compared to lumbar puncture. Acetaminophen co-administered with caffeine might be considered as first line management for post dural puncture headache in appropriate participants.
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