Direct Observation of Enhanced Nitric Oxide in a Murine Model of Diabetic Nephropathy.

Uncoupling of nitric oxide synthase (NOS) secondary to redox signaling is a central mechanism in endothelial and macrophage activation. To date studies on the production of nitric oxide (NO) during the development of diabetic complications show paradoxical results. We previously showed that recoupling eNOS by increasing the eNOS cofactor tetrahydrobiopterin (BH4) could restore endothelial function and prevent kidney injury in experimental kidney transplantation. Here, we employed a diabetic mouse model to investigate the effects of diabetes on renal tissue NO bioavailability. For this, we used in vivo NO trapping, followed by electron paramagnetic resonance spectroscopy. In addition, we investigated whether coupling of NOS by supplying the cofactor BH4 could restore glomerular endothelial barrier function. Our data show that overall NO availability at the tissue level is not reduced sixteen weeks after the induction of diabetes in apoE knockout mice, despite the presence of factors that cause endothelial dysfunction, and the presence of the endogenous NOS inhibitor ADMA. Targeting uncoupled NOS with the BH4 precursor sepiapterin further increases NO availability, but did not modify renal glomerular injury. Notably, glomerular heparanase activity as a driver for loss of glomerular barrier function was not reduced, pointing towards NOS-independent mechanisms. This was confirmed by unaltered increased glomerular presence of cathepsin L, the protease that activates heparanase

Tissue-dependent variation in NO free radical induction.

<p>(A) EPR spectrum of frozen kidney samples. The characteristic triplet structure of the mononitrosyl-iron complex (MNIC, double-headed arrow) centers around g  =  2.035 and represents the formation of local nitric oxide in 334–370 mg tissue. (B-D) Quantification of nitric oxide formation in kidney, liver and heart tissue, shown as mean pmol MNIC / mg wet tissue ± SD, n = 7–9. E) Plasma ADMA concentrations, shows as mean ± SD, n = 8. *P<0.05, compared with ApoE; #P<0.05 compared with DM. ApoE = ApoE KO mice, DM = diabetic apoE KO mice, DM + S = diabetic apoE KO mice + sepiapterin.</p

Increased NO levels affect endothelial glycocalyx non-uniformly.

<p>(A) Representative microscopic images of cationic ferritin (TEM; top), heparanase (HPSE, immunofluorescence; middle) and cathepsin L (CTSL; bottom) in glomeruli of apoE KO mice (apoE), diabetic apoE KO mice (DM) and diabetic apoE KO mice treated with sepiapterin (DM + S). (B) Quantification of endothelial cationic ferritin coverage in 6–8 capillary loops in 9 glomeruli of 3 mice, shown as mean percentage of total capillary length ± SD, (C) Quantification of glomerular heparanase expression, shown as mean area percentage ± SD. (D) Quantification of glomerular cathepsin L expression, shown as mean area percentage ± SD. *P<0.05 compared with ApoE, n = 6–8. Scale bars: 500 nm in TEM images; 20 μm in fluorescent and light microscopic images.</p

Experimental set-up for assessment of NO bioavailability.

<p>(A) male ApoE KO mice (<i>B6</i>.<i>129P2- Apoe</i>^{<i>tm1Unc</i>}<i>/J)</i> were injected with citrate buffer ± STZ. Diabetic mice received cholesterol enriched diet and insulin from week 8 onwards. At 18 week of age, diabetic mice were treated with sepiapterin or received normal drinking water for 4 weeks. Urine was collected upon commencing with the experimental procedure and after 2 and 4 weeks of treatment. At 22 weeks, plasma was collected and the mice were sacrificed.</p

Sepiapterin does not reduce albuminuria in diabetic apoE KO mice.

<p>(A) PAS-stained glomeruli of apoE KO mice (apoE), diabetic apoE KO mice (DM) and diabetic apoE KO mice treated with sepiapterin (DM + S), showing heterogeneous diabetic lesions 14 weeks after induction of diabetes with STZ (20). Scale bars: 20 μm. Sepiapterin did not affect mesangial area (B,C), nor blood glucose concentrations (D). Data are shown as mean ± SD, *P<0.05 compared with apoE, n = 8. (E) Albumin-creatinine ratios (ACR) at baseline, 2- and 4 weeks after treatment, as indicated by mean ± SEM, *P<0.05 compared with apoE, n = 14–23.</p

Glomerular Function and Structural Integrity Depend on Hyaluronan Synthesis by Glomerular Endothelium

Background A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function.Methods To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy.Results Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2.Conclusions Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrixthat is required for glomerular structure and function and lost in diabetic nephropathy.Diabetes mellitus: pathophysiological changes and therap

Schematic illustration on relation between glycocalyx accessibility and microvascular perfusion regulation.

<p><b>A</b>) Healthy state: Intact glycocalyx prevents red blood cells (RBC, red dots) from penetrating into its domain, reflected by a low perfused boundary region (PBR), and nicely aligned elongated RBC. The vessels are well perfused (higher tube hematocrit of microvessel and elongated shape of erythrocyte) resulting in a higher percentage of vessel segments with RBC present at any particular time point (high RBC filling percentage). <b>B</b>) Risk State: Altered composition of glycocalyx (lined dots) allows RBCs to penetrate deeper into the glycocalyx, closer to the anatomical border of lumen (endothelium), reflected by the high PBR. Due to the widening of RBC distribution width and volume, there is more space in between each RBC, as shown by decreased RBC filling percentage (less positive contrast per vascular segment per time point). Also, prolonged state of glycocalyx degradation leads to edematous and non-functioning vessels, leading to shorter vessel density per area of tissue (reduced valid microvascular density in risk PBR), depicted by the loss of bottom vessel.</p

Sidestream darkfield imaging derived variables characteristics.

<p>Data is presented as mean ± standard deviation.</p

A microscopic view on the renal endothelial glycocalyx

Item does not contain fulltextEndothelial cells perform key homeostatic functions such as regulating blood flow, permeability, and aiding immune surveillance for pathogens. While endothelial activation serves normal physiological adaptation, maladaptation of these endothelial functions has been identified as an important effector mechanism in the progression of renal disease as well as the associated development of cardiovascular disease. The primary interface between blood and the endothelium is the glycocalyx. This carbohydrate-rich gel-like structure with its associated proteins mediates most of the regulatory functions of the endothelium. Because the endothelial glycocalyx is a highly dynamic and fragile structure ex vivo, and traditional tissue processing for staining and perfusion-fixation usually results in a partial or complete loss of the glycocalyx, studying its dimensions and function has proven to be challenging. In this review, we will outline the core functions of the glycocalyx and focus on different techniques to study structure-function relationships in kidney and vasculature

Glycocheck algorithm on endothelial PBR determination and microvascular perfusion properties.

<p><b>A</b>) Red blood cells (RBC) are detected through reflection of light emitting diodes by hemoglobin. Images captured by the sidestream darkfield camera are sent to the computer for quality checks and assessment. The black contrast is the perfused lumen of the vessels. <b>B</b>) In each recording, the software automatically places the vascular segments (green), every 10 µm along the vascular segments (black contrast). <b>C</b>) After the acquisition, for the analysis, the software undergoes several quality check in the first frame of each recording (see text), to select vascular segments with sufficient quality for further analysis. Invalid vascular segments (yellow) are distinguished from the valid vascular segments (green). During the whole recording session of 40 frames, the percentage of time in which a particular valid vascular segment has RBCs present is used to calculate RBC filling percentage. <b>D</b>) Depiction of the concept of glycocalyx thickness by lateral RBC movement is shown here. <b>E</b>) For each vascular segment, the intensity profile is calculated to derive median RBC column width. <b>F</b>) Then, the distribution of RBC column width is used to calculate the perfused diameter, median RBC column width, and subsequently the perfused boundary region (PBR).</p