Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes

Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the TNF-α–mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps’alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps’alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes

A role for NPY-NPY2R signaling in albuminuric kidney disease

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential. Chronic kidney disease (CKD) is a major global healthcare concern, affecting over 10% of the general population, and frequently occurs secondary to other systemic disorders including diabetes, obesity, hypertension, and the metabolic syndrome. A common early hallmark of CKD is albuminuria, which not only reflects damage to the glomerular filtration barrier (GFB) in the kidney but also is an important independent risk factor for the progression to end-stage renal failure and cardiovascular disease (1⇓–3). Thus, strategies to prevent albuminuria have important therapeutic potential, particularly in the early stages of CKD progression. Podocytes are highly specialized epithelial cells of the glomerulus, lining the urinary side of the filtration barrier. Owing to their complex, dynamic structures and their ability to secrete (and adapt to) a number of growth factors, these cells have a central role in filtration barrier maintenance (4). As such, podocyte damage is a key driver of albuminuria and glomerular disease in numerous settings and occurs early in the pathogenesis of many albuminuric conditions (5⇓⇓⇓–9). While it is well-established that podocyte damage is a major cause of albuminuria (8), the pathways and molecules involved in podocyte injury are incompletely understood. We (10, 11) and others (12, 13) have highlighted the importance of podocyte insulin responses in maintaining glomerular function, and it is now evident that circulating factors associated with common systemic disorders, including diabetes, obesity, and the metabolic syndrome, can directly induce podocyte insulin resistance (14⇓⇓–17) and associated damage (15, 18). In this study, we analyzed the transcriptomes of insulin-sensitive and insulin-resistant podocytes with the aim of identifying molecules that are differentially regulated in podocyte damage, which may play a role in albuminuric kidney disease. This unbiased transcriptome analysis revealed that Neuropeptide Y (Npy) was the most highly down-regulated transcript in insulin-resistant vs. insulin-sensitive podocytes. Analysis of patient cohorts also revealed a significant reduction in glomerular NPY expression in both early and late-stage diabetic nephropathy (DN), as well as in several other human albuminuric conditions. This contrasts with the increased plasma and urinary levels of NPY that are observed in diabetes and CKD (19⇓⇓–22). This prompted us to further investigate the potential role of NPY (and NPY signaling) in the podocyte and glomerulus

Chronic exposure to laminar shear stress induces Kruppel-like factor 2 in glomerular endothelial cells and modulates interactions with co-cultured podocytes

Laminar shear stress (LSS), induced by flowing blood, plays a key role in determining vascular health by modulating endothelial behaviour and vascular tone. In systemic endothelium many of the beneficial effects of chronic LSS are mediated through the transcription factor Kruppel-like factor 2 (KLF2), but little is known regarding the role of chronic LSS in the renal glomerulus. We demonstrate that exposure of glomerular endothelial cells to chronic (>24 h) LSS of 10 dyn/cm2 increases phosphorylation of extra-cellular signal-related kinase 5 (ERK5) and increases expression of KLF2, leading to increased expression of the downstream molecules endothelial nitric oxide synthase (eNOS), thrombomodulin, endothelin-1 and nitric oxide. However, the proportion of eNOS which was phosphorylated at serine 1117 and threonine 495 residues was decreased. We demonstrated dependence of these effects on the ERK5 pathway by using the inhibitor UO126. We found high levels of KLF2 expression in human glomeruli confirming the relevance of our in vitro observations and, as KLF2 is specifically induced by chronic LSS, suggesting the physiological importance of shear stress in the glomerulus. Conditioned medium from glomerular endothelial cells under chronic LSS decreased podocyte monolayer resistance and increased phosphorylation of vasodilator-stimulated phosphoprotein. The latter effect was more pronounced using a novel insert-based direct co-culture system in which endothelial cells were exposed to chronic LSS. These data provide the first direct evidence of glomerular endothelial cell to podocyte cross-talk. © 2012 Elsevier Ltd.link_to_subscribed_fulltex

Estimation of GAG in GEnC supernatant. A

<p>Graph showing analysis of highly anionic GAG present in the supernatant following treatment of GEnC with controls versus H₂O₂. The experiment utilizes Alcian Blue dye that binds to the anionic residues of proteoglycans. Cumulative data from individual experiments shows significant increase in the quantity of alcian blue staining in the supernatant after H₂O₂. This confirms cleavage of GAG residues from the surface of GEnC after exposure to ROS (n = 3 experiments (individual experiment replicates = 8–12), p<0.05). <b>B</b> Chart showing comparison of 3H³-glucosamine labeled HS GAG fractions isolated from GEnC supernatant under control and post-H₂O₂ conditions. Results show marked increase in the HS GAG fractions in the supernatant compared to controls after treatment with H₂O₂ suggesting cleavage of HS GAG (n = 4; p = 0.0019; t test). <b>C</b> Chart showing comparison of 3H³-glucosamine labeled Hyaluronan GAG (non-sulphated) fractions isolated from GEnC supernatant under control and post-H₂O₂ conditions. Results show a trend but not a significant increase in the non sulphated/Hyaluronan GAG fractions in the supernatant compared to controls. (n = 4; p = 0.053; t test).</p

WGA lectin after ROS. A

<p>Fluorescence microscopy after labeling GEnC with WGA-FITC lectin and nuclear staining with DAPI. Left column: represents ‘control’ images (no treatment). Right column: represents images after treatment with100 µM of H₂O₂. The rows define the time periods: 1 h, 2 h and 5 h. These images show reduction in the binding of WGA-FITC lectin after treatment with H₂O₂ over time. <b>B</b> Bar chart showing quantitative comparisons of fluorescence intensity of WGA-FITC lectin between GEnC treated with vehicle only and H₂O₂ (100 µM) for 1 h, 2 h and 5 h. Fluorescence is quantified by using NIH Image J software. The chart shows significant reduction in the expression of WGA-FITC after treatment with H₂O₂ (n = 10, p = 0.01, ANOVA).</p

Transendothelial electrical resistance after ROS.

<p>Graph showing real time trans-endothelial electrical resistance (TEER) recordings of GEnC monolayers during exposure to H₂O₂. H₂O₂ is added at time point 0 and measurements are taken every 2 min. TEER (Y-axis) shown as a ratio of baseline recording versus time (X-axis). Results show a reduction in TEER after addition of H₂O₂ within the first 2 min. This effect is significant and peaks at 24 min followed by recovery by 60 min. There is complete resolution of changes in TEER by 90 min.</p