Diabetes is a main risk factor for kidney disease, causing diabetic nephropathy in close to half of all patients with diabetes. Metabolism has recently been identified to be decisive in cell fate... Show moreDiabetes is a main risk factor for kidney disease, causing diabetic nephropathy in close to half of all patients with diabetes. Metabolism has recently been identified to be decisive in cell fate decisions and repair. Here we used mass spectrometry imaging (MSI) to identify tissue specific metabolic dysregulation, in order to better understand early diabetes-induced metabolic changes of renal cell types. In our experimental diabetes mouse model, early glomerular glycocalyx barrier loss and systemic metabolic changes were observed. In addition, MSI targeted at small molecule metabolites and glycero(phospho)lipids exposed distinct changes upon diabetes in downstream nephron segments. Interestingly, the outer stripe of the outer medullar proximal tubular segment (PT_S3) demonstrated the most distinct response compared to other segments. Furthermore, phosphatidylinositol lipid metabolism was altered specifically in PT_S3, with one of the phosphatidylinositol fatty acid tails being exchanged from longer unsaturated fatty acids to shorter, more saturated fatty acids. In acute kidney injury, the PT_S3 segment and its metabolism are already recognized as important factors in kidney repair processes. The current study exposes early diabetes-induced changes in membrane lipid composition in this PT_S3 segment as a hitherto unrecognized culprit in the early renal response to diabetes. Show less
Wang, G.Q.; Heijs, B.; Kostidis, S.; Rietjens, R.G.J.; Koning, M.; Yuan, L.S.; ... ; Rabelink, T.J. 2022
Accumulating evidence demonstrates important roles for metabolism in cell fate determination. However, it is a challenge to assess metabolism at a spatial resolution that acknowledges both... Show moreAccumulating evidence demonstrates important roles for metabolism in cell fate determination. However, it is a challenge to assess metabolism at a spatial resolution that acknowledges both heterogeneity and cellular dynamics in its tissue microenvironment. Using a multi-omics platform to study cell-type-specific dynamics in metabolism in complex tissues, we describe the metabolic trajectories during nephrogenesis in the devel-oping human kidney. Exploiting in situ analysis of isotopic labeling, a shift from glycolysis toward fatty acid b-oxidation was observed during the differentiation from the renal vesicle toward the S-shaped body and the proximal tubules. In addition, we show that hiPSC-derived kidney organoids are characterized by a metabolic immature phenotype that fails to use mitochondrial long-chain fatty acids for energy metabolism. Further-more, supplementation of butyrate enhances tubular epithelial differentiation and maturation in cultured kid-ney organoids. Our findings highlight the relevance of understanding metabolic trajectories to efficiently guide stem cell differentiation. Show less
Understanding dynamic metabolic changes in complex biological samples often overlooks heterogeneity in cell composition. Wang et al. combine mass spectrometry imaging, isotope tracing, and... Show moreUnderstanding dynamic metabolic changes in complex biological samples often overlooks heterogeneity in cell composition. Wang et al. combine mass spectrometry imaging, isotope tracing, and multiplexed immunofluorescence microscopy to study metabolic dynamics in the kidney upon ischemia-reperfusion.A common drawback of metabolic analyses of complex biological samples is the inability to consider cell-to-cell heterogeneity in the context of an organ or tissue. To overcome this limitation, we present an advanced high-spatial-resolution metabolomics approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) combined with isotope tracing. This method allows mapping of cell-type-specific dynamic changes in central carbon metabolism in the context of a complex heterogeneous tissue architecture, such as the kidney. Combined with multiplexed immunofluorescence staining, this method can detect metabolic changes and nutrient partitioning in targeted cell types, as demonstrated in a bilateral renal ischemia-reperfusion injury (bIRI) experimental model. Our approach enables us to identify region-specific metabolic perturbations associated with the lesion and throughout recovery, including unexpected metabolic anomalies in cells with an apparently normal phenotype in the recovery phase. These findings may be relevant to an understanding of the homeostatic capacity of the kidney microenvironment. In sum, this method allows us to achieve resolution at the single-cell level in situ and hence to interpret cell-type-specific metabolic dynamics in the context of structure and metabolism of neighboring cells. Show less
Tiemeier, G.L.; Koning, R. de; Wang, G.Q.; Kostidis, S.; Rietjens, R.G.J.; Sol, W.M.P.J.; ... ; Rabelink, T.J. 2020
Differentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although... Show moreDifferentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial-like cells (hiPSC-derived endothelial cells [hiPSC-ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC-ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel-Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC-ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC-ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC-ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC-ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC-EC. Mechanistically, pro-VWF dimers require the lower pH environment of the trans-Golgi network for maturation and tubulation. These data show that while hiPSC-ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function. Show less
We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such... Show moreWe recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia. Show less
Wang, G.Q.; Tiemeier, G.L.; Berg, B.M. van den; Rabelink, T.J. 2020
The endothelial glycocalyx is critically involved in vascular integrity and homeostasis, by regulating vascular permeability, regulating mechanotransduction, and reducing inflammation and... Show moreThe endothelial glycocalyx is critically involved in vascular integrity and homeostasis, by regulating vascular permeability, regulating mechanotransduction, and reducing inflammation and coagulation. The turnover of the glycocalyx is dynamic to fine-tune these processes. This is in particular true for its main structural component, hyaluronan (HA). Degradation and shedding of the glycocalyx by enzymes, such as hyaluronidase 1 and hyaluronidase 2, are responsible for regulation of the glycocalyx thickness and hence access of circulating cells and factors to the endothelial cell membrane and its receptors. This degradation process will at the same time also allow for resynthesis and adaptive chemical modification of the glycocalyx. The (re)synthesis of HA is dependent on the availability of its sugar substrates, thus linking glycocalyx biology directly to cellular glucose metabolism. It is therefore of particular interest to consider the consequences of dysregulated cellular glucose in diabetes for glycocalyx biology and its implications for endothelial function. This review summarizes the metabolic regulation of endothelial glycocalyx HA and its potential as a therapeutic target in diabetic vascular complications. Show less
Wang, G.Q.; Tiemeier, G.L.; Berg, B.M. van den; Rabelink, T.J. 2020
In normal physiology, endothelial cells (ECs) form a vital barrier between the blood and underlying tissue controlling leukocyte diapedesis and vascular inflammation. Emerging data suggest that... Show moreIn normal physiology, endothelial cells (ECs) form a vital barrier between the blood and underlying tissue controlling leukocyte diapedesis and vascular inflammation. Emerging data suggest that neuronal guidance cues, typically expressed during development, have roles outside the nervous system in vascular biology and immune responses. In particular, Class III semaphorins have been reported to affect EC migration and angiogenesis. While ECs express high levels of semaphorin 3F (SEMA3F), little is known about its function in mature ECs. Here we show that SEMA3F expression is reduced by inflammatory stimuli and increased by laminar flow. Endothelial cells exposed to laminar flow secrete SEMA3F, which subsequently binds to heparan sulfates on the surface of ECs. However, under pro-inflammatory conditions, reduced levels of SEMA3F make ECs more prone to monocyte diapedesis and display impaired barrier function as measured with an electric cell-substrate impedance sensing system and a microfluidic system. In addition, we demonstrate that SEMA3F can directly inhibit the migration of activated monocytes. Taken together, our data suggest an important homeostatic function for EC-expressed SEMA3F, serving as a mediator of endothelial quiescence. Show less
Wang, G.Q.; Kostidis, S.; Tiemeier, G.L.; Sol, W.M.P.J.; Vries, M.R. de; Giera, M.; ... ; Rabelink, T.J. 2020
Objective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti... Show moreObjective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Kruppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with C-13-labeled glucose. Conclusions: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis. Show less
Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types... Show moreHuman induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality. Show less
Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types... Show moreHuman induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculaturehomeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality. Show less
Berg, B.M. van den; Wang, G.Q.; Boels, M.G.S.; Avramut, M.C.; Jansen, E.; Sol, W.M.P.J.; ... ; Rabelink, T.J. 2019
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... Show moreBackground 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. Show less
Leuning, D.G.; Witjas, F.M.R.; Maanaoui, M.; Graaf, A.M.A. de; Lievers, E.; Geuens, T.; ... ; Rabelink, T.J. 2019
The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being... Show moreThe bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell - derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney. Show less
Berg, B. van den; Wang, G.Q.; Cantelmo, A.R.; Kostidis, S.; Avramut, C.; Jansen, E.; ... ; Rabelink, T. 2017
