Chronic Kidney Disease and the Vascular Endothelium

C H A P T E R

22 Chronic Kidney Disease and the Vascular Endothelium Michael S. Goligorsky Departments of Medicine, Pharmacology and Physiology, Renal Research Institute, New York Medical College, Valhalla, NY, United States CKD progression, (c) outline pathways that explain the role of endothelial stem and progenitor cells in regenerative processes, and (d) propose strategies to alleviate endothelial dysfunction and, by doing so, slow the progression of CKD.

Abstract Although historically the contribution of the endothelium to chronic kidney disease (CKD) had been neglected, recent investigations provide conclusive evidence of its role in maintaining tissue homeostasis, supporting tissue regeneration, and, when dysfunctional, instigating development and progression of tissue fibrosis. These findings are of critical importance for understanding the development of nephrosclerosis and the progression of CKD. Three endothelial pathways involved in the progression of CKD include stressinduced premature senescence of endothelial cells, the endothelialemesenchymal transition, and the loss of the endothelial surface layer. These abnormalities are involved in the pathogenesis of proteinuria, a pro-inflammatory microenvironment, microvascular rarefaction, a profibrotic state, and failure of regeneration. Therapeutic strategies to overcome endothelial cell dysfunction and its renal consequences are discussed.

DEVELOPMENT AND LIFE-SPAN OF THE VASCULAR ENDOTHELIUM

Weighing 1 kg and covering a surface area of 7000 m2, the human vascular endothelium constitutes a large monolayer organ penetrating almost all compartments of the body. The main functions of this organ include regulation of vasomotion through production of endothelium-derived vasoactive substances, fine tuning of vascular permeability; and exchange of solutes, gaseous molecules, and macromolecules between the blood and the interstitium, regulation of coagulation and fibrinolysis, control of the trafficking of circulating immune-competent cells, and a recently discovered angiocrine-mediated regulation of tissue regeneration. Some or all of these functions may become compromised in CKD, leading to far-reaching consequences, such as predisposition to cardiovascular morbidity and progression of CKD. This chapter will (a) illustrate the role of the vascular endothelium in the induction and maintenance of CKD, (b) discuss established and potential mechanisms whereby the endothelium can affect Chronic Renal Disease, Second Edition https://doi.org/10.1016/B978-0-12-815876-0.00022-X

During early embryonic development, mesodermal cells migrate toward the extraembryonic yolk sac and create “blood islands.” The outer luminal layer of blood islands contains endothelial precursors, and the inner mass consists of hematopoietic precursors. The aortogonado-mesonephric region (AGM), which harbors endothelial progenitor cells (EPC), becomes the first hematopoietic organ due to the ability of EPC to give rise to hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC).1,2 This ability is conserved in mammals throughout adulthood, long after the disappearance of the AGM. The process of embryonic endotheliale hematopoietic transition in zebrafish occurs through a unique Runx1-dependent mechanism of endothelial cell bending and escaping the aortic ventral wall in the direction of the subaortic space.3 This process bears some similarities to the transition of adult endothelial cells (EC) into pericytes, as described in adipose tissue.4,5 EC insert or “dive” into the basement membrane, in the process transitioning to the pericyte, which later acquires the properties of MSC and preadipocytes. It is not known whether similar processes take place in the renal microcirculation. EPC and endothelial stem cells are represented in all vascular beds and play a role in vasculogenesis and

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angiogenesis.6 Solitary cells or small clusters of EPC are present in all three layers: adventitial, medial, and intimal. These c-Kitþ/VEGFR2þ/CD45 cells are clonogenic and can differentiate toward EC, smooth muscle cells (SMC), and fibroblasts.7 A small number of c-Kitexpressing EC (linCD31þ CD105þ Sca1þ CD117/cKitþ) reside in the adult blood vessel endothelium. This subpopulation can undergo clonal expansion in vivo and in vitro, whereas other EC have a very limited proliferative capacity.8,9 These c-Kitþ adult vascular endothelial stem cells (VESCs) comprise only 0.4% of all adult vessel wall lin-CD31þ CD105þ ECs. Transplantation of isolated VESCs confirmed that a single c-kitþ VESC can generate in vivo functional blood vessels that connect to the host circulation. By performing repeated rounds of cell isolation and in vivo serial transplantation, it has been demonstrated that VESCs also display longterm self-renewal capacity. Yoder and coworkers10 determined that the expression of neuropilin-1 (NP-1) in human pluripotent stem cells confers on them commitment toward endothelial lineage, which results in formation of NP-1þCD31þ cells highly resembling those obtained from the cord blood. Future studies are required to compare lin CD31þCD105þ Sca1þCD117/c-Kitþ with NP1þCD31þ cells to elucidate whether these are overlapping populations or distinct ones. Endothelium-dependent vasorelaxation and angiogenic competence are reduced in aging compared to young animals.11 A similar defect occurs in prematurely senescent Klotho mice.12 Caloric restriction rescues angiogenic competence.13 One of the downstream targets of caloric restriction, sirtuin-1, is robustly expressed in EPC and EC. Sirtuin-1 expression declines with age and following application of cardiovascular stressors.14 The mechanism of sirtuin-1 deficiency leading to premature senescence of EPC and EC is demonstrated by the stress-induced loss of integrity of lysosomal membranes and leakage of cathepsins, which are capable of directly degrading this deacetylase, as shown in in vitro studies.14 EPC isolated from the bone marrow of mice genetically engineered to lack endothelial sirtuin-1 exhibit higher rates of premature senescence and apoptosis even at young ages.14 Endothelial cell turnover is very slow under physiological conditions. In different vascular beds it has been estimated to vary from 2 months to 3 years.15 The disposal of damaged EC occurs as a result of the patrolling function of a noninvading subset of Ly6Clow monocytes. Intravital microscopy studies demonstrated that these monocytes crawl on the luminal surface of glomerular and peritubular capillaries and scavenge microparticles.16 In response to nucleic acid “danger” signaling, Ly6Clow monocytes exhibit prolonged dwell

times in glomerular and peritubular capillaries, more complex patrolling routes, attachment to damaged EC, and recruitment of neutrophils, which induce focal necrosis and disposal of cellular debris. Cells that escape this in situ disposal mechanism detach from their basement membranes and appear in the circulation.

STRUCTURAL COMPONENTS OF RENAL MICROVASCULATURE EC are heterogeneous. They have specialized functions in every organ. In addition to nutrient delivery, gas exchange, and removal of waste products, they produce diverse paracrine-trophic, angiocrine factors necessary for differentiation and regeneration of tissues as dissimilar as pancreatic acini, neurons, hematopoietic precursors, hepatocytes, or alveolar epithelia.17 Angiocrine factors relevant to the kidney are yet to be characterized. Glomerular and peritubular capillary endothelium is fenestrated. Fenestrae, most probably, lack diaphragms. Instead, EC are coated with an endothelial surface layer (ESL), consisting of an electron-dense, fluffy glycocalyx composed of covalently membrane-bound proteoglycans and the inner, luminal cell coat layer composed of charge-interacting proteoglycans, glycosaminoglycans, glycoproteins, and plasma proteins.18 Of note, these layers, in addition to the basement membrane and slit diaphragms in podocytes, are in part responsible for glomerular permselectivity, as demonstrated by enzymatic degradation or high-salt elution of glycosaminoglycans resulting in proteinuria, with other structures of the glomerular filtration barrier remaining intact. These structures remain similarly intact in transgenic mice overexpressing angiopoietin-2 in podocytes, characterized by apoptosis of glomerular EC and development of proteinuria.19 Salmon et al.20 demonstrated that old Munich-Wistar-Fromter rats exhibit a widespread loss of ESL, not only in fenestrated glomerular EC but in continuous mesenteric microvessels as well, and develop increased microvascular permeability and proteinuria. Glomerular EC and podocytes together with the basement membrane form a functional filtration and permselectivity unit. The endothelium maintains podocytes through secretion of platelet-derived growth factor (PDGF), while podocytes maintain the endothelium through release of VEGF to the glomerular basement membrane. Furthermore, increased production of endothelin-1 by stressed EC triggers shedding of the key component of slit diaphragms, nephrin, from podocytes.21 Therefore, any damage to each of the members

IV. PATHOPHYSIOLOGY

ANGIOGENIC INCOMPETENCE IN CKD

of this functional unit results in a defective performance of the other.

PRIMARY ENDOTHELIAL DYSFUNCTION LEADING TO KIDNEY DISEASE The cooperative behavior of EC and pericytes or podocytes plays a key role in the development of kidney disease induced by activation of and damage to the vascular endothelium. This is best illustrated by cases of development of kidney disease in preeclampsia, HUS, TTP, and complications of anti-VEGF treatments. In preeclampsia, which complicates 3e5% of pregnancies, the immune- and/or cytotrophoblastmediated activation of EC results in the imbalance of prostacyclin/thromboxane production, elevated production of asymmetric dimethylarginine (ADMA), reduced generation of annexin-V,22 increased shedding of the VEGF receptor-1 (soluble VEGFR1, sFlt-1), and endoglin.23 All these factors conspire to compromise the viability of EC, induce thrombophilia, impair vasomotion, and eventually affect the renal microcirculation exemplified by the appearance of proteinuria and hypertension. A similar mechanism may contribute to endothelial dysfunction in CKD. Di Marco et al.24 demonstrated that in patients with CKD plasma levels of sFlt-1 were elevated compared to healthy controls. Levels of sFlt-1 were found exclusively to be associated with renal function and degree of endothelial dysfunction, and they may predict cardiovascular risk. In TTP, exocytosis of WeibelePalade bodies and release of multimeric von Willebrand factor (vWF) are impaired, due to the defect in the metalloprotease ADAMTS13 (either due to genetic abnormalities as in UpshaweSchulman syndrome, or due to autoimmune production of anti-ADAMTS13 antibodies), which normally cleaves ultralarge multimers of vWF. Defective cleavage results in formation of vWF multimeric “strings” on the surface of EC and in the circulation, leading to platelet aggregation and disseminated thrombophilia,25 followed by the development of proteinuria and renal insufficiency. Damage to the endothelium in HUS is mediated by members of the Shiga toxin family, which bind to their specific receptor globotriaosylceramide on the surface of EC. After receptor-mediated endocytosis, they inactivate 28S ribosomal RNA and inhibit protein synthesis.26e28 Despite these differences, there is growing realization that both syndromes have common roots, as Shiga toxin was found to induce release of vWF from EC and impair its cleavage in vitro and in vivo, producing a TTP-like syndrome in mice lacking ADAMTS13.29 In both cases endothelial dysfunction plays a paramount role. Both disorders only rarely result in CKD. Perhaps

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this can be explained by the fact that the acutely developing endothelial dysfunction is reversible on removal of the causative agent, while persistent activation of the endothelium may lead to the development of chronic disease. Therapeutic use of anti-VEGF antibodies in cancer patients revealed development of proteinuria as their on-target side effect.30 Analogous functional and morphologic perturbations are observed in genetically engineered mice lacking VEGF production by podocytes. These findings reinforce the role of VEGF in the maintenance of glomerular endothelial architecture and the existing cross talk between the endothelium and the neighboring podocytes.

ANGIOGENIC INCOMPETENCE IN CKD Studies by Bohle et al. demonstrated microvascular rarefaction at the sites of tubulointerstitial fibrosis.31,32 These observations were confirmed and mechanistically expanded in a series of studies by Johnson’s group,33 giving rise to the idea that microvascular rarefaction and tubulointerstitial fibrosis are causally linked. Such a link has been also established in glomerulosclerosis. In a rat renal ablation model, injury and activation of EC was associated with a biphasic response, characterized by an early hypertrophic phase, followed in 3 weeks by increased expression of fibronectin, laminin B1, angiotensinogen, and TGF-b1 RNA transcripts, all becoming widespread among endothelial and mesangial cells in sclerotic areas 2.5 months after ablation.34 This scenario is typical of the angiogenic wound-healing response to injury, which is triggered by proinflammatory mediators, causing local release of VEGF by platelets and ischemic parenchyma, followed by increased microvascular permeability with the leakage of matrix proteins (some of which have antiangiogenic properties) and culminating in vascular drop-out and tissue scarring. This process is partially recapitulated in rat models of focal segmental glomerulosclerosis and other human glomerulopathies. Kriz et al.35 described the development of synechia between glomerular capillaries and Bowman’s capsule. Because synechia contain perfused glomerular capillaries, this leads to filtration of plasma proteins into the paraglomerular and peritubular spaces, proliferation of interstitial fibroblasts, and eventual remodeling toward glomerulosclerosis, tubulointerstitial fibrosis, and formation of atubular glomeruli. The biphasic response of the renal microvascular endothelium to injury, namely, the initial proliferative, angiogenic phase and the later antiangiogenic microenvironment predisposing to obliteration of vascular beds explains the divergent results of several

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22. CHRONIC KIDNEY DISEASE AND THE VASCULAR ENDOTHELIUM

therapeutic strategies. For instance, in the early proliferative stages of diabetic nephropathy, antiVEGF treatments may be beneficial, whereas stimulation of angiogenesis may become preferable during fibrotic remodeling in CKD. In most cases the second phase predominates, as is the case with anti-GBM nephritis, where within 3e8 weeks after induction of injury, peritubular capillaries are found to be rarefied, EC undergo apoptotic cell death, and fibrosis ensues.36 The loss of the glomerular capillary endothelium occurs within a similar time course. These findings are consistent with the conclusion that glomerular capillary regression due to the injury and angiogenic incompetence lead to glomerular sclerosis.37 Similar observations were made in other models of renal disease, as well as in the aging kidney, where the loss of endothelial nitric oxide synthase and peritubular capillaries is associated with the development of tubulointerstitial fibrosis.38 Severe acute renal ischemia leads to the gradual loss of peritubular capillaries in the inner stripe of the outer medulla even before the development of manifest tubulointerstitial disease.39 In humans with different types of chronic tubulointerstitial disease, rarefaction of peritubular capillaries occurs in association with a variable pattern of VEGF expression. Increased VEGF expression in morphologically intact areas and decline in sclerotic glomeruli have been described.40 Persistent, cyclosporine A-resistant rejection of transplanted kidneys differs from the treatment-responsive cases in that the former exhibits the loss of peritubular capillaries and proliferation of myofibroblasts, leading to progressive interstitial fibrosis.41 The main process responsible for ablation of the microvasculature involves the following steps. Damaged EC precipitate cessation of normal blood flow and perturb the flow-dependent shear stresse induced activation of eNOS, leading to further cell damage and apoptosis. These damaged and apoptotic EC induce local platelet adhesion and attract macrophages, which in turn recruit other leukocytes, eventually disposing of cell debris.15 That leaves behind a decellularized basement membrane, referred to as a string vessel or empty basement membrane tube. The remaining scaffold of the basement membrane is rich in endothelial and pericyte growth factors, such as VEGF, basic fibroblast growth factor, and PDGF. These growth factors guide repopulation of string vessels with EC, leading to the deposition of a new layer of basement membrane material, potentially restoring blood flow to the area. Restoration of the endothelial lining of string vessels occurs by proliferation of EC and is assisted by EPC.42 Sequential rounds of recellularization of string vessels result in the appearance of thickened basement

membranes, a frequent morphologic companion of renal disease. Why does the rarified microvasculature in CKD not undergo self-repair via angiogenic or vasculogenic processes? Sprouting angiogenesis is initiated by the gradient in VEGF-A and Notch receptor, which guides the pathfinder “tip” EC to navigate within the interstitium and direct the “stalk” EC to the growing vessel branch.2 Endothelial SIRT1 exerts an inhibitory effect on signaling by the Notch intracellular domain, which is usually expressed in stalk cells of sprouting angiogenic vessels, thus resulting in enhanced angiogenesis. Reduced NAD abundance in aging cells leads to reduced SIRT1 activity. This can be reversed by supplementation with an NAD precursor, nicotineamide mononucleotide.43 Other guidance cues, such as class 3 semaphorins, netrins, and SLIT proteins assist in orchestrating this process. Forming sprouts undergo lumenization and mature by recruiting mural cells, pericytes and SMC, to stabilize and maintain their structure. Bone marrow-derived EPC were proposed to contribute to angiogenesis, but their direct contribution through engraftment of growing vessels has recently been questioned. This multistep angiogenic process can be disrupted at multiple, but as of yet not precisely identified, stages. Endothelial dysfunction is the main contributor to the insufficiency of angiogenesis to meet metabolic requirements. EC exposed to diverse cardiovascular risk factors exhibit impaired ability to form angiogenic sprouts even in the presence of VEGF-A.44,45 Angiopoietin-1 deficiency further contributes to microvascular rarefaction.46 This imbalance between the demand and supply leads to the observed microvascular rarefaction, patchy tissue hypoxia, and fibrosis (Figure 22.1).

Endothelium-dependent pathways of CKD progression EC Senescence

Endo-MT

Loss of ESL

SASP Impaired angiogenesis

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Pro-inflammatory state

Myofibroblasts

Increased vascular permeability

Hemodynamic compromise Microvascular rarefaction

Matrix deposition and accumulation

Tissue hypoxia Reduced MMP-14

FIGURE 22.1 Endothelium-dependent pathways of chronic kidney disease progression. Endo-MT, endothelialemesenchymal transition; ESL, endothelial surface layer; SASP, senescence-associated secretory products.

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ENDOTHELIAL CELL DYSFUNCTION IN CKDdTRANSCRIPTOMIC AND METABOLIC ANALYSES

ENDOTHELIAL CELL DYSFUNCTION IN CKDdTRANSCRIPTOMIC AND METABOLIC ANALYSES Multiple lines of evidence indicate that endothelial cell dysfunction (ECD) develops in CKD. Flowdependent relaxation of conduit vessels, a function of endothelium-derived relaxing factors, mainly NO production, is suppressed in patients and animals with CKD. Different modifications of testing endotheliumdependent vasorelaxation have become the standard for diagnosing ECD, together with other surrogate biomarkers such as markers of oxidative stress (8-isoPGF2a and oxidized LDL), markers of inflammation (high-sensitivity CRP, lipoprotein-associated PLA2, soluble ICAM-1, IL-6, von Willebrand factor), the inhibitor of eNOS ADMA, circulating procoagulants, and others.47 Diverse traditional and nontraditional risk factors for cardiovascular disease (CVD), such as ADMA, advanced glycation end products, and prooxidants, all accumulating in CKD, conspire to induce ECD by eNOS uncoupling (Figure 22.2). Cardiovascular microarray gene screens of EC with inhibited or uncoupled

Noxious stimuli

Vascular endothelium

RISK FACTORS • LDL/oxLDL • Hypercholesterolemia • ADMA • Hyperglycemia • Hyperhomocysteinemia • AGEs • Smoking • Genetic factors

eNOS revealed that this condition is associated with upregulation of the receptor for oxidized LDL (LOX1),48 induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase leading to endothelial lipidosis,49 upregulation and redistribution of a gap-junctional protein connexin-43, resulting in perturbed transmission of endothelium-derived hyperpolarizing factor to vascular SMC,50 increased synthesis of collagen XVIII51, and accumulation of its antiangiogenic fragment, endostatin, which actuates endothelialemesenchymal transition (Endo-MT).52 Transcriptomic analyses of iliac arteries from renal transplant recipients and renal arteries from healthy living kidney donors revealed 15 differentially expressed gene transcripts with upregulation of mRNAs associated with apoptosis, NF-kB signaling, smooth muscle contractility, HIF-3a, and vimentin, among others.53 ECD is associated with profound metabolic abnormalities. Such abnormalities can not only initiate but also tend to aggravate preexisting ECD. Screening renal microvascular isolates obtained from mice with inhibited eNOS revealed (using 2-D electrophoresis, in-gel digestion and mass-spectrometry analyses) at

Endo-MT Fibrosis

Warburg effect, reduced mitochondrial biogenesis, impaired autophagy FUNCTIONAL EFFECTS • Superoxide anions • Peroxynitrite • Proinflammatory state • Procoagulant state • Profibrotic state • Impaired lysosomal permeability

Perturbed cell cycle

Premature senescence

Vascular regression

Apoptosis Loss of stem cell competence

No attrition of telomeres

Reversible Stage FIGURE 22.2

Subverted Autophagy

Irreversible Stage?

Mechanisms of endothelial dysfunction, premature senescence and microvascular rarefaction. A long list of traditional and nontraditional risk factors illustrates the vulnerability of endothelial cells, which respond to these stressors by evoking a cascade of reactions summarized under the rubric “Functional Effects.” These result in impaired intermediary metabolism with a switch toward a Warburg-like normoxic glycolysis, reduced mitochondrial biogenesis, and subverted autophagy, a result of lysosomal dysfunction. Cell cycle arrest and development of premature senescence of endothelial cells follows. Notably, this type of senescence is not associated with attrition of telomeres and, if the effects of noxious stimuli and risk factors are eradicated, the cells have the potential to reverse to a normal state. The point-of-no-return is not well-defined, but perhaps is determined by the persistence of noxious stimuli, loss of stem cell competence, endothelialemesenchymal transition, and progressive microvascular drop-out. The details of this sequence of events are provided in the text. Endo-MT, endothelialemesenchymal transition.

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least 13 nonredundant differentially expressed proteins with high level of confidence. Five of those are specific for mitochondria, and two downregulated proteins, aconitase-2 and enoyl-coA-hydratase-1, are components of the Krebs cycle.54 This deficiency of key enzymes is associated with reduced mitochondrial mass, mitochondrial oxidative stress, and a switch to normoxic glycolysis (Warburg type of metabolic hypoxia seen in chronic uncoupling of eNOS) to support energy metabolism. Moreover, by supplying cultured cells with the metabolic intermediate downstream of the deficient aconitase-2da-ketoglutarate (which enters the Krebs cycle bypassing the enzymatic bottleneck)dit becomes possible to restore energy metabolism and prevent cell death or premature senescence. These findings raise the question whether it could be possible to restore EC metabolism in ECD by supplementing animals with glutamine. This question has been addressed in follow-up metabolomic studies of isolated renal microvasculature and plasma of mice chronically receiving an eNOS uncoupler with and without glutamine supplementation. This treatment ameliorated vasculopathy (as judged by restored endothelium-dependent vasorelaxation) and decreased proteinuria.55 In addition, metabolomic studies conducted using liquid chromatographyemass spectrometry analyses disclosed multiple metabolite abnormalities developing in ECD and restored by glutamine supplementation. Among those were elevated lysophospholipids and hippuric acid, and reduced levels of glutamine/glutamate, which were normalized after glutamine supplementation.55 Hence, metabolic abnormalities affect EC functions. Correcting these abnormalities leads to amelioration of ECD and vasculopathy, both of which contribute to the progression of CKD. Another metabolic aberration typical of CKD consists of the activation of glycocalyx-degrading enzymes such as heparanases, hyaluronidases, and ADAM17, which leads to structural and functional damage to the endothelial glycocalyx, the structure that is normally responsible for the mechanotransduction of flow parameters to eNOS, regulation of permeability, deterrence of leukocytes, protection from oxidative stress, and harboring growth/survival factors.56e58

PREMATURE ENDOTHELIAL CELL SENESCENCE IN CKD A role of telomere attrition and cell senescence in aging and diseased kidneys has been initially promoted by Halloran’s group.59 It has become clear from studies by Chen et al.60 that stress-induced premature senescence (SIPS) of EC occurs even in the presence of relatively unaffected telomeres. A diverse group of stress

signals, such as prooxidants, ADMA, and nonenzymatically glycation-modified proteins induce cell cycle arrest, SIPS, and eventual apoptosis in low-passage cultured cells and in young mice. A flow-chart depicting the role of SIPS in microvascular drop-out is shown in Figure 22.2. It emphasizes the fact that SIPS can be reversed after the withdrawal of the offending stressor. If the stressor persists, SIPS becomes irreversible, and EC may undergo apoptosis, which culminates in microvascular rarefaction. Senescent EC not only disrupt the function of the endothelial lining of the vessels but also affect the neighboring cells by their secretome, collectively designated as senescence-associated secretory products (SASP). SASP contain TGF-alpha, galectin-3, IGFBP-3, -4, and -6, and MIC-1.61 Dysfunctional senescent EC also release collagen XVIII and its C-terminal antiangiogenic fragment, endostatin.52 High-resolution mass spectrometric analysis of the secretome of EPC disclosed 133 proteins, some known as membranebound, others as secreted.62 Specifically, soluble forms of VEGF receptors, adhesion molecules, semaphorin 3F and TGF-b, CD109, members of the roundabout (robo) family, and endothelial markers were detected. Mass spectrometry screen of the secretome of colonyforming units, precursors of mature EC, identified 272 nonredundant proteins, of which 124 were also found in cultured EPC.63 Secretory products included MMP9, IL-8, MIF, various cathepsins and protease inhibitors, S100 proteins A11, A8 and A4, PAI-2 and apolipoprotein E, as well as a proangiogenic and prosurvival factor, thymidine phosporylase. These investigations explain the observed incipient shift from “cell-based therapy” to “cell-free therapy”. Several successful and on-going clinical trials, conducted mostly in patients with CVD, are underway. Regeneration of the microvasculature is further impaired by the production of antiangiogenic substances, such as endostatin, and developing incompetence of EPC. EST has been described as an interactive partner of another profibrogenic factor, transglutaminase 2 (TG2), an enzyme cross-linking extracellular matrix proteins, rendering them resistant to proteolytic degradation, which is elevated in kidney disease and aging.64 Individually EST and TG2 suppress angiogenesis. Transgenic mice overexpressing EST show renal interstitial fibrosis at a young age. Injection of TG2 in the intact kidney produces increased cross-linking within 24 hours, and increased matrix accumulation was detected after two weeks. Subcapsular injection of TG2 or EST in kidneys of young mice not only induced fibrosis but also increased the proportion of prematurely senescent cells. In addition, obliteration of the microvasculature occurs via Endo-MT.

IV. PATHOPHYSIOLOGY

LYMPHATIC ENDOTHELIUM

ENDOTHELIAL-MESENCHYMAL TRANSITION Endo-MT is a physiological developmental stage occurring during embryonic formation of heart valves and septa. In adulthood, Endo-MT is implicated in pulmonary hypertension, vein graft failure, atherosclerosis, and metastatic spread of malignant cells. Endo-MT is a major contributor to vascular drop-out and development of tubulointerstitial fibrosis, as detected in three different models of renal diseasedunilateral ureteral obstruction, streptozotocin-induced diabetic nephropathy, and a mouse model of Alport syndrome.65,66 About 30e50% of interstitial fibroblasts were found to originate from the endothelium. TGF-b is a major mediator of EndoMT, and BMP-7 is a factor counteracting Endo-MT, as demonstrated using an endothelial cell fate-tracing technique. Actions of TGF-b are mediated via activin receptor-like kinases 1 and 5 (Alk1 and Alk5). Activation of Alk1, selectively expressed on EC, results in cell migration, proliferation, and angiogenesis, while stimulation of Alk5 induces (via Smads 2/3 phosphorylation) the transcription of SM22a, fibronectin, and PAI-1, which mediate differentiation along the smooth muscle/mesenchymal phenotype, leading to formation of myofibroblasts. The balance between activation of these two Alk pathways is regulated by endoglin, another specific endothelial TGF coreceptor. Prolonged activation with TGF-b results in the escape of Alk1 signaling and predominant signaling via Alk 5, thus promoting Endo-MT.67 A recent study of Endo-MT showed that energy-supplying mitochondrial b-oxidation of long-chain fatty acids (FAO) in EC is inhibited by TGF-b signaling.68 Reduced activity of FAO results in a fall of acetyl-CoA levels and impaired acetylation of SMAD7, leading to the liberation of SMAD2. Endo-MT is also a contributing factor in the acute-tochronic kidney disease continuum and the development of chronic graft dysfunction.69,70 The role of endothelial dysfunction in kidney transplantation is further emphasized by a recent study on enhancing protection of EC using a Corline Heparin Conjugate (CHCÔ ), which improved early outcomes in preclinical investigations.71 Gene microarray analysis of cultured EC treated with an inhibitor of nitric oxide synthase revealed upregulation of collagen XVIII and its antiangiogenic fragment endostatin, a finding confirmed in vivo in mice chronically treated with an NOS inhibitor.52 Enhanced generation of endostatin in these animals leads to the development of Endo-MT and eventual rarefaction of renal microvasculature, thus further compounding vascular and parenchymal pathology. EC exposed to diverse stressors respond with lysosomal dysfunction, leakage of cathepsins, and degradation of SIRT1. SIRT1

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depletion, in turn, leads to downregulation of MMP-14 and accumulation of ECM. While the secretory products of intact EC contribute to the maintenance of the surrounding parenchyma, the dysfunctional endothelium secretes a host of profibrogenic factors that activate resident fibroblasts, as well as further worsening endothelial dysfunction, Endo-MT, and microvascular rarefaction.72

LYMPHATIC ENDOTHELIUM Lymphatic EC originate as sprouts from the embryonic veins and, perhaps, the adjacent mesenchyme, then sprout, branch and proliferate to form the lymphatic network, which drains into lymph nodes and eventually into the venous circulation at subclavian veins. The first podoplanin-positive lymphatic EC appear in the hilus of a developing kidney. From there they form tubular structures that branch and invade renal parenchyma. Terminal lympatics, blind-ended capillaries, collect and evacuate protein-enriched fluid, lymphocytes and antigen-presenting dendritic cells from the interstitium, thus participating in tissue homeostasis and immune surveillance.2 VEGF-C, and to a lesser extent VEGF-D, fibroblast growth factor, hepatocyte growth factor, PDGF, and insulin-like growth factors are necessary for sprouting and maintenance of the lymphatic architecture. Lymphatic capillaries lack pericyte coverage and have a discontinuous basement membrane and discontinuous button-like cellecell junctions, allowing the entry of fluid, lymphocytes, and dendritic cells.73 In chronic inflammation, infiltrating macrophages produce VEGF-C that induces lymphangiogenesis, which in turn provides an outlet for the resolution of inflammatory infiltrates and the reduction of edema. In the rat remnant kidney model, fibrotic interstitial areas are characterized by a massive proliferation of lymphatic vessels. If specific markers of lymphatic endothelium such as podoplanin are not used for characterization, these cells could be readily mistaken for vascular endothelium.74 In the setting of renal transplantation, CCL21 produced by host lymphatic vessels serves as a guidance cue for CCR-7eexpressing dendritic cells.75 These cells elicit antigen recognition and immune response. Therefore, strategies designed to curtail lymphangiogenesis may be beneficial for graft survival. The emerging understanding of the role of lymphatics in cardiovascular homeostasis is attributed to the fact that these vessels are present in the adventitia of arteries where they accompany vasa vasorum. During progression of atherosclerosis, plaque areas develop dysfunctional lymphatic vessels, which by interfering with the normal processes of eliminating inflammatory cells and lipids contribute to their expansion.76

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The lymphatic endothelium has been linked to the maintenance of blood pressure, with the failure of lymphangiogenesis resulting in development of hypertension in animals with high-salt consumption.77 Salt load results in the elevation of osmotic pressure of the skin interstitium, attracting macrophages that produce VEGF-C and stimulate lymphangiogenesis. This in turn restores tissue homeostasis and helps maintain the blood pressure. Disruption of this adaptive lymphangiogenic mechanism leads to the development of salt-sensitive hypertension, even though the expression of eNOS is elevated. These seminal studies establish the macrophageelymphatics axis as an extrarenal regulator of extracellular volume and blood pressure homeostasis. The relationship between the lymphatic microvasculature and CKD remains to be fully explored, and it awaits investigation into the role of this system in maintaining interstitial pressure and renal function in health and disease.

MICROVASCULAR AND TISSUE REGENERATION: ROLE OF STEM AND PROGENITOR CELLS The identification of EPC78 and their potential to regenerate blood vessels resulted in a body of experimental evidence confirming their role in diverse diseases. Specifically, adoptive transfer of EPC has been shown to improve the course of several cardiovascular and renal diseases. Recent studies, however, have questioned the direct involvement of stem cells in general and EPC in particular in regenerative processes. Using genetic fate tracing technology, it has been documented that bone marrow-derived or circulating cells do not contribute to regeneration of a distal phalanx in an adult mouse. The germ layer and lineage-restricted stem/progenitor cells are responsible for the regeneration.79 It has been concluded that endothelial stem/progenitor cells involved in adult angiogenesis must be local, nonhematopoietic, and noncirculating, tissue resident cells. Furthermore, it has been demonstrated7 that c-Kitþ adult VESCs reside in the vascular wall. The importance of the vascular endothelium for tissue regenerative processes has been amply illustrated in a study by Rafii’s group,80 which provides evidence for angiocrine signals generated during vascular regeneration through production of EGF-like laminin fragments. The latter foster growth of the pulmonary epithelia. Whether analogous processes takes place in the kidney remains unknown, although the recently discovered product of EC and platelets, SCUBE1 protein containing several EGF-like repeats, has been shown to be upregulated after the injury and to promote regeneration of tubular epithelial cells.81

THERAPEUTIC STRATEGIES TO AMELIORATE ENDOTHELIAL DYSFUNCTION Sir William Osler opined “The physics of a man’s circulation are the physics of the waterworks of the town in which he lives, but once out of gear, you cannot apply the same rules for the repair of the one as of the other.” Indeed, this calls for an in-depth understanding of the metabolic abnormalities associated with the affected endothelium, a field of knowledge that still remains in its infancy. Although the functional abnormalities have been elucidated, the molecular basis for developing endothelial dysfunction in CKD still remains obscure. The first glimpses on proteomic and metabolomic disparities between normal and dysfunctional endothelium, as detailed above, constitute the backbone for the rational design of therapeutic strategies. Some wellestablished, traditional therapeutic interventions are considered.82

Inhibitors of Angiotensin-2 Action One of the mainstay therapies directed to slow the progression of CKD, ACEIs and ARBs, exert their action through inhibition of NADPH oxidase, preservation of eNOS function and bradykinin levels, thus improving EC dysfunction.

HMG-CoA Reductase Inhibitors Statins elicit their effect on EC by reducing oxidative stress and improving eNOS function, independent of their lipid-lowering effects.82,92

PPAR-a Agonists Fibrates improve endothelial cell function via reduction of oxidative stress and NF-kB activation.93

Antioxidants Tempol and ebselen protect the endothelium by preventing eNOS uncoupling, thus restoring endothelial functions. A synthetic triterpenoid, bardoxolone methyl, an activator of the antioxidant Keap1-Nrf2 pathway, has been advocated as a potential therapeutic agent restoring endothelial dysfunction83 and improving renal function in CKD patients with type 2 diabetes.84 Yet, the study of more than 2000 patients with type 2 diabetes and stage 4 CKD showed no benefit of bardoxolone methyl therapy over placebo.85 A possible therapeutic role of an endogenous antioxidant, lipoic acid, in vascular and renal protection against elevated levels of

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angiotensin II-induced injury has been demonstrated in transgenic rats harboring human renin and angiotensinogen genes.86 Several well-rationalized experimental strategies for vascular protection have emerged. These are briefly noted below.

mTOR Inhibitor Rapamycin may improve endothelial dysfunction by preventing SIPS of EC.94

cytoprotection, leading to attenuation of oxidative stresseinduced endothelial injury.

Activation of Endothelin Type B Receptors Endothelin type B receptors, expressed predominantly by EC, mediate activation of endothelial nitric oxide synthase. Intrarenal infusion of these receptors in sarafotoxin 6c insulinopenic streptozotocin-injected rats improved renal hemodynamics.91

SUMMARY Activators of Sirtuins Resveratrol and newly developed analogues should have a place in the prevention of endothelial cell senescence and the restoration of metabolic abnormalities. Based on earlier studies of sirtuin 1 activation by resveratrol, a number of small-molecule SIRT1 activators have been synthesized and are being currently tested.87 Sirtuin-activating compounds (STACs) exert their effect by allosteric activation of this deacetylase. Three generations of STACs include, in addition to resveratrol, quercetin and butein (first generation), SRT 1720, 1460, and 2183 (second generation), and STAC-5, -9, and -10 (third generation), all extending lifespan and/or health-span in preclinical settings. These compounds are presently undergoing clinical trials. Dietary restriction, which induces SIRT1, acts via mTOR signaling and nicotine amide dinucleotide (NAD)edependent pathways accompanied by a shift toward oxidative metabolism,88 both representing novel modes of rejuvenation therapy. NADþ is a cofactor for activation of several sirtuins. NADþ bioavailability is reduced in disease states and aging.89 A precursor of NADþ, nicotinamide, is being evaluated as a therapy to correct NADþ deficiency and improve sirtuin activity.

Glutamine Supplementation Glutamine supplementation remains in experimental stages.54

Suppression of the JNK Pathway Suppression of the JNK pathway, which prevents vascular dysfunction, can be achieved through activation of adenosine monophosphate kinase by chronic pretreatment with 5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside, acadesine, N1-(b-D-Ribofuranosyl)5-aminoimidazole-4-carboxamide (ICAR) or metformin.90 Their effect is mediated via activation of PGC-1a and improved mitochondrial biogenesis and

Endothelium-dependent pathways of fibrogenesis play critical roles in the maintenance and progression of CKD. The mechanics of the process involve microvascular rarefaction, which itself is a result of the confluence of a multitude of pathogenic factors, such as Endo-MT, SIPS and associated abnormal secretory profiles, impaired angiogenesis and curtailed regeneration of obliterated microvascular beds, an aberrant secretome of dysfunctional EC, and enhanced degradation of endothelial glycocalyx. Strategies to alleviate endothelial dysfunction and improve the renal microcirculation must be tested in animal studies and clinical trials. Although the accumulated knowledge leaves no doubt regarding the participation of the endothelium in the pathogenesis of CKD, there remains a long translational journey to use these findings to improve outcomes in patients.

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QUESTIONS AND ANSWERS Question 1 A 25-year-old prima para is followed in the outpatient clinic. Until the present visit all physiological and laboratory data were within the normal range. She is in the third trimester of pregnancy. Her blood pressure is found to be elevated at 158/95 mm Hg. Dipstick of her urine shows 2þ protein. Her BUN and S[Cr] are in the upper normal range, as is her uric acid level. She denies any recent intercurrent infections. You strongly suspect development of preeclampsia. Being aware of the role played by vascular endothelium in this syndrome, you ask yourself: What are the mediators of preeclampsia? A. B. C. D.

Increased serum concentration of the soluble Flt-1 Increased serum concentration of VEGF Decreased serum concentration of ADMA All of the above

Answer: A Choices B and C are incorrect because the opposite occurs in preeclampsia: decreased levels of VEGF and increased levels of ADMA. Therefore, Choice D is automatically eliminated. The only correct choice is A, as the cleavage of Flt-1 receptor on EC and elevated levels of its soluble fragment are characteristic findings in preeclampsia, as well as other chronic kidney diseases.

Question 2 A 59-year-old truck driver is seen by you in the outpatient clinic. He has been referred by his family physician, who on a routine check-up found elevated blood pressure and proteinuria. During your examination and analysis of the laboratory data you come across S[Cr] of 5.3 mg/dL. You immediately perform a renal ultrasound and find that both kidneys are shrunken to 7.9 cm in length. Since you are well aware of the endothelial contribution to nephrosclerosis, you ask yourself: What are the known endothelium-dependent mediator(s) of fibrosis? A. Activation of matrix metalloproteinases B. Inhibition of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) C. Epithelialemesenchymal transition (EMT) D. Endo-MT Answer: D Choices A and B are incorrect because neither activation of MMPs nor inhibition of TIMP-1, which also results in the activation of MMPs, could be considered mechanisms of fibrosis. EMT has been proposed as a mechanism of fibrosis; however, this choice is incorrect

because it is not an endothelium-dependent mediator of fibrosis. Hence, the only correct choice is DdEndoMT.

Question 3 Considering lymphatic EC, which of the following statements is correct? A. Terminal lymphatic capillaries are blind-ended B. VEGF-C is the main growth factor for lymphatic endothelium C. Dysfunctional lymphatic EC in the vascular wall contribute to progression of atherosclerosis D. All of the above Answer: D Although our knowledge of lymphatic endothelial cells in general and renal lymphatics in particular is rather scanty, all three statements are correct. The answer is D.

Question 4 A 63-year-old woman with CKD and eGFR of 45 mL/ min/1.73 m2 is admitted to the hospital with chest pain and T-wave inversions on ECG. Having controlled her chest pain and while you are waiting for the results of biomarker tests, you search through your memory for established relations between CKD and cardiovascular disease (CVD). Which of the following statements is correct? A. Endothelial cell dysfunction is in part responsible not only for the progression of CKD but also for the increased cardiovascular morbidity and mortality in CKD patients compared to the general population without kidney disease B. The main therapies for endothelial cell dysfunction include statins, ACEIs, ARBs, and PPAR-alpha agonists C. Exercise and calorie restriction regimens may improve endothelial function D. All of the above Answer: D You are aware of the fact that stage 3 CKD is associated with a dramatic increase in cardiovascular death. Endothelial cell dysfunction is a nearly constant companion of CKD. This makes choice A a correct one. Indeed, the therapeutic modalities mentioned in choice B are correct and new modalities are on the way. This shows choice B is also correct. There is a plethora of data obtained in humans with endothelial cell dysfunction, as diagnosed using impaired endotheliumdependent vasorelaxation, elevation in levels of markers

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QUESTIONS AND ANSWERS

of chronic inflammation and oxidative stress, which demonstrate beneficial effects of exercise and caloric restriction on dysfunctional endothelium. Hence, your choice of C is also correct. Having correctly answered all three choices, you have logically selected as the best answer choice Ddall of the above.

Question 5 Which of these statements correctly characterize renal glomerular EC? A. They are fenestrated B. They are coated with glycocalyx, removal of which can lead to albuminuria C. EC and podocytes form a functional unit

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D. All of the above Answer: D EC of glomeruli and peritubular capillaries are fenestrated, as are EC in endocrine glands or sinusoidal cells in the liver. Therefore, Answer A is correct. Indeed, EC are coated with glycocalyx, and its removal from glomerular EC leads to albuminuria. This makes Answer B correct. Glomerular EC and podocytes, together with the glomerular basement membrane, represent a glomerular filtration unit. Damage to any portion of this unit leads to dysfunction of the other components. For this reason Answer C is also correct. Having correctly answered all three choices, you have necessarily selected as the best answer choice Ddall of the above.

IV. PATHOPHYSIOLOGY