- Email: [email protected]
The endothelium as part of the integrative glomerular barrier complex
commentary
see basic research on page 72
The endothelium as part of the integrative glomerular barrier complex Bo¨rje S. Haraldsson1,2 A new study by Xu et al. presents compelling evidence for an important role of the glomerular endothelium in acute kidney injury. They show that lipopolysaccharide reduces the endothelial surface layer, resulting in mild albuminuria, reduced glomerular filtration rate, and fewer endothelial fenestrae. Tumor necrosis factor-a (TNF-a) is identified as instrumental in these lipopolysaccharide effects through the TNF-a type 1 receptor. The study highlights that the glomerular endothelium has a key role in the maintenance of the glomerular filtration barrier. Kidney International (2013) 85, 8–11. doi:10.1038/ki.2013.317
Since the discovery of nephrin as the culprit for the Finnish-type congenital nephrotic syndrome and as a major component of the slit diaphragm, most genetic forms of proteinuria and focal segmental glomerulosclerosis have been found to be due to mutations in structural proteins of the podocyte slit membrane or the attached podocyte cytoskeleton.6 Other hereditary diseases have been shown to be due to mutations of GBM proteins.7 However, far less is known about mechanisms that contribute to proteinuria in acquired kidney disease, which is more common and unfortunately still lacks highly effective forms of therapy. With this as background, the glomerular endothelium and cross-talk between glomerular cells and their various communications are gaining new and growing interest. THE PAPER BY XU et al.
Most patients with kidney disease suffer from injury to their glomerular structures. Xu and co-workers1 (this issue) show that damage to the glomerular endothelium is crucial for the development of acute kidney injury after injection of lipopolysaccharide (LPS) to mice. Before discussing their data, let us examine our current knowledge of the glomerular barrier. COMPONENTS OF THE GLOMERULAR BARRIER
Figure 1 shows the four main elements of the glomerular barrier: the endothelial surface layer (ESL), the endothelium, the glomerular basement membrane (GBM), and the podocyte.2 Actually, there is a fifth component, the mesangial cell, which indirectly contributes to the filtration unit by providing the scaffold for the capillary loops, has contrac1 Institute of Medicine, University of Gothenburg, Nephrology, Sahlgrenska University Hospital, Gothenburg, Sweden and 2Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA Correspondence: Bo¨rje S. Haraldsson, Institute of Medicine, University of Gothenburg, Nephrology, Sahlgrenska University Hospital, Bruna Stra˚ket 5, SE-413 45 Gothenburg, Sweden. E-mail: [email protected]
8
tile and phagocytic properties, and communicates with the endothelial cells and with the podocytes.3 Indeed, as seen in Figure 1, all the glomerular cells are in close proximity and constantly exchange growth hormones and other signaling molecules. The glomerular filtration area is huge, so that even at the highest filtration rates, the flow velocities are low across the GBM. As a consequence, most larger molecules can easily diffuse back and forth, and against the filtration flow and across the gel-like GBM between glomerular cells.2 Theoretically, the first of these four selective filtration permeability barriers, the ESL, needs to be at least as selective as the last, the podocyte, in order to avoid concentration-polarization effects, which would result in a buildup of proteins within the layers, leading to ‘clogging’ of the filtration unit.2 The idea that the endothelium plays an important role in the glomerular barrier was proposed four decades ago.4 However, for the next two decades researchers considered the GBM to be the only important component of the glomerular membrane.5 For the past decade, the field has been, and still is, dominated by the exploration of podocytes, and for good reasons.
Recently the mild and transient form of proteinuria, which may occur during sepsis and with LPS administration, was mostly attributed to direct LPS effects on podocytes.8 Xu et al. now report that LPS not only lowered the glomerular filtration rate (GFR) and caused albuminuria but also induced important alterations in the glomerular endothelium and its surface layer (ESL).1 Thus, evaluation by electron microscopy revealed that LPS markedly reduced the density of endothelial fenestrations from 3.6 per micrometer of glomerular capillary in the wild-type control mice to 0.6 per micrometer at 24 h after LPS. As many effects of LPS are mediated by tumor necrosis factora (TNF-a), they then examined the potential signaling through TNF-a using mice with genetic deletion of the type 1 receptor. LPS injection in TNF receptor 1 knockout (Tnfr1–/–) mice did not reduce GFR or induce proteinuria, and the number of endothelial fenestrae remained similar to that in control, uninjected mice. Regarding possible functional effects of the reduced number of fenestrae after LPS, the authors speculate that it may reflect a reduced hydraulic conductance and hence GFR. Although this is a plausible explanation, one must not Kidney International (2014) 85
commentary
forget that in sepsis GFR is reduced mainly because of hemodynamic changes including a low blood pressure. In wild-type mice, LPS injection not only reduced the number of glomerular endothelial fenestrae but also at the same time increased the diameter of the fenestrae from 64 to 195 nm, while the 76-nm diameter of fenestrae in Tnfr1–/– mice injected with LPS remained close to that in uninjected controls. Despite the LPS-induced albuminuria, no changes could be detected with electron microscopy in the podocytes’ morphology, slit diaphragm, number or size of foot processes, and so on. The mRNA and protein levels of vascular endothelial growth factor (VEGF) were found to be low upon LPS injection, albeit it was measured in total kidney tissue and not specifically in glomeruli. The expression of VEGF receptor 2 was not altered. LPS injection caused a threefold increase in the expression of heparanase in wild-type mice but not in Tnfr1–/– mice. Heparanase specifically removes heparan sulfate from heparan sulfate glycoproteins, which contribute to the glomerular filtration barrier.2 In addition, LPS was found to reduce the amount of sialic acidcontaining proteoglycans in the ESL as analyzed by use of wheat germ agglutinin, a lectin that specifically binds sialic acid. All of these changes were diminished in Tnfr1–/– mice, and the effects were mimicked by infusions of TNF-a. Thus, the endothelium and its surface layer seem to play important roles in the development of the glomerular functional changes of LPS-induced acute kidney injury and involve predominantly the type 1 TNF-a receptor.1 How then do the findings of the study by Xu et al.1 fit into the context of previous work in the field? It has been known for two decades that in the endothelium the TNFR1 mediates most of the response to TNF-a. Moreover, the endothelial cells in most organs seem to be affected by TNF-a during sepsis, resulting in capillary leak.9 However, lately the mild and transient proteinuria induced by LPS has been attributed predominantly to direct and LPSmediated effects on podocytes and, as such, has been used as a ‘podocyteKidney International (2014) 85
Podocyte Urine GBM
Ang-1 Ang-2 ANGPTL4 sFLT-1
HGF VEGF TNF-α ET-1, IL-1 ANGPTL3 IGFBP-rP1 No
EGF ET-1 MMP-9
IGF Midkine
TGF-β Mesangial cell
PDGF No ESL
Blood
Endothelial cells
Figure 1 | The integrative glomerular barrier complex. The integrative glomerular barrier complex (IGBC) is composed of the glomerular endothelial surface layer (ESL), the endothelial cells, the glomerular basement membrane (GBM), and the podocyte, with its foot processes and their slit membranes. The mesangial cell is also a vital component of the IGBC. The ESL has two components: the covalently bound ‘glycocalyx’ and the more loosely attached ‘cell coat.’ The interstitial milieu surrounding the three cell types, podocytes, endothelial cells, and mesangial cells, contains a number of factors that are produced by one or more of the cell types. The factors rapidly diffuse across the interstitial space, including the GBM. Thus, podocytes produce factors that affect the properties of the glomerular endothelial cells, which in turn release other compounds that are required for optimal podocyte and mesangial cell function. All three cell types participate in this intricate and complex balance of power, with an inherent capacity of mutual annihilation. Ang, angiopoietin; ANGPTL, angiopoietin-like protein; EGF, epidermal growth factor; ET-1, endothelin-1; HGF, hepatocyte growth factor; IGFBP-rP1, insulin-like growth factor-binding protein-related protein 1; IL-1, interleukin-1; MMP-9, matrix metalloproteinase-9; NO, nitric oxide; PDGF, platelet-derived growth factor; sFLT-1, soluble fms-like tyrosine kinase-1; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.
specific’ tool to elicit albuminuria.8 On the basis of the present data of Xu et al.,1 this exclusively podocyte-based explanation for LPS-induced glomerular function has to be modified to also include changes in the glomerular endothelial cells (GECs) as contributing to the altered permselectivity that occurs in response to LPS or TNF-a in the glomerulus. THE ROLE OF THE ENDOTHELIUM AND ITS SURFACE LAYER IN THE GLOMERULAR FILTRATION BARRIER
Primary GEC studies have provided considerable knowledge about the properties of these cells.10 In terms of permeability of these fenestrated cells, the focus has been on their surface
layer.2 Thus, So¨rensson et al. studied the proteoglycan synthesis of GECs,11 and Singh et al. could reproduce the results in immortalized human GECs.12 However, these studies mostly describe the inner glycocalyx component of the ESL. To study the intact ESL, in vivo studies are required, and these have shown that enzymatic removal of the ESL induces albuminuria.13–16 Recently, the composition of the delicate and elusive ‘cell coat’ layer of the ESL was revealed by an in vivo ion exchange approach and mass spectrometry.17 It is now evident that the endothelium is also affected early in experimental kidney disease resulting from podocyte damage, such as the adriamycininduced nephrosis in mice.18,19 The 9
commentary
paper by Xu et al.1 further provides support for the importance of the endothelium and its surface layer for the glomerular barrier. GLOMERULAR CELL–CELL COMMUNICATION AND THE BALANCE OF POWER
As fascinating as the expanding story of the glomerular endothelium may be, it may just be part of an even greater mystery—that of glomerular cell–cell communication. The exciting era started with the discovery by Susan Quaggin’s group of VEGF production by the podocyte and its essential role in the adjacent endothelial cell’s function and survival.20 These studies led to the identification of this cross-talk as playing an essential role in eclampsia21 and the further evolution of a deeper understanding of the soluble VEGF receptor22 and the mechanism for proteinuria as a serious side effect of anti-VEGF therapy for carcinomas.21 Furthermore, recent work indicates that this exciting VEGF story may represent only the tip of the iceberg of glomerular cell cross-talk and communication. For example, podocytes can affect the adjacent endothelium via several secreted molecules such as angiopoietins, angiopoietin-like protein 3 (Angptl3), Angptl4,23 insulin-like growth factorbinding protein-related protein 1,24 and endothelin-1.25 Mesangial cells can also affect podocytes by a number of signaling molecules, such as midkine, transforming growth factor-b, nitric oxide, cytokines, and chemokines.3 Interestingly, the endothelial cells also produce a number of growth hormones, such as epidermal,26 insulin-like, hepatocyte, and platelet-derived growth factors,27 that may affect both mesangial cells and podocytes.28 Although the in vivo significance of the cross-talk of these various mediators remains to be fully established, it clearly points to the increasing recognition of intraglomerular cell–cell communication as maintaining the intricate glomerular balance of power required for the maintenance of the structural and functional integrity of this intricate filtration unit. 10
TOWARD A CONCEPT OF AN INTEGRATIVE GLOMERULAR BARRIER COMPLEX
Are we witnessing a paradigm shift in our understanding of the glomerular barrier? This may well be so. Consider the ingenious design of the glomerular filter, with cells on each side of the ‘membrane’ that communicate with each other to maintain an almost perfect size-selective filtration membrane during a century of human life. So in spite of the strong support for a podocentric view provided by the genetic forms of proteinuric renal diseases, support is also accumulating for contributing roles of the endothelial and mesangial cells in the maintenance of the filtration unit, especially in acquired proteinuric glomerular diseases such as diabetes.29 It may be time to modify the concept of ‘the single most important component’ of the glomerular filter and to embrace a more all-encompassing concept of an integrative glomerular barrier complex. At present, we are just beginning to explore the details of the cross-talk between different cell types in the glomerulus. Fortunately, as is so often the case in science, novel tools may allow us to study the in vivo cell–cell communications within the glomerulus by use of different fluorescent probes expressed by different glomerular cells and their visualization in real time by multiphoton laser microscopy. I believe that a better understanding of the various glomerular cell–cell communications will not only enlighten us about the intricacies of glomerular function in health and disease, but will thereby also point toward new therapeutic avenues, which are so desperately needed for our patients with glomerular diseases.
2.
3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
15.
16.
DISCLOSURE
The author declared no competing interests.
17.
ACKNOWLEDGMENTS
The Swedish Research Council (project no. 09898) and the Wenner-Gren Foundation supported this work.
18.
REFERENCES
19.
1.
Xu C, Chang A, Hack BK et al. TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis. Kidney Int 2013; 85: 72–81.
Haraldsson B, Nystro¨m J, Deen WM. Properties of the glomerular barrier and mechanisms of proteinuria. Physiol Rev 2008; 88: 451–487. Schlondorff D, Banas B. The mesangial cell revisited: no cell is an island. J Am Soc Nephrol 2009; 20: 1179–1187. Ryan GB, Karnovsky MJ. Distribution of endogenous albumin in the rat glomerulus: role of hemodynamic factors in glomerular barrier function. Kidney Int 1976; 9: 36–45. Kanwar YS, Linker A, Farquhar MG. Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion. J Cell Biol 1980; 86: 688–693. McCarthy HJ, Bierzynska A, Wherlock M et al. Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 2013; 8: 637–648. Miner JH. The glomerular basement membrane. Exp Cell Res 2012; 318: 973–978. Reiser J, von Gersdorff G, Loos M et al. Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest 2004; 113: 1390–1397. von Drygalski A, Furlan-Freguia C, Ruf W et al. Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor. Arterioscler Thromb Vasc Biol 2013; 33: 769–776. Ballermann BJ. Regulation of bovine glomerular endothelial cell growth in vitro. Am J Physiol 1989; 256: C182–C189. So¨rensson J, Bjo¨rnson A, Ohlson M et al. Synthesis of sulfated proteoglycans by bovine glomerular endothelial cells in culture. Am J Physiol Renal Physiol 2003; 284: F373–F380. Singh A, Satchell SC, Neal CR et al. Glomerular endothelial glycocalyx constitutes a barrier to protein permeability. J Am Soc Nephrol 2007; 18: 2885–2893. Jeansson M, Haraldsson B. Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular barrier. Am J Physiol Renal Physiol 2006; 290: F111–F116. Meuwese MC, Broekhuizen LN, Kuikhoven M et al. Endothelial surface layer degradation by chronic hyaluronidase infusion induces proteinuria in apolipoprotein E-deficient mice. PLoS One [online] 2010; 5: e14262. Salmon AH, Ferguson JK, Burford JL et al. Loss of the endothelial glycocalyx links albuminuria and vascular dysfunction. J Am Soc Nephrol 2012; 23: 1339–1350. Dane MJ, van den Berg BM, Avramut MC et al. Glomerular endothelial surface layer acts as a barrier against albumin filtration. Am J Pathol 2013; 182: 1532–1540. Friden V, Oveland E, Tenstad O et al. The glomerular endothelial cell coat is essential for glomerular filtration. Kidney Int 2011; 79: 1322–1330. Jeansson M, Bjo¨rck K, Tenstad O et al. Adriamycin alters glomerular endothelium to induce proteinuria. J Am Soc Nephrol 2009; 20: 114–122. Sun YB, Qu X, Zhang X et al. Glomerular endothelial cell injury and damage precedes that of podocytes in adriamycin-induced nephropathy. PLoS One [online] 2013; 8: e55027. Kidney International (2014) 85
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Eremina V, Sood M, Haigh J et al. Glomerularspecific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 2003; 111: 707–716. Eremina V, Jefferson JA, Kowalewska J et al. VEGF inhibition and renal thrombotic microangiopathy. N Engl J Med 2008; 358: 1129–1136. Jin J, Sison K, Li C et al. Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function. Cell 2012; 151: 384–399. Clement LC, Avila-Casado C, Mace C et al. Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoidsensitive nephrotic syndrome. Nat Med 2011; 17: 117–122. Matsumoto T, Hess S, Kajiyama H et al. Proteomic analysis identifies insulin-like growth factor-binding protein-related protein-1 as a podocyte product. Am J Physiol Renal Physiol 2010; 299: F776–F784. Daehn IS, Zhang T, Casalena G et al. Role of endothelin-1 in podocyte-to-endothelial
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cross-talk in podocytopathies. Kidney Week 2011 (annual meeting), 8–13 November 2011. American Society of Nephrology: Philadelphia, PA, 2011, pp FR-OR297. Miyazawa T, Zeng F, Wang S et al. Low nitric oxide bioavailability upregulates renal heparin binding EGF-like growth factor expression. Kidney Int 2013; 84: 1176–1188. Lindahl P, Hellstro¨m M, Kale´n M et al. Paracrine PDGF-B/PDGF-Rb signaling controls mesangial cell development in kidney glomeruli. Development 1998; 125: 3313–3322. Obeidat M, Obeidat M, Ballermann BJ. Glomerular endothelium: a porous sieve and formidable barrier. Exp Cell Res 2012; 318: 964–972. Weil EJ, Lemley KV, Mason CC et al. Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy. Kidney Int 2012; 82: 1010–1017.
see basic research on page 142
In and out of the bone: can the osteocyte escape skeletal jail and yet regulate mineralization? Junichiro J. Kazama1 and Masafumi Fukagawa2 An increase in the levels of fibroblast growth factor 23 (FGF23) develops in the early phase of chronic kidney disease (CKD). However, the initial trigger of CKD–mineral and bone disorder (CKD–MBD) is a matter of debate. Fang and associates provide evidence, in a mouse model of early CKD, that osteocytes and osteocyte-like cells play an important role at the earliest stage of CKD–MBD. The osteocytes may become a target of intervention in CKD–MBD. Kidney International (2013) 85, 11–12. doi:10.1038/ki.2013.296
Chronic kidney disease–mineral and bone disorder (CKD–MBD) is a systemic disturbance of mineral metabolism that comprises three elements: 1 Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan and 2Division of Nephrology, Endocrinology, and Metabolism, Tokai University School of Medicine, Isehara, Japan Correspondence: Masafumi Fukagawa, Division of Nephrology, Endocrinology, and Metabolism, Tokai University School of Medicine, 143 ShimoKasuya, Isehara, Kanagawa 259-1193, Japan. E-mail: [email protected]
Kidney International (2014) 85
laboratory abnormalities, bone metabolic abnormalities, and soft tissue mineralization including vascular calcification. Recently, this syndrome has attracted much attention, in particular because of its possible link with cardiovascular disease.1 However, the earliest disease stage at which CKD–MBD starts developing still remains obscure. Fang and colleagues2 (this issue) attempted to tackle this issue using ldlr–/– high-fat-fed mice with chronic kidney insufficiency but not associated with hyperphosphatemia. They used inulin clearance in these animals to
accurately estimate glomerular filtration rate and categorized them into humanlike CKD stages by comparing the clearance data with those of normal mice. They thus successfully reproduced a very early CKD condition equivalent to human CKD stage 2. They found increased circulating levels of fibroblast growth factor 23 (FGF23) and sclerostin in this early-CKD animal. It has been believed that both FGF23 and sclerostin are produced by osteocytes.3 FGF23 is one of the most likely molecular candidates that link abnormal mineral metabolism and cardiovascular disease in early CKD.4,5 An early enhancement of sclerostin production may account for the decrease in bone formation2 in this model, as well as in another CKD model reported previously.6 Interestingly, Fang et al.2 also found expression of FGF23 and local klotho in the aorta of sham-operated animals. In the process of vascular calcification, it has been recognized that osteoblastic lineages characterized by Runx2 expression appear in vascular tissue that play a central role in its development, resembling ectopic bone formation.7 However, their finding indicates that resident cells in the aortic wall naturally have an osteocyte-like phenotype. However, both the expression of FGF23 and that of local klotho were reduced, while the circulating klotho level was increased in the early CKD. They assumed that the increased FGF23 immunoreactivity found in the aortic media of early CKD animals might represent the complex of FGF23, FGF receptor, and circulating klotho, which plays a protective role in the vasculature. However, they failed to provide further convincing data on this issue. The key word common to lesions in both types of tissues is ‘osteocyte.’ Although osteocytes account for the overwhelming majority of bone resident cells, their physiological and/or pathophysiological roles remain far from being well known. It has been difficult to analyze their functions because of their anatomical property, namely, that they are buried in calcified tissue. Bone tissue consists of an aggregation of tube-shaped units termed 11
see basic research on page 72
The endothelium as part of the integrative glomerular barrier complex Bo¨rje S. Haraldsson1,2 A new study by Xu et al. presents compelling evidence for an important role of the glomerular endothelium in acute kidney injury. They show that lipopolysaccharide reduces the endothelial surface layer, resulting in mild albuminuria, reduced glomerular filtration rate, and fewer endothelial fenestrae. Tumor necrosis factor-a (TNF-a) is identified as instrumental in these lipopolysaccharide effects through the TNF-a type 1 receptor. The study highlights that the glomerular endothelium has a key role in the maintenance of the glomerular filtration barrier. Kidney International (2013) 85, 8–11. doi:10.1038/ki.2013.317
Since the discovery of nephrin as the culprit for the Finnish-type congenital nephrotic syndrome and as a major component of the slit diaphragm, most genetic forms of proteinuria and focal segmental glomerulosclerosis have been found to be due to mutations in structural proteins of the podocyte slit membrane or the attached podocyte cytoskeleton.6 Other hereditary diseases have been shown to be due to mutations of GBM proteins.7 However, far less is known about mechanisms that contribute to proteinuria in acquired kidney disease, which is more common and unfortunately still lacks highly effective forms of therapy. With this as background, the glomerular endothelium and cross-talk between glomerular cells and their various communications are gaining new and growing interest. THE PAPER BY XU et al.
Most patients with kidney disease suffer from injury to their glomerular structures. Xu and co-workers1 (this issue) show that damage to the glomerular endothelium is crucial for the development of acute kidney injury after injection of lipopolysaccharide (LPS) to mice. Before discussing their data, let us examine our current knowledge of the glomerular barrier. COMPONENTS OF THE GLOMERULAR BARRIER
Figure 1 shows the four main elements of the glomerular barrier: the endothelial surface layer (ESL), the endothelium, the glomerular basement membrane (GBM), and the podocyte.2 Actually, there is a fifth component, the mesangial cell, which indirectly contributes to the filtration unit by providing the scaffold for the capillary loops, has contrac1 Institute of Medicine, University of Gothenburg, Nephrology, Sahlgrenska University Hospital, Gothenburg, Sweden and 2Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA Correspondence: Bo¨rje S. Haraldsson, Institute of Medicine, University of Gothenburg, Nephrology, Sahlgrenska University Hospital, Bruna Stra˚ket 5, SE-413 45 Gothenburg, Sweden. E-mail: [email protected]
8
tile and phagocytic properties, and communicates with the endothelial cells and with the podocytes.3 Indeed, as seen in Figure 1, all the glomerular cells are in close proximity and constantly exchange growth hormones and other signaling molecules. The glomerular filtration area is huge, so that even at the highest filtration rates, the flow velocities are low across the GBM. As a consequence, most larger molecules can easily diffuse back and forth, and against the filtration flow and across the gel-like GBM between glomerular cells.2 Theoretically, the first of these four selective filtration permeability barriers, the ESL, needs to be at least as selective as the last, the podocyte, in order to avoid concentration-polarization effects, which would result in a buildup of proteins within the layers, leading to ‘clogging’ of the filtration unit.2 The idea that the endothelium plays an important role in the glomerular barrier was proposed four decades ago.4 However, for the next two decades researchers considered the GBM to be the only important component of the glomerular membrane.5 For the past decade, the field has been, and still is, dominated by the exploration of podocytes, and for good reasons.
Recently the mild and transient form of proteinuria, which may occur during sepsis and with LPS administration, was mostly attributed to direct LPS effects on podocytes.8 Xu et al. now report that LPS not only lowered the glomerular filtration rate (GFR) and caused albuminuria but also induced important alterations in the glomerular endothelium and its surface layer (ESL).1 Thus, evaluation by electron microscopy revealed that LPS markedly reduced the density of endothelial fenestrations from 3.6 per micrometer of glomerular capillary in the wild-type control mice to 0.6 per micrometer at 24 h after LPS. As many effects of LPS are mediated by tumor necrosis factora (TNF-a), they then examined the potential signaling through TNF-a using mice with genetic deletion of the type 1 receptor. LPS injection in TNF receptor 1 knockout (Tnfr1–/–) mice did not reduce GFR or induce proteinuria, and the number of endothelial fenestrae remained similar to that in control, uninjected mice. Regarding possible functional effects of the reduced number of fenestrae after LPS, the authors speculate that it may reflect a reduced hydraulic conductance and hence GFR. Although this is a plausible explanation, one must not Kidney International (2014) 85
commentary
forget that in sepsis GFR is reduced mainly because of hemodynamic changes including a low blood pressure. In wild-type mice, LPS injection not only reduced the number of glomerular endothelial fenestrae but also at the same time increased the diameter of the fenestrae from 64 to 195 nm, while the 76-nm diameter of fenestrae in Tnfr1–/– mice injected with LPS remained close to that in uninjected controls. Despite the LPS-induced albuminuria, no changes could be detected with electron microscopy in the podocytes’ morphology, slit diaphragm, number or size of foot processes, and so on. The mRNA and protein levels of vascular endothelial growth factor (VEGF) were found to be low upon LPS injection, albeit it was measured in total kidney tissue and not specifically in glomeruli. The expression of VEGF receptor 2 was not altered. LPS injection caused a threefold increase in the expression of heparanase in wild-type mice but not in Tnfr1–/– mice. Heparanase specifically removes heparan sulfate from heparan sulfate glycoproteins, which contribute to the glomerular filtration barrier.2 In addition, LPS was found to reduce the amount of sialic acidcontaining proteoglycans in the ESL as analyzed by use of wheat germ agglutinin, a lectin that specifically binds sialic acid. All of these changes were diminished in Tnfr1–/– mice, and the effects were mimicked by infusions of TNF-a. Thus, the endothelium and its surface layer seem to play important roles in the development of the glomerular functional changes of LPS-induced acute kidney injury and involve predominantly the type 1 TNF-a receptor.1 How then do the findings of the study by Xu et al.1 fit into the context of previous work in the field? It has been known for two decades that in the endothelium the TNFR1 mediates most of the response to TNF-a. Moreover, the endothelial cells in most organs seem to be affected by TNF-a during sepsis, resulting in capillary leak.9 However, lately the mild and transient proteinuria induced by LPS has been attributed predominantly to direct and LPSmediated effects on podocytes and, as such, has been used as a ‘podocyteKidney International (2014) 85
Podocyte Urine GBM
Ang-1 Ang-2 ANGPTL4 sFLT-1
HGF VEGF TNF-α ET-1, IL-1 ANGPTL3 IGFBP-rP1 No
EGF ET-1 MMP-9
IGF Midkine
TGF-β Mesangial cell
PDGF No ESL
Blood
Endothelial cells
Figure 1 | The integrative glomerular barrier complex. The integrative glomerular barrier complex (IGBC) is composed of the glomerular endothelial surface layer (ESL), the endothelial cells, the glomerular basement membrane (GBM), and the podocyte, with its foot processes and their slit membranes. The mesangial cell is also a vital component of the IGBC. The ESL has two components: the covalently bound ‘glycocalyx’ and the more loosely attached ‘cell coat.’ The interstitial milieu surrounding the three cell types, podocytes, endothelial cells, and mesangial cells, contains a number of factors that are produced by one or more of the cell types. The factors rapidly diffuse across the interstitial space, including the GBM. Thus, podocytes produce factors that affect the properties of the glomerular endothelial cells, which in turn release other compounds that are required for optimal podocyte and mesangial cell function. All three cell types participate in this intricate and complex balance of power, with an inherent capacity of mutual annihilation. Ang, angiopoietin; ANGPTL, angiopoietin-like protein; EGF, epidermal growth factor; ET-1, endothelin-1; HGF, hepatocyte growth factor; IGFBP-rP1, insulin-like growth factor-binding protein-related protein 1; IL-1, interleukin-1; MMP-9, matrix metalloproteinase-9; NO, nitric oxide; PDGF, platelet-derived growth factor; sFLT-1, soluble fms-like tyrosine kinase-1; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.
specific’ tool to elicit albuminuria.8 On the basis of the present data of Xu et al.,1 this exclusively podocyte-based explanation for LPS-induced glomerular function has to be modified to also include changes in the glomerular endothelial cells (GECs) as contributing to the altered permselectivity that occurs in response to LPS or TNF-a in the glomerulus. THE ROLE OF THE ENDOTHELIUM AND ITS SURFACE LAYER IN THE GLOMERULAR FILTRATION BARRIER
Primary GEC studies have provided considerable knowledge about the properties of these cells.10 In terms of permeability of these fenestrated cells, the focus has been on their surface
layer.2 Thus, So¨rensson et al. studied the proteoglycan synthesis of GECs,11 and Singh et al. could reproduce the results in immortalized human GECs.12 However, these studies mostly describe the inner glycocalyx component of the ESL. To study the intact ESL, in vivo studies are required, and these have shown that enzymatic removal of the ESL induces albuminuria.13–16 Recently, the composition of the delicate and elusive ‘cell coat’ layer of the ESL was revealed by an in vivo ion exchange approach and mass spectrometry.17 It is now evident that the endothelium is also affected early in experimental kidney disease resulting from podocyte damage, such as the adriamycininduced nephrosis in mice.18,19 The 9
commentary
paper by Xu et al.1 further provides support for the importance of the endothelium and its surface layer for the glomerular barrier. GLOMERULAR CELL–CELL COMMUNICATION AND THE BALANCE OF POWER
As fascinating as the expanding story of the glomerular endothelium may be, it may just be part of an even greater mystery—that of glomerular cell–cell communication. The exciting era started with the discovery by Susan Quaggin’s group of VEGF production by the podocyte and its essential role in the adjacent endothelial cell’s function and survival.20 These studies led to the identification of this cross-talk as playing an essential role in eclampsia21 and the further evolution of a deeper understanding of the soluble VEGF receptor22 and the mechanism for proteinuria as a serious side effect of anti-VEGF therapy for carcinomas.21 Furthermore, recent work indicates that this exciting VEGF story may represent only the tip of the iceberg of glomerular cell cross-talk and communication. For example, podocytes can affect the adjacent endothelium via several secreted molecules such as angiopoietins, angiopoietin-like protein 3 (Angptl3), Angptl4,23 insulin-like growth factorbinding protein-related protein 1,24 and endothelin-1.25 Mesangial cells can also affect podocytes by a number of signaling molecules, such as midkine, transforming growth factor-b, nitric oxide, cytokines, and chemokines.3 Interestingly, the endothelial cells also produce a number of growth hormones, such as epidermal,26 insulin-like, hepatocyte, and platelet-derived growth factors,27 that may affect both mesangial cells and podocytes.28 Although the in vivo significance of the cross-talk of these various mediators remains to be fully established, it clearly points to the increasing recognition of intraglomerular cell–cell communication as maintaining the intricate glomerular balance of power required for the maintenance of the structural and functional integrity of this intricate filtration unit. 10
TOWARD A CONCEPT OF AN INTEGRATIVE GLOMERULAR BARRIER COMPLEX
Are we witnessing a paradigm shift in our understanding of the glomerular barrier? This may well be so. Consider the ingenious design of the glomerular filter, with cells on each side of the ‘membrane’ that communicate with each other to maintain an almost perfect size-selective filtration membrane during a century of human life. So in spite of the strong support for a podocentric view provided by the genetic forms of proteinuric renal diseases, support is also accumulating for contributing roles of the endothelial and mesangial cells in the maintenance of the filtration unit, especially in acquired proteinuric glomerular diseases such as diabetes.29 It may be time to modify the concept of ‘the single most important component’ of the glomerular filter and to embrace a more all-encompassing concept of an integrative glomerular barrier complex. At present, we are just beginning to explore the details of the cross-talk between different cell types in the glomerulus. Fortunately, as is so often the case in science, novel tools may allow us to study the in vivo cell–cell communications within the glomerulus by use of different fluorescent probes expressed by different glomerular cells and their visualization in real time by multiphoton laser microscopy. I believe that a better understanding of the various glomerular cell–cell communications will not only enlighten us about the intricacies of glomerular function in health and disease, but will thereby also point toward new therapeutic avenues, which are so desperately needed for our patients with glomerular diseases.
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DISCLOSURE
The author declared no competing interests.
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ACKNOWLEDGMENTS
The Swedish Research Council (project no. 09898) and the Wenner-Gren Foundation supported this work.
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see basic research on page 142
In and out of the bone: can the osteocyte escape skeletal jail and yet regulate mineralization? Junichiro J. Kazama1 and Masafumi Fukagawa2 An increase in the levels of fibroblast growth factor 23 (FGF23) develops in the early phase of chronic kidney disease (CKD). However, the initial trigger of CKD–mineral and bone disorder (CKD–MBD) is a matter of debate. Fang and associates provide evidence, in a mouse model of early CKD, that osteocytes and osteocyte-like cells play an important role at the earliest stage of CKD–MBD. The osteocytes may become a target of intervention in CKD–MBD. Kidney International (2013) 85, 11–12. doi:10.1038/ki.2013.296
Chronic kidney disease–mineral and bone disorder (CKD–MBD) is a systemic disturbance of mineral metabolism that comprises three elements: 1 Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan and 2Division of Nephrology, Endocrinology, and Metabolism, Tokai University School of Medicine, Isehara, Japan Correspondence: Masafumi Fukagawa, Division of Nephrology, Endocrinology, and Metabolism, Tokai University School of Medicine, 143 ShimoKasuya, Isehara, Kanagawa 259-1193, Japan. E-mail: [email protected]
Kidney International (2014) 85
laboratory abnormalities, bone metabolic abnormalities, and soft tissue mineralization including vascular calcification. Recently, this syndrome has attracted much attention, in particular because of its possible link with cardiovascular disease.1 However, the earliest disease stage at which CKD–MBD starts developing still remains obscure. Fang and colleagues2 (this issue) attempted to tackle this issue using ldlr–/– high-fat-fed mice with chronic kidney insufficiency but not associated with hyperphosphatemia. They used inulin clearance in these animals to
accurately estimate glomerular filtration rate and categorized them into humanlike CKD stages by comparing the clearance data with those of normal mice. They thus successfully reproduced a very early CKD condition equivalent to human CKD stage 2. They found increased circulating levels of fibroblast growth factor 23 (FGF23) and sclerostin in this early-CKD animal. It has been believed that both FGF23 and sclerostin are produced by osteocytes.3 FGF23 is one of the most likely molecular candidates that link abnormal mineral metabolism and cardiovascular disease in early CKD.4,5 An early enhancement of sclerostin production may account for the decrease in bone formation2 in this model, as well as in another CKD model reported previously.6 Interestingly, Fang et al.2 also found expression of FGF23 and local klotho in the aorta of sham-operated animals. In the process of vascular calcification, it has been recognized that osteoblastic lineages characterized by Runx2 expression appear in vascular tissue that play a central role in its development, resembling ectopic bone formation.7 However, their finding indicates that resident cells in the aortic wall naturally have an osteocyte-like phenotype. However, both the expression of FGF23 and that of local klotho were reduced, while the circulating klotho level was increased in the early CKD. They assumed that the increased FGF23 immunoreactivity found in the aortic media of early CKD animals might represent the complex of FGF23, FGF receptor, and circulating klotho, which plays a protective role in the vasculature. However, they failed to provide further convincing data on this issue. The key word common to lesions in both types of tissues is ‘osteocyte.’ Although osteocytes account for the overwhelming majority of bone resident cells, their physiological and/or pathophysiological roles remain far from being well known. It has been difficult to analyze their functions because of their anatomical property, namely, that they are buried in calcified tissue. Bone tissue consists of an aggregation of tube-shaped units termed 11