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Abdollahzadeh F, Moghadasali R (2021) A comprehensive survey on optimal components of extracellular matrix for kidney development and function. Arch Clin Nephrol 7(1): 018-032. DOI: 10.17352/acn.000052The extracellular matrix (ECM) is an essential component of the network nearby the cells to guarantee their physiological function and homeostasis. Choosing the most appropriate ECM for mammalian cells, including human cells, is one of the most important criteria for qualifying the in vitro experiments of tissue engineering and functional analyses. While most previous studies examined several ECM components to optimization of ECMs proper for many mammalian cells, a comprehensive study with an integrated conclusion in this scope is still lacking.c The current review will discuss the factors which should be taken into account for designing or optimizing an optimal ECM for the development and maintenance of the kidney tissues. Besides, the components of the previously reported ECMs were compared to each other.
ADPKD: Autosomal Dominant Polycystic Kidney Disease; CKD: Chronic Kidney Disease, DN: Diabetic Nephropathy; ECM: Extracellular Matrix; FN: Fibronectin; GAG: Glycosaminoglycan; Gps: Glycoproteins; HSPG: Heparan Sulfate Proteoglycan; LIF: Leukemia Inhibitory Factor; LRTC: Label-Retaining Tubular Cells; PG: Proteoglycan; SP: Side Population; Sgag: Sulfated Glycosaminoglycan; TGF: Transforming Growth Factor; TN: Tenascin
ECM as a cell-free compound in the organs is physical scaffolding of the cellular components. ECM has physicochemical signals that are needed for tissue morphogenesis and homeostasis. Moreover, synthetic ECM could be established for in vitro and ex vivo experiments such as differentiation [1-3], development [4], hemostasis [2,4,5], regeneration [6] and supporting the phenotype of tissues or organs [1,6]. Furthermore, ECM affects cell behavior through various molecular interactions [7]. ECM may contain fibrous and non-fibrous constituents and involved in structural and biochemical protection of its surrounding cells, balancing growth factor, binding to integrins, and cell surface receptors for molecular signal transduction [8].
In normal conditions, ECM is the main part of cell organization scaffold, a base for epithelial cells [9], basement membrane, surrounding capillary and neural cells [2]. Aberrations in ECM structure or functions have been implicated in some pathological states such as fibrosis, cancer [10], and diabetic nephropathies. Furthermore, ECM gives useful information about diagnosis and prognosis of some diseases such as congenital uretero-vesical and uretero-pelvic junction obstruction. Further physiological functions were also attributed to ECM, the inhibitory effect of collagenase on branching morphogenesis has been identified [11]. β-D-xyloside could inhibit chondroitin sulfate to induce branch distribution of ureteric bud (UB) in organotypic cultures [12]. Decomposition of GAGs by heparitinase and chondroitinase or their inhibition by sodium chloride inhibits the uretral growth and branching without any interference in nephron formation [13,14]. In ECMs without GAGs, HGF and BMP4 could partly rescue the growth and elongation of the surrounding cells to form UB [13,15].
Organotypic culture of embryonic kidney showed that heparin or heparin sulfate reduces nephron number, while chondroitin sulfate has a reverse effect. There is an essential gene linking the heparan sulfate to growth factors for survival [16]. ECM types directly affect the label-retaining tubular cells (LRTC) phenotype in the culture and response of collagen gel to FGF and HGF. This effect leads to converting these cells to tubular-like cells or tubular cysts. Thus ECM can be used for tubulogenesis for in vitro culture optimization [17]. In vitro culturing of Madin-Darby Canine Kidney) MDCK( cells on collagen but not on matrigel induces tubular structure while HGF could not induce this process. Several ECM proteins have inhibitory roles in tubular formation and some proteins such as entactin, fibronectin (FN), and laminin facilitate the formation and elongation of tubules. Vitronectin, collagen IV and heparin sulfate inhibit HGF-mediated tubulogenesis [18]. Natural ECM and kidney scaffolds such as renal rhesus monkey also have been used for pluripotent stem cells (PSCs) and human embryonic stem cell (hESC) culture. These scaffolds have organizational, spatial, cell migration, and differentiation effects in engineered [19]. Renal lineage markers have been expressed by growth factors induction, in decellularized scaffold without cytokine or growth factor stimulation [19]. ECM that is secreted by cortex, medulla, and papilla kidney cells can create intact sheets of decellularized, Hydrogels and solubilized ECM.They are the main three forms of the ECM regulated by Kidney Stem Cell (KSC) function [20], and Side Population (SP). Leukemia Inhibitory Factor (LIF), induces differentiation of SP cells into several cell populations in collagen gel but not in a synthesized polymer that has no cell adhesion factor [21].
A vast variety of researches have studied the effects of ECM on kidney development and function. However, many of these studies give information about kidney morphogenesis, branching, diseases, and repair, and comprehensive research on the importance and efficacy of the ECM materials on cultured cells or organoids is still lacking.
ECM is deformed in various diseases especially during fibrosis and chronic kidney disease (CKD). Renal ECM has known as a non-invasive biomarker for fibrosis diagnosis (despite the renal biopsy as the gold standard diagnosis), also can be used as a scaffold for degenerations in regenerative medicine. Information of the construction and functional properties of these ECMs can help scientists to optimize ECMs for developing new media for cell or tissue culturing. This information also provide an insight for develop approaches of early diagnosis and prognosis. In this review we present a comprehensive study on ECM roles in tissue remodeling, branching morphogenesis as well as its involvement in some kidney diseases.
During embryogenesis, the behavior of various cell populations is modulated by the effects of the micro-environmental signals [22]. Kidney-specific ECM is composed of a complex network of collagen fibers, proteoglycans, glycoproteins, and growth factors which together form the basement membrane and interstitial space of renal cells throughout glomeruli, tubulo-interstitium and vessels. These dynamic structures provide mechanical support to the renal cells, as well as regulate the differentiation of neighboring cells. Therefore, they have a key role in kidney development and physiological function [23]. Mechanosensitive cell-surface receptors include integrins, sense the biophysical properties of their surrounding ECM [24]. With this information, such signals from the ECM can play a role in determining the of the cells shape. The ECM stiffness regulates differentiation into each three embryonic germ layer [22,25].
Studies indicated that the utility of kidney-derived ECM increased the viability, proliferation, vascularization and maturation of in vitro kidney constructs [26]. Kidney-specific ECM has a wide variety of soluble cytokines and growth factors and chemokines that scattered throughout the ECM and coordinate with fibrous proteins to organize cellular behavior [27] Figure 1.
During embryonic development renal branching occurs as a result of UB progression into mesenchyme that is surrounded by ECM. This ECM Combination around the branch units varies from time to time. Branching also is a sample of multiple roles of ECM in morphogenesis. Types of ECM including Proteoglycans, GAGs, collagens, and many other GPs are involved and regulate kidney development. ECM can affect on cellular binding and stop branching or cell surface receptors as a regulator for signaling and branching. ECM also with activation or blocking of growth factors and cytokines can act as vital mechanical traces for branching.
Studies indicated that Wnt family, FGF2, and TGFβ have a critical role in renal ECM remodeling and branching morphogenesis. GAG chains in vitro with effect on Wnt family, FGF2, TGFβ rescue mesenchyme from apoptosis and leads to EMT of these cells. Wnt4 prevents UB branching and tubulogenesis with deprivation of GAG chains [28-30]. The breakdown of ECM can also lead to activation of important signaling molecules such as Areg, Wnts, TGFβ and FGF, which affect branching [31]. TGFβ can bind to various integrins and other ECM molecules like fibrillin, vitronectin and FN, some when this action occurs via heparin sulfate [32] TGFβ signaling may be regulate the affinity of integrins for relevant ECM binding. Proteolytic cleavage of ECM by MMPs, plasmin, urokinase, elastase, thrombin and cathepsin can stop of TGFβ effects [33] TGFβ and FGF lead to EMT cells and tubule formation interactions of the actin cytoskeleton with the cytoplasmic integrin needs to TGF-β activation [34]. TGFβ signaling can be adapted by mechanical affected via the composition and rigidity of the ECM, and the mobility of cells and tissues in contact with ECM. TGF-β could function as an inducer, beginning a variety of de-differentiating events that might be further controlled by other agents in the cellular environment [35]. Studies indicated that Rho kinase is required for assembly of microfilaments and normal branching [36,37]. Basement membrane ECM prevents apoptosis of cultured mesangial cells induced by serum ablation from media, supporting the efficacy of this ECM as well as with the ECM of the type I collagens. Exclusion of matrix-derived signals such as knock out of ẞ1 integrin increases mesangial cells apoptosis [9]. Diabetic nephropathy is diagnosed by the abnormal ECM accumulation and the phenotypic change of mesangial cell [9] Tables 1,2.
GPs, as a core construct of basement membrane, are essential for epithelial-mesenchymal interactions and regulation of tissue growth. These interactions lead to nephron differentiation during kidney development [38]. There are collagens and non-collagenous GPs such as laminins, Tenascins (TNs) and FNs [39]. TN, nidogen, FN are mesenchymal ECM proteins, and type IV collagen, laminin and proteoglycan (PG) are integral basement membrane proteins which regulate metanephric development. Interactions between laminin, collagen IV, nidogen, perlecan and other basement are crucial for cell behavior regulation via basement membrane [40]. Cell adhesion molecules (CAM) and integrin receptors play an important role in these processes.
Nidogen is a critical component of renal basement membrane. Nidogen binding to laminin prevent its degradation [41-43]. It’s binding to collagen type IV due to basement membrane assembly is the most essential factor for tissues development and functionality during embryogenesis [23,38]. Knock out of nidogen-1 no change was reported in phenotype, but deletion of 56 amino acid from the nidogen-binding domain of this protein was lethal for mice at birth, due to renal agenesis that lead to locally restricted ruptures in the basement membrane of the elongating Wolffian duct [23]. Nidogens are also important for maintaining the mesangial spaces and basement membrane. During sclerosis of mesangial space or thickening of basement membrane, glomerular nidogen is increased [38]. Although nidogens have a role in the basement membrane formation, but they are not strictly required for GBM formation [44].
Glomerular ECM, containing both mesangium and glomerular basement membrane (GBM). ECM is essential for survival and apoptosis of mesangial cells whose aberration causes expansion and elimination of the mesangial cells, occurred in glomerulus sclerosis. In physiological conditions its major components are FN, collagen type IV (α1 and α2 chains, but not α3 and α5), collagen type V, laminin α, α1, α2, chondroitin sulphate and heparan sulphate proteoglycans (perlecan, collagen type XVIII, but not agrin and nidogen) [37]. The small proteoglycans decorin, biglycan, fibromodulin and lumican rather localized in the tubular interstitium [23,45]. GBM includes collagen type IV (α1.α1.α2 and α3.α4.α5-heterotrimers), collagen type V, FN, Heparan Sulfate Proteoglycan (HSPG) : agrin, perlecan , collagen I, laminin α5α2α1 and nidogen [38]. GBM is thicker compared to most other basement membranes and acts as a selective filtration barrier between the vascular system and the urinary space. The α3, α4 and α5 collagen (IV) chains are often expressed in the adult GBM [46]. The distribution of up-regulated collagen type IV was equivalent in the GBM, mesangium and interstitium. Collagen type IV has also been considered as a symptom of glomerular sclerosis and interstitial fibrosis [47]. ECM can affect the viability and apoptosis of several cell lineages contain glomerular mesangial cells. Several glomerular diseases are influenced by abnormal expansion of the mesangial ECM, which might be due to glomerular wound effects. In a rat glomerular nephrectomy model, it was shown that apoptosis and ECM accumulation are affected by deleting the glomerular cells due to glomerular sclerosis progression. The basement membrane of the Bowman’s capsule includes collagen IV (α1.α1.α2 and α5.α5.α6 hetero-trimers), laminins, nidogen, and HSPGs [38].
The ECM of the tubulo-interstitial part includes basal membranes of peritubular capillaries, tubules, and interstitial space [38]. Interstitial ECM includes different collagens (types I, III, IV, V, VI, VII, VIII and XV), glycose aminoglycans (such as hyaluronan), polysaccharides, and GPs (such as FN, versican, biglycan, and decorin). A major non-fibrous group of ECM in the interstitial space of any organ or tissue is proteoglycan with some properties such as binding, hydration, and force resistance [48]. Hyaluronic, playing role in force resistance through water absorbing and supplying flexibility around cells [48].
Studies showed that ECM proteins have important role in renal pathophysiology. In focal segmental glomerulosclerosis (FSGS), collagen 3, 4, and parietal epithelial cells specific matrix (LKIV69) increased. In mesangial sclerosis (Pierson-Syndrome) increase of collagen IV (α1.α1.α2), V, FN, nidogen, laminin, Collagen I, III, decorin, biglycan was observed. In thickening of GBMs (Alport syndrome) increase of collagen IV α3.α4.α5, Collagen I, III, collagen VI, VII, XVII, XV, perlican, nidogen, laminin was observed with no change in agrin. In thickening of tubular basement membrane as a result of fibrosis, increase of collagen IV α1.α1.α2, α3.α4.α5 α5.α5.α6, perlican, with no chang in agrin was observed. In interstitial fibrosis, increase of Collagen I, II, III, IV, V, VI, VII, XV, FN, biglycan, versican, decorin was recognized [23]. During fibrosis, the formation of scar tissue in the interstitial space is the result of extremely accumulation of ECM components [49].
In Diabetic Nephropathy (DN), mechanical stress on glomerular cells by glomerular hypertension and hyperfiltration can increase transcription of Transforming Growth Factor (TGF)-β1 and decrease MMP activity. Furthermore, changes in GBM via increase of laminin, FN, collagen IV concentration has been seen. In diabetic nephropathy also collagen IV glycation and crosslinking increase and agrin, perlecan, and collagen XVIII concentration decrease [50]. In renal fibrosis disease, an increase in decorin, collagen type 1, and biglycan levels on mesengial matrix has been reported [51].
Biglycan could act as an initiator and regulator of inflammation in the kidney [52]. Biglycan and decorin expression was up-regulated in the glomeruli of IgA Nephropathy (IgAN) [53]. Elevated levels of hyaluronan in several kidney diseases such as rat model of ischemia-reperfusion injury also diabetic nephropathies in human, renal transplant rejection and kidney stone formation was observed [54]. In patients with different proteinuric nephropathies, versican expression was increased in areas with marked tubulointerstitial fibrosis, versican may have an important role during CKD progression [55].
Collagen is the main structural protein in the ECM and also abundant protein in mammals. Collagens are classified into both fibrillar and non fibrillar forms and can be assembled into netted networks and sheets [49]. There are several types of collagen in the human body and over 90% of the content of collagen is type I collagen. Other types of collagen also play critical roles in development, maintaining of their surrounding cells and cellular migration, chemotaxis, development and cell adhesion [1,3]. Furthermore, tensile strength could be facilitated by collagen fibers [56]. Collagens contain the main structural element of the interstitial ECM. Most interstitial renal matrix changes are related to collagen type I, III, but collagen IV is the most ECM proteins in GBM [57].
In physiological conditions collagen type III is normally expressed at low levels in the interstitium but not in the glomeruli. During fibrosis the expression levels of Collagen type I and III are increased in the interstitium and glomeruli [58,59]. In pathological conditions such as fibrotic glomeruli, Collagen type I localized in tubulointerstitial space and arterial walls, with decorin and biglycan [23]. In a model of DN, a reduction in the extent of glomerulosclerosis was observed with inhibition of collagen I expression which is mostly expressed in typical glomerulosclerosis scar [38]. During kidney development, collagen type IV is a major component of the GBM because of it is produced by both podocytes and endothelial cells during glomerulogenesis. Collagen type IV often produced by glomerular podocytes in the fully mature GBM [45]. Collagen IV is capable of binding to other collagen type IV molecules in the basement membrane. The α3, α4 and α5 chains of Collagen IV are the most expressed in the adult GBM [46,60] Mutations in the gene of α5 collagen IV chain were reported to be associated with the X-linked of Alport syndrome in human that is characterized by advance glomerular injury. Aberrations of α3 collagen (IV) gene in the human cause the Alport syndrome that has been shown to be associated with the absence of α4 and α5 chains in mice. While mutations in the α5 chain of collagen is common X-linked form of the disease [49]. Mutations in the genes encoding α3 and α4 chains can lead to the autosomal resilient and autosomal dominant forms of Alport syndrome and tenuous basement membrane nephropathy. Two autoimmune diseases which cause kidney glomerulonephritis were shown to affect collagen type IV [44]. Increased collagen type IV expression was observed in chronic transplant nephropathy [61] and can be bioandicator for glomerular sclerosis and interstitial fibrosis diagnosis [47].
The transmembrane collagen type XVII has been lately detected in the GBM, and its deficiency leads to elimination of podocyte foot processes [44,62]. Collagen XVIII is similar to perlecan and agrin, as a collagen/proteoglycan with heparan sulfate side chains [62,63]. Collagen XVIII has generally the features of collagen fibrils, in some properties such as its distribution, non-denaturation, non-covalently-linked oligomers, and sensitivity for collagenase digestion. It also has attributes of an HSPG, such as long heparitinase-sensitiv carbohydrate chains and a great negative net charge. Accumulation of Collagen XVIII was observed in basal laminae of several organs including kidney. Expression of collagen XVIII HSPG in the kidney of chick embryo has been determined [63]. XVIII HSPG may also has a dual function in the Wnt signaling pathway, by antagonizing binding properties of the Wnt ligands to the receptors of the pathway. Wnt-11 is detected at the ureter tip when the collagen XVIII is not present, because the expression of Wnt-11 in the UB is controlled by this type of collagen through its heparan sulfate GAG chains [56,64].
FN, vitronectin, TN and other ECM proteins, but not all of them bindins to many integrins by adhesive Arg-Gly-Asp (RGD) sequence. It seems the ECM component has a password that is translated by the contact cells and due to different cell behaviors by affecting on adhesion, polarity, migration and other intracellular signals that regulate cell survival, proliferation and differentiation [60]. Integrin α8α1 is expressed at the first step of metanephric development by the mesenchymal cells surrounding the UB. Integrin α8-deficient mice induces at the birth, renal agenesis or dysgenesis, also deprivation of UB branching or ureter formation. In the mice lacking integrin α3 subunit, glomeral basal membrane is abnormal that is lethal at the time of birth due to abnormal development of kidney.
Laminin is a protein similar to FN in specification with three subunits: α, β, and γ that considered as fiber forms GPs. Laminins ECM are functional by oligosaccharides that can bind to other laminin molecules or proteins such as collagens, which facilitate the ECM structures formation. To date, 14 different laminin isoforms of five different α chains have been identified [65]. A null mutation in the gene encoding of laminin subunit α4 can cause progressive glomerular and tubulointerstitial fibrosis [66]. Defects in the laminin ẞ and α pause the basal glomeral connection and defects in the ẞ2 chain also cause proteinuria because of ablation of glomeral filtration and abnormal foot process [67]. Laminin β2 subunit gene mutations can cause Pierson syndrome, that characterized by premature death due to renal failure. This defect is similar to minor changes in nephrotic syndrome with unknown etiology [68]. Laminin as a network is essential for protection of the basement membrane integrity and present in the mature GBM [69]. After collagen, the highest amount of kidney ECM composition is related to laminins constitutions [70]. Laminins can also bind to their surrounding cells via the integrin receptors which are localized on the cell surfaces [71]. Mice with a hypomorphic mutation in the gene for the laminin α5 subunit has been shown polycystic kidney disease [56]. In autosomal dominant polycystic kidney disease (ADPKD), renal cystic cells have an increase in proliferation rate and surrounded by an abnormal ECM. Express of Laminin 5 (Ln-5, a α3ß3γ2 trimer), has been disrupted in the peri-cystic ECM of ADPKD kidneys. This complication could be rescued by overexpression of laminin β1 gene, suggesting a tight relationship between the laminins β1 and β2 [72]. In addition, deletion of laminin α5 from the genome of mice prevents the transition from LM-111 to the mature LM-521 that is occurred due to breakdown of the GBM and failure of glomerular vascularization [73].
TN is a mesenchymal GP of ECM that plays key role in epithelial-mesenchymal interactions during fetal development [74]. TN is a large oligomeric GP that has strong expression in embryonic kidney and important role in nephrogenesis [75]. Their functions typically oppose those of FN and are largely associated with anti-adhesive effects but extend beyond cellular architecture [76]. TN is probably a component of the normal mesangial matrix and is a ubiquitous component of the expanded glomerular ECM in pathologic conditions according to morphologic subtypes. During development of the mouse kidney, mesenchymal cells convert into epithelial cells. TNs could not be detected in the mesenchyme until kidney tubule epithelia begin to form. However, their expression were observed around the condensates and s-shaped bodies in the early stages of tubulogenesis. In an in vitro culture system, the formation of new epithelium induces the expression of TNs in the nearby mesenchyme. During postnatal development, the expression of TNs decreases. In adult mouse kidney, the expression of TN genes in medullary stroma but not in cortex section were observed. Induction of TN-C increase rates of re-epithelialization and wound closure [77]. The expression of TNs are downregulated when tubulogenesis is inhibited. TN molecules interacts with FNs, proteoglycans, S-shaped bodies and tubules [37]. No renal impairments have been determined in TN deficient knock-out mice [78]. Mesangial cells are a source of TGF [79]. Increase of TGF expression occurs in some types of glomerular diseases [80]. TGF induces TN synthesis [81]. Furthermore, coagulation play a role in TN synthesis. Studies indicated that thrombin has mitogenic effect on endothelial and mesangial cells. In this presses, the synthesis of TN enhances in both smooth muscle cells and mesangial cells. TN synthesis in glomeruli may be modulated by several factors including growth factors, hemodynamics and the coagulation process [82].
FNs is an essential protein for cell-cell and cell-matrix interactions and mesenchymal cell condensation [83]. FNs is multifunctional ECM GPs that interacts with other ECM components such as collagen, fibrin, heparin, and hyaluronic acid. FNs also interacts with integrin receptors on the cell surface [84,85]. FNs participates in cell behavior, development, wound healing, and tumorigenesis [86,87]. Targeted null mutation in the genes for fibronectin and either two major integrin receptors of fibronectin, displays distinct phenotypes and they are lethal early in development [88]. ECM components such as fibronectin and laminin have been shown to stimulate tubulogenesis. Expression of HGF and c-met stimulates fibronectin gene expression and branching morphogenesis in renal epithelial cells [89]. In embryonic kidneys at E12, E14, and E16 days of gestation, fibronectin in the un-differentiated mesenchyme especially in the tips, stalks of UB branches, interstitium, around the glomerulo tubular structures and blood vessels is expressed. Maximum expression of it is at E12 and decreases with advanced gestation [90]. Immunostaining analysis of embryonic kidney showed that FN proteins expressed in whole basal membranes and mesangial matrix [91]. In a three-dimensional culture of UB cells containing collagen type I gel, fibronectin induces cord and tubule formation in a dose-dependent pattern. FN induces UB cells branching and tubulogenesis through interaction with multiple integrin receptors [90]. Expression of Fn-EIIIA and a -SMA showed very similar patterns in the renal parenchyma in the late embryo and early developing kidney. There is a nearly parallel localization of these two proteins in the glomerular mesangium and peritubular interstitium [92].
Hensins are the higher order multimer in ECM which regulate terminal differentiation of renal epithelial cells. This differentiation is performed by conversion of intercalated cells via polymerization of hensin by galectin 3 [93]. Polymerization of hensin as a final step of epithelial cell differentiation is acted by the integrins ẞ6 and ẞ4 in medulla of the kidney where they are important for kidney structure. The best characterized ligand for integrin ẞ6 and ẞ4 is laminin-332 (also known as laminin-5). Hensins are also ligands of these receptors and could interfere with laminin-332.
Nephronectins allow the kidney development via integrin α8β1-regulated stimulation of GDNF expression. In absence of integrin α8β1, invasion by the UB into the metanephric is inhibited, resulting in renal agenesis. Therefore nephronectin is essential ligand that engages integrin α8β1 during early kidney development [94]. Nephronectin is detected in the UB epithelium and forms a complex with integrin α8β1 in vivo [95]. Embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia. Gdnf expression decreases in both nephronectin and α8β1integrin-null mutants specifically in the metanephric mesenchyme at the time of UB invasion [96].
Osteopontins are a category of proteins which are related with repair processes in the kidney. These protein are upregulated in regenerating tubules in different animal models of ischemia/reperfusion and gentamicin-induced tubular necrosis. In gentamicin-induced tubular necrosis, osteopontins are expressed individually in proliferating cells of proximal and distal tubules regenerative. Osteopontin-deficient mice upon acute renal ischemia, display renal dysfunction that is much more severe than that of healthy mice [97]. Antibodies against osteopontin inhibit kidney morphogenesis in organotypic culture, but no evident kidney defects were observed in osteopontin-deficient mice. It seems that a compensatory mechanism is involved in the in vivo developmental stages [43].
Nidogens are a family of sulfated monomeric GPs located in the basement membrane [98]. Two nidogens have been identified in humans: nidogen-1 and nidogen-2. Nidogen-1 was thought to be central in the assembly processes of basement membrane [45]. Connecting the networks formed by collagen type IV, laminins and perlecan [99]. Entactin/nidogen (E/N) was present in all renal basement membranes and was distributed throughout the expanse of the GBM with localization along its epithelial sight. E/N distribution was similar to that of novel collagen IV chain α3 in the GBM. In the mesangium, E/N was distributed mainly around the mesangial region that is encompassed by the GBM, while classical collagen chain αl (IV) is present widely throughout the mesangium. During the development of nephron, E/N is present in basement membranes of the UB, primitive renal vesicle and S-shaped body. Often E/N co-localized with laminin ẞ2 chain. E/N dispensation in basement membranes is not altered in diseases that morphologic changes of basement membrane structure has been occurred (e.g. cystic kidney disease, Alport’s familial nephritis, thin membrane disease) or those that changes in filtration and permeability are occurred (e.g. idiopathic and congenital nephrotic syndrome) [100-102]. In the knockouts of nidogen 1 or nidogen 2 showed no kidney phenotype with normally developed GBM [103]. Upregulation of nidogens in the mesangial matrix and the GBM was described in patients with glomerular diseases, that is, diabetic nephropathy, IgA-nephropathy, and lupus nephritis [104].
Proteoglycans include heparan sulfate, chondroitin sulfate, keratin sulfate and hialoronic acid are non-fiber forms. Proteoglycans and heparan sulfate molecules mostly are present in basement membrane and cell surfaces [6]. Perlican or heparan sulfate exists in UB basement membrane. Pelican, agrin and collagen XVIII are the core construct of HS-bearing species in the ECM, especially basement membrane. Cell surface proteoglycans, heparan bearing and chondroitin sulfate chains are also present within and around the mesenchymal cells. Two groups of the proteoglycans (syndecans and glypicans) play roles in nephrogenesis. Proteoglycans may be required for glial-derived neurotrophic factor (GDNF) synthesis or for GDNF (an essential mesenchymal effector of ureteric budding from the Wolffian duct) attachment to the ECM [23]. Heparin, heparan sulfate, and chondroitin sulfate, all required for UB branching, maintenance of Wnt-11 on UB tips [63].
The non-integrin ECM receptors, syndecans are important in growth factor signaling. Syndecans bind to growth factor ligands via their heparan and chondroitin sulfate glycosaminoglycan side chains. Syndecans bind to several growth factors including FGF, PDGF, EGF, HGF and VEGF [105]. Surface expression of syndecans may activate multiple molecular signaling pathways and increase growth factor binding attachment to their receptors [106]. Syndecan-1 switches from bFGF binding to FGF in a temporally regulated manner [107]. syndecans as a transmembrane proteins, may have a duel function during early kidney morphogenesis. They might be involved in cell aggregation through their adhesive capabilities and also in proliferation of the induced mesenchymal cells through binding to growth factor [108].
Glypicans which are glycophosphatidylinositol anchors which bind to cell surfaces [12]. Cell surface proteoglycans (syndecan-1, glypican-3, 5, 6) that expressed during early kidney development can bind to growth factors, increasing their localization and inducing their signaling properties. For example, HGF, (FGFs) are known to bind to heparan sulfate, and (TGFβ) to betaglycan core protein [109]. Mutation in the glypican-3 gene was observed in the patients with X-linked Simpson-Golabi-Behmel syndrome and renal dysplasia [110]. In this mutation (by treatment of sodium chlorate inhibitor of GAG sulfation or heparitinase and chondroitinase) developmental process of nephron could not be altered. In some patients and in vitro studies is determined that GPC3 inhibits cell proliferation and cell survival in specific tissues during development [111]. It has been recently reported that GPC3 regulates body size. Glypican-3, controls cell proliferation in maturing. In mice with collecting duct glypican-3-deficient, increase of branching and proliferation due of decreased endostatin ability to inhibit growth factor-mediated [111].
Versican is a chondroitin sulphate proteoglycan, the largest member of the modular proteoglycans. Versican has an important role in maintaining of the ECM integrity by interacting with hyaluronan, collagen I, and FN [112]. In healthy kidney, versican expressed in the tubulointerstitial space and the blood vessels, but not in the glomeruli. Versican is an anti-adhesion molecule [113]. In kidney diseases, both in animal models and patients, versican was upregulated in interstitium during fibrosis, and it was expressed de novo in glomerular crescents. High versican-expression is associated with progressing of CKD [55].
As a small leucine rich proteoglycan, decorins can inhibit TGF-β to prevent fibrous scar formation and repair muscle healing after injury. Decorin have key role as a modulator for matrix assembly and activator for growth factor [114]. Decorin, biglycan, and fibromodulin, act as potent regulators of TGF-β [115,116]. They are weakly expressed in the mesangial matrix and rather localized in the tubular interstitium [117]. In normal renal arterial vessels, decorin and biglycan are expressed throughout whole layers [115]. In pathological conditions, decorin and biglycan were shown to be up-regulated in glomeruli [59]. They have high expression in the interstitium in various renal diseases and fibrosis in animal models and patients [118]. Decorin and biglycan also have an important role in collagen fibrillogenesis and either bound to the ECM [119]. Both ECM-bound decorin and biglycan effect on collagens and regulate ECM-assembly. They are considered ECM derived promotors of inflammatory response by effect on toll-like-receptors [120,121]. Decorin has known anti-fibrotic properties in kidney by blocking of TGF-β with interfering by its signaling [116]. Its use as an anti-fibrotic molecule has been observed in a rat model of glomerulonephritis [122]. Decorin exerts an anti-apoptotic activity on tubular epithelial and endothelial cells and can induce fibrillin-1 expression, by binding the insulin-like growth factor type I (IGF-I) receptor [123]. Biglycan can also interact with TGF-β,[116] however, no acts biglycan treatment on pulmonary fibrosis [116]. Biglycan was lately found to induce autophagy in macrophages via TLR4 and CD44, due to limit inflammation and ischemia-reperfusion induced kidney injury in mice [124].
GAGs are classified into four groups including heparan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid. GAG chains stimulate UB branching [80-83] and development, through affecting the activity of Wnt family, FGF2 and TGFβ signaling. They stimulate decreasing the apoptosis rate in mesenchymal cells while increasing the growth and branching of UB. They act by affecting the combination of FGF2 and TGFβ, FGF2, LIF, [125]. They induce nephrogenesis and tubulogenesis by influencing the Wnt-4 and Wnt-6 ligands [126], and required for UB branching specially heparan sulfate via bind to HGF and FGF. Inhibition of chondroitin sulfate proteoglycan by β-D-xyloside in organotypic culture leads to disruption of UB branching [65]. In cultured without GAG chains, HGF and BMP4 could partially rescue growth and elongation of the UB [15]. Mutation in the heparan sulfate 2-Osulfotransferase gene (one essential gene of heparan sulfate, for its interaction with different growth factors) is showed that it is needed for mesenchymal condensation around the UB and initiation of branching morphogenesis. Mice homozygous for this gene allele die in the neonatal period, because of bilateral renal agenesis [21]. Incubation of embryonic kidney in organotypic culture with heparin or heparan sulfate chains reduces the number of nephrons, where as chondroitin sulfate chains have an opposite effect [127]. Hyaluronans, as the largest group of GAGs, present at the tip of nascent branching ureter bud. It is important for growing the UB-derived cell lines in 2D culture. Hyaluronan, supports branch formation in cultures of a UB-derived cell line via its receptor CD44, which is localized at the tip of nascent branching ureter bud [128]. Perlecans are a ubiquitous heparan sulfate proteoglycan, found in basement membranes, which binds multiple regulatory factors. They are bonded with (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and Interleukin-2 (IL-2), to concentrate them and formation of morphogen gradients [129]. Agrins are the major heparan sulphate proteoglycans of the GBM in healthy kidneys, while perlecan is an abundant component of other basement membranes [119]. In healthy kidneys both agrin and perlecan exist in mesangial matrix but not expressed in interstitium even in disease condition [130]. Both agrin and perlecan contribute to the electric charge of the GBM but seems not to be important for glomerular filtration [131]. In a rat model of chronic transplant dysfunction, perlecan was significantly induced in the GBM and the mesangial matrix [130]. In IgA-nephropathy, perlecan was specifically upregulated and great expression correlated with a better outcome of patients Agrins and perlecan are used in conditionally immortalized human podocyte cell line in 2D cultures [132].
ECM with cell adhesion, motility and polarity mediated has important roles in differentiation and morphogenesis. ECM component can be leader for differentiation signals according to the temporal-local regulation expression and dictating particular cells fates. Laminin has been shown to promote specific fates in different tissues. ECM components, as a mixture of substrates including of laminin, FN, and collagen induce differentiation of adult stem cells, embryonic stem cells and multipotent embryonic precursor cell populations in vitro. Several ECM proteins along with basement membranes also mediate terminal differentiation of epithelial cells in different segments of the nephron and collecting system [93].
hPSCs (including human embryonic stem cells (hESCs) and human inducible pluripotent stem cells (hiPSCs) have cultured onto plates pre-coated with GelTrex matrix which is virus free and include collagen IV, laminin, entactin, and heparin sulfate proteoglycans. This agent is appropriate for long term culture and maintenance of hPSCs [133-135].
The stem cell niche consist of a wide range of elements including biochemical and biophysical signals, cell- cell, and cell- ECM interactions [136]. Matrigel contain 60% Laminin, 30% collagen IV, 3% Heparan Sulfate, 1% entactin, 0.2% vitronctin, and growth factors is a common component that provides a scaffold and support of signaling pathways. These signaling pathways via basement membrane ligands are necessary for cell attachment and survival, in vitro modeling of cellular behaviors such as differentiation, apoptosis, capillary formation, cancer growth and invasion [35]. Some studies indicated that Matrigel bed is appropriate for generation of branched renal tubular structures [137-140]. Narayanan and colleagues differentiated hESCs onto various ECM such as laminin, FN, collagen IV, and Matrigel to find the best coating ECM for demonstration of efficient differentiation. They demonstrate that Matrigel as the best platform for the directed differentiation into functional renal proximal tubular cells [138].
Song and colleagues differentiated hiPSCs to functional renal podocyte cells. They cultured iPSCs in the presence of Activin A, BMP7, and Retinoic Acid (RA) on 0.1% gelatin-coated plates in 10 days. They showed that the differentiated cells had a morphology similar to mature kidney podocytes with cytoplasmic extensions and tight junction-like structures between adjacent cells [141].
Many studies indicated that laminins are good choice for culturing several PSCs with different origins. Laminin-511, laminin-511 E8 fragment, laminin-521 or type I collagen (as a control) coating was examined for their ability to support hiPSCs adhesion under serum-free conditions. In this study, researchers determined that hiPSCs only poorly adhered to collagen I, surfaces coated with laminin 511, laminin-511 E8 fragment, or laminin 521 were all efficient for the attachment and propagation of dissociated hiPS cells. Based on its higher cell binding efficiency and lower cost the laminin 511 E8 fragment selected for all subsequent studies [67]. The protocols for differentiation of PSCs into mature phenotypes usually used the media on 2D laminin substrates [70]. To in vitro modeling of cystogenesis, primary ADPKD cells isolated from patient’s kidneys. Cultured cells secrete Ln-5 that incorporated to the peri-cystic ECM. In monolayers, purified Ln-5 induces Erk activation and proliferation of ADPKD cells, while upon EGF stimulation, blocking of Ln-5, reduces the sustained Erk activation and inhibits proliferation. In three-dimensional (3D) gel culture, addition of purified Ln-5 stimulates cell proliferation and cyst formation, whereas blocking endogenous Ln-5 strongly inhibits cyst formation [71].
The type of ECM proteins strongly influenced the phenotype of (BrdU) label-retaining tubular cells (LRTC). In 3D collagen gel culture, LRTCs formed tubule-like or tubulo-cystic structures in response to growth factors (HGF, FGF) that are inducer for kidney cell tubulogenesis in vitro, and participate in renal regeneration in vivo. LRTCs that seeded on laminin coated plate are spherical or spindle shape (fibroblastic) with attachment and separation without clustering. While LRTCs that seeded on FN are spherical with clustering without separation and attachment. LRTCs that cultured on collagen 1 Gel have morphology similar to laminin [17]. Several study indicated that laminin is a critical ECM component for differentiation of PSCs into podocyte [142,143]. Some studies indicated that ECM proteins have critical role in generation of tubular structures from tissue derived cells. In one study, adult kidney stem/progenitor cells isolated from the S3 segment of adult rat kidney nephrons. These cells can reconstitute a 3D kidney-like structure in the presence of growth factors (b-FGF, HGF, EGF and BMP-7) onto Matrigel after 3 weeks [144]. Another study indicated that kidney side population (SP) colonies cells embedded into type I collagen gel or thermoreversible gelation polymer (TGP) could be formed island-like colonies and tube-like structures [140].
Several studies showed that ECM have key role in kidney organoid vascularization and differentiation. Guo and colleagues showed that kidney- specific ECM promoted the differentiation capability of multipotent human renal stem cells into tubular-like structures within the 3D kidney constructs [26]. Takebe and colleages explored the effect of ECM stiffness on self-organization and cell density of kidney organoids onto a 3D Matrigel culture. Data showed that soft environmental states (about 10-20 kPa) improved cell condensation and self-organization [145]. Assessment of the effect of ECM softness on maturations efficiency into kidney cells indicated that the use of soft polyacrylamide hydrogel substrate (about 1 kPa) or soft substrates with mechanical properties similar to an early embryonic niche such as chick embryo chorioallantoic membrane (CAM) promoted the expression of mesodermal and kidney-specific markers. These 3D renal-micro-tissues had more differentiated structures compared to aggregates that growed on rigid substrates [146,147].
Decellularized kidneys have shown great progress in tissue engineering. ECM composition in kidney tissue mainly consists of collagen IV (37% by mass of all proteins), laminins constituting 8.6%, HSPG 3%, vitronectin 2.4%, fibronectin 0.4% and elastin 0.3 [37]. Kidney specific ECM from (cortex, medulla, and papilla) is used for necessary differentiating kidney stem cells into region-specific cell types. Component of these kidney region was different. Sulfated glycosaminoglycan (sGAG) in papilla was significantly more than cortex and medulla. Kidney stem cells cultured on papilla-specific ECM consistently displayed lower proliferation, higher metabolic activity, and differences in cell morphology, and structure formation as compared to stem cells on cortex and medulla-specific ECM. Region-specific ECM can use as an effective substrate for the in vitro cultivation or the delivery of therapeutic kidney stem cells and their derivatives or in kidney repair and regeneration [20,148]. Matrigel, has used as a reconstituted basement membrane derived from the Engelbroth Holm-Swarm (EHS) mouse sarcoma [149] that mimics mechanical and biochemical properties of ECM. This agent used to facilitate cellular self-organization, successful implantation of artificial tissue include kidney and bladder in patients [150]. Atala and colleagues showed that artificial bladders are composed of autologous cells (isolated from the patient) seeded on collagen or composite collagen and polyglycolic acid scaffolds. The using of decellularized biopsies of different regions of porcine kidneys including cortex, medulla, and papilla as the hydrogels and sheets of ECM showed that kidney stem cells had significantly higher metabolic activity when cultured on papilla ECM [149]. In the intact ECM was observed potential to support proliferation of epithelial and endothelial cells, which consequently resulted in urine producing kidneys [150-153].
There have been major signs of progress in our understanding of cellular components and ECM components during kidney development and diseases. During developmental processes, the behavior of various cell populations is modulated by the effects of the micro-environmental cues including ECM. Kidney ECM provide mechanical support to the renal cells, as well as regulate the differentiation and cell fate determination of neighboring cells. Thus, they have a critical role in developmental and physiological function of kidney. Therefore, choosing the most appropriate ECM for renal cells is one of the most important criteria for qualifying the in vitro experiments of tissue engineering and regenerative medicine. The application of kidney-specific ECM for regenerative medicine is still in its infancy, and further studies are needed to uncover the signaling pathways involved in differentiation of renal cells.
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