Keywords

16.1 Introduction

Clinical therapy is the mainstay of treatment of severely diseased due to trauma, accidents, or congenital defects, to facilitate tissue repair or regeneration. Tissue or organ transplantations are the first choice for such kinds of diseases which came as an important breakthrough in the medical field. While these treatments have been revolutionary and lifesaving, major problems exist with these techniques like donor deficit, lifelong requirement of immunosuppressants, and other hazardous complications. One of the common instances of such treatments is patients suffering from renal disease which is tremendously affecting individuals’ physical as well as mental well-being (Xie et al. 2018). In a way, kidneys are custodian of the human body. Using their tiny nephrons, the kidneys regulate fluid balance in the body to keep our blood healthy and are indispensable for tissue homeostasis. However, the efficiency of the kidneys decreases as the age increases. Though diabetes and hypertension are the main causes that are accountable for kidney diseases, there are also several other factors that ultimately result in loss of renal structure and function (Levin et al. 2017). Compared to other organs in the body, the disease affliction in renal tissue occurs at two different levels the acute renal disease and/(or) chronic renal disease. Acute kidney injury acts as a predisposing factor for the chronic kidney disease ending in end-stage renal disease. The occurrence of reported cases of acute kidney injury (AKI) has increasing at an alarming rate in recent years (Sawhney et al. 2017). Accordingly, the frequency of chronic kidney disease is also progressing that has global implications for health and disease (Heung et al. 2016). As per the study of Global Burden of Disease (GBD) 2017, the frequency of CKD has mounted to 27% from the year 1990 to 2017 (James et al. 2018). Thus, CKD is certain to be one of the most challenging health problems of this century.

The pathogenesis of CKD is based on the deposition of extracellular matrix which leads to fibrosis and finally to end-stage renal disease. Hence, it is a major public health problem and has a considerable impact on billions of individuals worldwide. At present, there is no special treatment for patients suffering from renal interstitial fibrosis other than hemodialysis and renal transplantation. Since renal replacement therapy is a high-priced procedure and most of the sufferers are prevented from being given access to this type of therapy (Liyanage et al. 2015). Therefore, it is high time to look for adequate treatment modalities to delay the renal disease progression.

Amongst the numerous causes of kidney failure, unilateral ureter obstruction is one of the potential factors that can be blamed for this disaster. It causes subacute kidney injury peculiarly in infants and children. Ureter obstruction is a serious problem and happens when the flow of urine is prevented due to some blockage in the ureter, the consequence of which is cellular apoptosis and necrosis as an end sequalae to extracellular matrix deposition (Zhang et al. 2018). In order to understand the procedure concerned with the development of kidney fibrogenesis to kidney fibrosis wide variety of models have been explored (Fogo 2001; Rabe and Schaefer 2016). Unilateral Ureter Obstruction (UUO) is scrutinized as one of the extensively studied models for renal injury as it sits at the interface linking AKI and CKD (Wongmekiat et al. 2013). This model has a fair advantage over other models as it generates fibrosis and extracellular matrix development expeditiously. Moreover, by using this model severity and the span of obstruction can be altered according to the requirement (Tan et al. 2007). Additionally, the presence of contralateral kidney in this model can be considered as a control. Human obstructive nephropathies and UUO animal model show many resemblances in terms of manner of causation which puts this model in a favorable position for studying the mechanism of human nephropathy (Klahr and Morrissey 2002; Lopez-Novoa et al. 2010).

The most common causes overall are (Fig. 16.1):

  • Extramural: Compression of ureter, prostatomegaly, abnormal growth of renal vessels, neoplasm of adjacent structures like uterus, cervix, ovaries, etc.

  • Intramural: Ureteric stricture, congenital stenosis, carcinoma of ureter.

Fig. 16.1
figure 1

Causes of unilateral ureter obstruction. The figure represents the most common causes of unilateral ureter obstruction

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16.2 Obstructive Nephropathy Pathophysiology

Ureter obstruction, although initiated by acute kidney injury can complicate the course and consequently results in end-stage renal disease depending upon the degree and span of obstruction. Ureter obstruction exhibits triphasic design of renal blood flow and pressure changes. Diminishing GFR is the hallmark of acute unilateral obstruction. Immediately after the onset of the first stage of acute unilateral obstruction, which is just after an hour, there is increase in the RBF, accompanied by high renal tubular pressure and collecting system pressure which is imparted to the Bowman’s capsule. However, a compensatory response of contralateral non-obstructed kidney intended to maintain GFR. After 2–4 h of obstruction, the pressure remains elevated but renal blood flow and GFR begins to decline due to persistent obstruction. Since the pressure filtration system in the glomerulus is reduced, there is a further decline in RBF and GFR (Farris and Colvin 2012). Leukocyte infiltrate starts appearing in the peritubular interstitium of the injured kidney after 4 h of unilateral obstruction and reaches peak at fourteenth day of obstruction. Leukocyte is accompanied by high renal tubular pressure and collecting system pressure which is imparted to the Bowman’s capsule. Leucocyte infiltrate mainly composed of macrophages and T lymphocytes. The leucocyte infiltrate which is recruited at the peritubular interstitium gets accumulated possibly by the production of inflammatory cytokines and growth factors (Chevalier 2006; Li et al. 2018). TNF-α trigger recruitment of leucocytes in the direction of kidney injury mainly at the tubular region of the kidney (Lee et al. 2014).

If the obstruction is not relieved and is maintained then the established kidney lesion may become converted into a progressive destructive lesion which is sorted into three major headings: nephritis, apoptosis of tubular cells, and fibrosis of renal interstitium (Kido et al. 2017; Chaabane et al. 2013). In the development of obstructive nephropathy, there are diverse groups of cells involved like Ang II, TGFβ, TNF-α, connective tissue growth factor (CTGF), various cytokines, renin-angiotensin system (RAS), nuclear factor-ĸB (NFĸB), fibroblasts, and several proteins. Of these AngII and TGF-β1 are of main focus as they play a substantial role in the advancement of kidney diseases (Fig. 16.2).

Fig. 16.2
figure 2

Principal pathogenic mechanisms of unilateral ureter obstruction. The figure depicts the fundamental mechanisms and the primary molecules involved in the pathophysiology of unilateral ureter obstruction

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Acute inflammation is a vital part of body’s immune response to injury for the purpose of initiating tissue repair. However, if this response lingers on it may eventually start damaging tissues and organs as is the case of UUO. One of the prime molecules that is responsible for inflammation during urinary obstruction is AngII and is a prominent mediator for stimulation of number of genes that have a role in renal injury (Chevalier and Cachat 2001). After UUO, RAS system gets triggered, and the after-effect of RAS activation is the migration of NF-κB to the nucleus where it induces expression of genes responsible for inflammation (Abbas et al. 2018). A vicious circle exists between NF-κB and RAS; and amidst NF-κB and TNF-α (Hosseinian et al. 2017). Wnt/β-catenin signaling regulates RAS genes (Wang et al. 2018a). RAS gene promotes fibrosis by activating two pathways, TGF-β/Smad 2/3 complex signaling pathway and Wingless/Int/β-catenin signaling pathway. There is a reinforcing loop since fibrosis promotes activation of RAS and it further turns on the activation of above-mentioned signaling pathways, TGF-β/Smad 2/3 complex and Wingless/Int/β-catenin pathways. Chief molecule of RAS system is angiotensin II (Ang II), and most of the physiologic and pathophysiologic functions of angiotensin II are because of the activation of its two receptors, Ang II AT1 (type 1) receptor and Ang II AT2 (type II) receptor (Touyz and Schiffrin 2000; Zhuo et al. 2013).

Apoptosis is a process of programmed cell death that occurs in multicellular organisms when a cell intentionally decides to die. Cellular homeostasis is regulated by a balance among cell proliferation and apoptosis. This is how appropriate cell numbers are maintained in healthy organs. Various forms of cell injury result in an increased rate of apoptosis leading to cell atrophy. UUO is one of the forms of renal injury which results in tubular atrophy due to extensive apoptosis. Mounting evidence indicate that dysregulated apoptosis and EMT have a significant role during UUO-induced kidney injury (Gobe and Axelsen 1987). As a result of EMT, tubular epithelial cells lose their polarity from apical-basal to front-rear and assume a mesenchymal cell phenotype. This architectural rearrangement is the stimulus which prompt cells for the commencement of apoptosis. Tubular cell apoptosis can be perceived after 24 h of obstruction and is at its peak after 12 days of obstruction. Another key factor that is playing a part in apoptosis is mechanical stretching of tubular cells (Chevalier et al. 2010). Onchoprotein Bcl-2, which is well known for its antiapoptotic function is a regulator of apoptosis. Downregulation of Bcl-2 is an indicator of apoptosis and its expression is diminished in case of dilated tubular cells after UUO (Ucero et al. 2014). It is evident that AngII is a potent pleiotropic molecule that plays a significant role in the commencement of apoptosis by stimulating other factors responsible for the regulation of apoptosis-like TGF-β1, Fas ligand, and caspase activity (Misseri et al. 2004). Elevated level of TGF-β due to sustained obstruction stimulate the production of ROS which is another important mechanism responsible for apoptosis (Hosseinian et al. 2017). Tubular cell apoptosis starts rapidly and is anticipated to amplify with time in case of sustained obstruction (Sun et al. 2013).

Obstructive uropathy consequently leads to kidney fibrosis which gradually terminates in end-stage renal disease if not treated timely. It relentlessly causes amassing of extracellular matrix (ECM) and gradually leads to degeneration of renal function (Soji et al. 2018). This ECM is predominantly produced by α-smooth muscle actin, expressing activated myofibroblasts (Duffield et al. 2013). Apart from α-smooth muscle actin, other proteins that serve as operators of fibrosis progression are type I, type III, and type IV collagen, fibronectin, and glycosaminoglycans (Farris and Alpers 2014). A potentially significant task is played by myofibroblasts in kidney fibrosis; however, their source of production and activation is still debatable. One school of thought presumes them to originate from the local renal fibroblasts itself but according to the study done by Strutz et al., they may arise as a result of EMT causing increased expression of fibroblast markers by the renal tubular epithelial cells under diseased conditions (Strutz et al. 1995). In 2015, two back-to-back studies resolved this argument and proffered additional understanding of the budding role of EMT in the advancement of kidney fibrosis (Grande et al. 2015; Lovisa et al. 2015). Thus, EMT is one of the driving forces behind fibrosis in renal progressive diseases, particularly in UUO model (Hu et al. 2015). Clinical studies have proposed that the acquisition of the α-SMA-positive phenotype by tubular epithelial cells may be secondary to EMT (Zhao et al. 2016).

16.3 Role of Epithelial-Mesenchymal Transition (EMT) in Streaming Kidney Fibrosis

Elizabeth Hay was credited for the first time for introducing the process of epithelial-mesenchymal transition (EMT) using a murine model (Hay 1995). Since it is a reversible process, therefore later, the term transformation was replaced by transition which was concurred in the first meeting of The EMT International Association (TEMTIA) held in Australia in 2003. Fundamentally, the process of EMT involves the transformation of epithelial cells into mesenchymal cells by undergoing manifold biochemical alterations like loss of apical-basal polarity and cell-cell cohesiveness and attain migratory and invasive properties (Kalluri and Weinberg 2009). The mesenchymal cells thus formed have front-rear polarity, spindle-shaped form, and favor cell ECM interaction rather than cell-cell adhesions (Kalluri and Weinberg 2009).

EMT, an evolutionary conserved developmental program is seen during embryogenesis where some of the epithelial cells become pliable and acquire the potential to move to and fro amidst epithelial and mesenchymal states by the process of EMT and MET (Lee et al. 2006). It was further realized that the activation of this program occurs not only during development but also under conditions of wound healing and pathological stress contributing to fibrosis and carcinomas (Kalluri and Weinberg 2009; Piera-Velazquez et al. 2011; Ribatti 2017). As a result, the rising concept of EMT has received a great deal of attraction in the last recent years due to its role not just only in embryology but in pathology as well.

Wound healing is body’s innate response to tissue injury and is a dynamic process. Researchers worldwide have identified the role of various cells during tissue repair. Myofibroblasts are one of them which play a significant role during wound healing by degrading the damaged tissue besides synthesizing the provisional ECM (Duffield 2010). After the accomplishment of the wound healing process, myofibroblasts undergo apoptosis and are thus vanished from the injured site. However, sometimes the inflammatory phase is prolonged and the wounds instead of going through the stages of healing remain confined, most probably in the inflammatory phase. Under such instances, myofibroblasts continue to produce fibers, consequently leading to organ fibrosis and ultimately organ destruction (Thannickal et al. 2004).

As mentioned above, that the origin of fibroblasts during fibrosis is a highly debatable issue. Earlier it was assumed that one of the major causes of fibrosis is the persistent generation of interstitial fibroblast which gets transformed into myofibroblasts resulting in scarring of functional tissue (Kriz et al. 2011). However, studies on fibrosis have revealed that a remarkable segment of the myofibroblasts has been contributed by the process of EMT (Iwano et al. 2002). Kidney is one such organ where the role of EMT has been witnessed in conferring fibrosis (Liu 2010). In a model of unilateral ureter obstruction-induced kidney injury, EMT has been observed as a chief process that contributes to fibrosis (Chevalier et al. 2009; Yuan et al. 2015; Lan et al. 2014).

EMT took on a more recognizable form 18 years ago in a research conducted using a mouse model having anti-glomerular membrane disease and was found that kidney epithelial cells abnormally producing fibroblast specific protein (FSP1) (Strutz et al. 1995). It was thus speculated by Strutz et al. that some of epithelial cells gets transformed into fibroblasts during fibrosis. This finding was further certified by Iwano that a considerate number of interstitial fibroblasts are due to epithelial cells of the renal tubule having lac Z as a reporter gene in unilateral ureter obstruction-induced kidney fibrosis in a mouse model (Iwano et al. 2002). These are some of the landmark studies which clearly exemplify the significant part played by EMT in the causation of chronic renal fibrosis in various experimental models.

There are multiple factors responsible for activating EMT in pathological and physiological states (Lim and Thiery 2012). The foremost important factors which play a significant role in triggering EMT includes TWIST, SNAIL, and ZEB (Lamouille et al. 2014; Skrypek et al. 2017; Kishi et al. 2015; Craene and Berx 2013). These factors repress the expression of E-cadherin, thereby leading to loss of cell-cell adhesions and contributing to initiation of EMT (Peinado et al. 2007). CTGF, IGF, and EGF are also some of the other factors which appear to play important role in induction of EMT and fibrosis (Lim and Thiery 2012; Skrypek et al. 2017). Besides these, TGF-β1 is considered as the principal profibrotic cytokine and myofibroblasts as the dominant cells responsible for generating fibrotic ECM (Ucero et al. 2014; Xia et al. 2018). TGF-β has also proven undoubtedly to be one of the most significant players responsible for inducing EMT (O’Connor and Gomez 2014).

Role of TGF-β is also witnessed in patients who developed fibrosis due to unilateral ureter obstruction and increased expression of TGF-β is seen in renal biopsy of the patients (Sato et al. 2003). In some of the in vitro studies also it was found that the epithelial cells attain the characteristics of mesenchymal cells phenotype when kidney epithelial cell lines were treated with TGF-β (Lamouille et al. 2014; O’Connor and Gomez 2013; Xu et al. 2009). In yet another parallel study, increased expression of α-SMA was observed when rodent mesenchymal cell line MT-9 and a porcine kidney epithelial cell line, LLC-PK1 were treated with TGF-β (Yamate et al. 2005).

Therefore, inhibiting the signaling of TGF-β can be a central target to halt the activation of EMT so as to prevent fibrosis. TGF-β mainly functions through Smad dependent/independent pathway and the signaling molecules predominantly responsible are Smad 2 and Smad 3 (Wang et al. 2005). Upon activation, SMAD proteins form a complex which migrates to the nucleus, ultimately inducing the transcription of their target genes (Saitoh and Miyazawa 2012; Griggs et al. 2017; Hewitson et al. 2017).

It has been revealed by various experiments that BMP-7 plays a significant role in kidney fibrosis by counteracting the action of TGF-β (Long et al. 2013). BMP-7 has an inhibitory effect especially on Smad-3 (Luo et al. 2010), and functions by decreasing the accumulation of ECM and favoring its degradation (Li et al. 2015). Likewise, in UUO model of mouse, deficiency of BMP-6 elevated kidney fibrosis independent of BMP-7 (Dendooven et al. 2011). Thus, not just BMP-7, BMP-6 can also be considered as a potential therapeutic tool (Yan et al. 2009).

Thus, there is enough documentation that EMT plays a key role in renal fibrogenesis by promoting activation and mobilization of multiple fibrogenic cells. The process is mediated through a distinctive signaling pathway which may act as viable therapeutic targets. Therefore, identification of these EMT markers and inhibition of their expression can become central target for antifibrotic strategies. Although a great deal of research has been performed on the application of EMT markers, there is still a great deal that must be achieved in this field so as to use these markers clinically. Moreover, there are no significant differences between the markers of different types of EMT that are used in development as well as in pathology (Popov and Schuppan 2010; Zeisberg and Duffield 2010).

16.4 Intricate Structural Arrangement of Kidney

The mammalian kidney arises from intermediate mesoderm (IM) and passes to three advanced phases during the course of development, pronephros, mesonephros and metanephros. The pronephros and mesonephros gradually degenerate at early stages of the development and the metanephros takes the form of functional and permanent adult kidney (McCampbell and Wingert 2012). Metanephric kidney is produced by two embryonic structures, the ureteric bud and the metanephric mesenchyme (Saxén 1987). The metanephric mesenchyme forms a population stem cell capable of self-renewal called as cap mesenchyme (CM) (Little et al. 2007; Brunskill et al. 2008; Mugford et al. 2009; Yu et al. 2012). Kidney development proceeds when a bundle of CM goes through EMT to form renal vesicles. These renal vesicles formed by CM are the progenitors of nephrons (Saxén 1987; Dressler 2006; Schedl 2007). Nephrons are the structural and functional elements of the kidneys which produce urine and removes wastes from the body. It comprises of three main parts, the glomerulus, tubules, and duct (Reilly et al. 2007).

When the development of adult metanephros kidney is accomplished, it bears an intricate and branched architecture with considerable cellular heterogeneity therefore, their potential of cell renewal is slow and restricted (Reilly et al. 2007). Still, they have the potential to regenerate to a certain extent which further relies on the magnitude of injury. Whenever the kidney gets damaged, the tubules get affected the most as they are highly vulnerable to injury. Following acute kidney injury, the kidney tubules, however, are capable of reestablishing their function. It is proposed that dominating role is played by the kidney stem cells in the repair process which are chiefly present in interstitium or tubules of the kidney. However, this is a remarkably challenging task. As already mentioned above, the kidneys are some of the most important organs having an intricate structure, comprising around 25 different types of cells dispersed in various compartments (Dressler 2006). Therefore, it is surprisingly hard to come up with a precise location of kidney stem cells and to explore their role in tissue repair (Humphreys 2009).

The scenario is totally different when the damage is severe enough, as in the case of chronic injury, which leads to destruction of nephrons and eventually tissue fibrosis. Kidney fibrosis is usually irreversible and consequently triggers toward end stage of the disease. Lifelong dialysis or kidney transplantation is most often required to tackle such problems. Presently, treatment options for renal fibrosis are usually staged as limited (Decleves and Sharma 2014). Therefore, understanding the milieu of the disease is particularly important in order to prevent or revert the progression of the disease. The recent advances in the field of regenerative medicine have motivated many researchers to propose nonrenal stem cells as a versatile treatment for replacement and repair of damaged tissues. In recent years, application of stem cells, specifically mesenchymal stem cells, have proved to be a preferred choice for various disorders (Bianco et al. 2013).

16.5 Stem Cells: Overview

As long as the success of tissue regeneration is concerned, cell source selection plays a very crucial role. It is particularly important to choose an appropriate cell and learn their intricacies to facilitate their effectiveness and success. Since the progress of tissue regeneration relies on the choice of the cell so it becomes mandatory, that the cells should be able to fulfill some of the fundamental requirements before they can be applied clinically. To mention a few, predominantly they should be able to home to the target tissue and should be able to release some signaling molecules for neo-tissue formation. Scientists have targeted almost all the cells in the body for research purposes. Many used autologous chondrocytes, i.e., cells or tissues obtained from the same individual for knee replacement (Mayhew et al. 1998). While for heart valve engineering some utilized nonspecific cell types, including dermal fibroblasts (Shin-Oka et al. 1997). However, while working with such early cell sources, researchers had to confront many challenges due to their severe shortcomings like they got tailored with age and their low yield.

A breakthrough came in the area of tissue regeneration by the probability of using stem cells which paved way for the researchers to design new strategies in the field of regenerative medicine. Stem cells are capable of restoring and repairing damaged tissue. As a consequence, stem cells have come up as promising alternative cell sources for tissue regeneration. Stem cells are considered among the top choices by the researchers not only because of their self-renewal capabilities but also because of their easy accessibility, expansibility, and their potentiality of differentiation (Blanpain and Fuchs 2014).

Together, both embryonic (ESCs) as well as adult (ASCs) stem cells are considered as good sources of stem cells that can be utilized for the applications of tissue regeneration (Bernstein and Srivastava 2012). Both types of cells have their own advantages and disadvantages.

ESCs are isolated from inner cell mass of blastocyst stage of embryo. They are pluripotent cells, i.e., that are able to differentiate into all derivatives of the three primary germ layers—ectoderm, endoderm, and mesoderm—but their use is highly restricted due to ethical controversies associated with them. Isolating the inner cell mass results in destruction of the blastocyst which raises ethical issues. Besides the ethical concerns, there is a technical problem of histocompatibility and their potential to produce teratomas also has to be addressed. All these controversies linked with embryonic stem cells highly prevent their use from participating in the field of tissue engineering.

On the contrary, there are no ethical issues associated with the isolation of adult mesenchymal stromal/stem cells as they reside in the non-embryonic somatic tissues so the destruction of the blastocyst is not involved. The adult stem cells are multipotent cells which have much more lineage restrictions in terms of differentiation potential; but despite that, they succeed in dealing the difficulties that are linked with the embryonic stem cells like ethical issues, negligible chances of tissue rejection, and avoiding teratoma formation. All these advantages together, make the adult stem cells a preferred source for research. Over the past 10 years, the field of tissue regeneration has been benefitted by the substantial usage of stem cells. Having unique biological properties mesenchymal stem/stromal cells (MSCs) amongst the adult stem cells have been explored widely for research (Kuppe and Kramann 2016).

16.5.1 Mesenchymal Stem Cells Introduction

Both the clinicians and researchers have shown keen interest in the mesenchymal stem cells for their immense potential to enhance tissue regeneration. All stem cells, regardless of their source, share unique properties, such as: they can transform into cells of different types including osteoblasts, adipocytes, chondroblasts, and cells of the visceral mesoderm (Wu et al. 2017a; Shi et al. 2012; Ma et al. 2014). Furthermore, they also keep the capacity of differentiating into the non-mesoderm lineages (Choi et al. 2018; Wan Safwani et al. 2017). Bioactive macromolecules secreted by MSCs are immune-privileged in nature which is also one of the important requirements in the field of tissue repair. Besides, MSCs have the capacity to migrate toward sites of injury and tumor microenvironments. All these properties of stem cells make them potent enough to repair or regenerate any injured tissue and therefore scientists are fascinated to the use of stem cells.

The timeline of the isolation of stem cells marks all the way back in 1967, when Friedenstein and his team, first isolated MSCs from stroma of bone marrow and reported them as plastic-adherent, fibroblast-colony-forming unit cells. These isolated stem cells from stroma of the bone marrow had enormous replicative propensity, great tendency to differentiation into osteoblasts, chondrocytes, adipocytes when cultured in vitro and also had the ability to support hematopoietic microenvironment when individual fibroblast-colony-forming unit cells were in vivo re-transplanted (Friedenstein et al. 1968). Such cells which are presently known as “mesenchymal stem cells” were termed by Arnold Caplan in 1991 (Caplan 1991).

Bone marrow stroma is not the sole source for the isolation of MSCs but there are several other alternative sources from which they can be harvested like: adipose tissue (Wankhade et al. 2016), amniotic fluid (Baulier et al. 2014; Sedrakyan et al. 2012), umbilical cord blood (Bieback et al. 2004), and renal progenitors (Bussolati and Camussi 2015; Pleniceanu et al. 2018) by means of various noninvasive approaches. They can proliferate to enough number for tissue and organ regeneration as they have enormous capacity for self-replication. Owing to these unique properties of MSCs like multi-lineage differentiation potential, immunoregulatory properties, migratory capacity, and ready availability, scientists are taking keen interest in exploring these unique subsets of cells for their potential use in regenerative medicine and tissue engineering.

16.5.2 Benchmarks for Isolation of Stem Cells

Prime issue confronted by the researchers in singling out the MSCs is the existence of varied protocols for harvesting MSCs encompassing multiple laboratories. Therefore, the International Society for Cellular Therapy (ISCT) in 2006 framed a standardized protocol for the selection of MSCs and specified them in accordance with the following parameters:

  • Morphologically mesenchymal stem cells should be fibroblast-like cells defined as colony-forming-unit fibroblast (CFU-F) and should be adherent to plastic under standard culture conditions (Dominici et al. 2006).

  • MSCs must display the following cell surface markers: CD44, CD73, CD90, and CD105, and diminished levels of MHC-I, and must omit the following set of markers: CD11b, CD14, CD 31, CD34, CD45, and MHCII (Dominici et al. 2006).

  • Stem cells should be able to transform in vitro into osteocytes, chondrocytes, and adipocytes (Dominici et al. 2006).

However, based on the above criteria, it is still not possible to isolate MSCs’ population which are homogenous in nature and still produce diverse cells. Research is still required to further sort this issue out.

16.5.3 Biological Attributes of Mesenchymal Stem Cells for Kidney Restoration

The mesenchymal stem cells act by multiple mechanisms in restoration of renal injury. On reaching the injured site they differentiate into the renal cells and bring about repair by immunomodulation and also by their paracrine activity (Fig. 16.3).

Fig. 16.3
figure 3

Main processes of mesenchymal stem cell therapy. The figure represents the potential properties of stem cells involved in the treatment of kidney diseases

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16.5.3.1 Differentiation

Owing to the multipotent nature of the stem cells they are capable of differentiating into bones, cartilage, fat, tendon, muscle etc. when cultured. In addition to that, MSCs have tremendous plasticity of trans-differentiation once infused into the injured site. Increasing evidence of their differentiation potential is mainly from in vitro reports in contrast to in vivo studies (Weng et al. 2003; Singaravelu and Padanilam 2009; Wong et al. 2014). However, some in vivo studies have been successful in displaying the differentiation capability of MSCs into renal cells. It was demonstrated in a mouse model through laser-scanning microscopy that GFP-tagged bone marrow MSC differentiated into mesangial cells of kidney glomerulus (Imasawa et al. 2001). In yet another research, Li et al. by using ischemic model of mouse further reported the trans-differentiation of infused MSCs toward renal tubular epithelium thereby contributing to tissue recovery (Li et al. 2010). Furthermore, in another model of mouse, it was exhibited that hASC show trans-differentiation into renal tubular epithelial cells at an advanced stage of AKI (Li et al. 2010). Recently, a body of researchers transplanted stem cells from bone marrow of rat into female rats and after 2 days MSCs were found differentiated into embryonic cells (Zou et al. 2016). However, it has been seen in some studies that human bone marrow-derived mesenchymal stem cells, when transplanted into mouse embryo, are capable not only of differentiating into specific renal cells but are also capable of differentiating into a complete nephron (Yokoo et al. 2005). Thus, it has been revealed through several findings that MSC exhibits such traits that can assist in tissue repair/regeneration. Thus, mesenchymal stem cell therapy provides better environment to regenerate damaged cells via differentiating into the renal specific cells and also induce the resident stem cells to regenerate to specific cell types.

16.5.3.2 Homing

Homing is defined as migration of endogenous host cells from their site of storage to a distant organ. Mesenchymal stromal cells, due to their multipotent differential ability, can be directed to migrate to the target areas which can be a tumor site, inflammatory site or even a damaged tissue. Homing of the MSCs may be influenced by the pathological and physiological conditions thus making control of their homing a complex matter.

A team of researchers carried out successful trials and showed the homing of MSCs to the injured kidney after ureter obstruction (Ozbek et al. 2015). Another strong evidence was provided by the researchers where they showed through bioluminescence imaging that the micro-RNA secreted by MSC home the kidney injured by ureter obstruction (Wang et al. 2016). However, in various renal injury models, no proof of homing of cells to injured kidney were detected although therapeutic effect was prominent. In a model of acute kidney injury, there was no proof of MSCs after 7 days of their infusion (Cheng et al. 2013). Thus, it is a highly debatable issue whether the homing of the infused MSCs to the site of injury is important for their healing action. Most of the studies have shown the clinical efficacy of MSC intravenous delivery but at the same time, following intravenous delivery route of MSC bulk of them are entrapped chiefly in spleen, liver, and lungs (Fischer et al. 2009; Iwai et al. 2014; Tang et al. 2015; Zanetti et al. 2015). As a result, cell count is declined and thus are incapable in reaching the damaged site. Thus, concerted efforts in this area of research are required to ensure maximum homing of cells to the damaged site.

16.5.3.3 Immunomodulation

A breakthrough came with the findings that MSCs have the efficiency to tailor the immune response of an organism which demonstrated that MSCs are immunomodulatory in function (Wang et al. 2018b; Gao et al. 2016). To present that MSCs are immunosuppressive in nature, was first time documented from studies with baboons (Bartholomew et al. 2002) that revealed that activation and proliferation of T cells of our immune system can be repressed by MSCs. Following the first report, consecutive studies were conducted using animal and human models manifesting immunomodulatory property of stem cells. The ability of MSC to dampen the immune response relies on interaction of MSC with the immune cells in conjunction with secretion of soluble factors (Wu et al. 2017b; de Witte et al. 2018). The immune system of a body is the one that has its fair share of controversies against successful outcomes of tissue regeneration applications. It has been shown by various clinical studies that immunomodulation is one of the inherent properties of MSCs and they bear the tendency to:

  • Diminish the immune response of T-cells, B-cells, and macrophages (Contreras et al. 2016)

  • Modulate the function of T-regulatory cells (Contreras et al. 2016)

  • Vanquish the activation of dendritic cells and natural killer cells (Mattar and Bieback 2015).

  • Furthermore, they reduce the production of proinflammatory cytokines (Ge et al. 2010; Eggenhofer et al. 2013).

In general, T-regulatory cells and macrophages have been implicated to play a leading role in maintaining the facets of immunomodulatory ability of MSCs (Riquelme et al. 2018; Chang et al. 2012; Goncalves et al. 2017). Thus, due to their immune-privileged status, the role of MSC is just not limited to therapeutic mechanism but are effective across species barriers also (Gieseke et al. 2010). Currently, studies have shown that even the dead and fragmented MSCs retain their immunosuppressive potential (Luk et al. 2016; Koniusz et al. 2016; Nargesi et al. 2017). Apart from interacting with the cells of the immune system they also have the potential to modulate kidney functions like renal blood flow, survival of endothelial cells, and permeability of capillary cells (Kramann and Humphreys 2014). In a recent study, it has been demonstrated that kidney injury has been ameliorated after infusion of MSCs procured from bone marrow in obstructive nephropathy model by modulating the function of podocytes (Xing et al. 2019).

16.5.3.4 Paracrine Activity

It is no surprise that MSC’s differentiation potential and homing at the injured site is strongly correlated to the success of tissue regeneration but it has also been observed that another important mechanism must be responsible for the application of MSC, and paracrine effect has proved to be a potential significant player. Studies have shown that when a tissue gets injured, MSCs secrete a plethora of bioactive molecules like enzymes, growth factors, chemokines, cytokines inclusive of extracellular vesicles, exosomes, and micro-vesicles which are attracted to the damaged tissue to modify its behavior (Andrzejewska et al. 2019). It has also been observed that bioactive factors released by MSCs have proregenerative, antifibrotic, antimicrobial, anti-apoptosis, and antioxidation properties indicating that most of the benefits of MSCs can be attributed to its paracrine effect during tissue injury (Maguire 2013; Haynesworth et al. 1996; Patschan et al. 2006; Tögel et al. 2005; Gnecchi et al. 2006; Kim et al. 2019).

Freshly, it has been shown in various studies that majority of the benefit of MSCs is due the microvesicles secreted by them (Sedrakyan et al. 2017; Bruno et al. 2009; Ranghino et al. 2017). Investigators observed that paracrine activity of extracellular vesicles derived from MSCs relies on the secretion of genes from them that are responsible for angiogenesis (Eirin et al. 2018). In various clinical studies, it has been evidenced that cytokines responsible for inflammation such as TNF-α, IFN-γ, and IL1b are diminished, whereas those suppressing inflammation such as TGF-α and bFGF are highly escalated in kidneys treated with MSCs derived extracellular vesicles (Tögel et al. 2007; Uccelli et al. 2008; Rabb 2005; Hu and Zou 2017).

The potency of extracellular vesicles has been demonstrated in various forms of acute as well as chronic kidney injury. There are some convincing studies in UUO animal model of kidney injury in which the renal injury has been mended by the paracrine effect of EV of MSC (He et al. 2015). Recently, attenuation of kidney fibrosis with prominent decline in the expression of TGFb1, TGFbR1, and collagen IV has been demonstrated in unilateral ureter obstruction-induced kidney injury model by extracellular vesicles secreted by MSCs (Wang et al. 2016). Consequently, evidence suggests that it is the paracrine action of the MSCs that is responsible for conferring renoprotection.

Paracrine effect of MSCs has a profound effect on tissue regeneration and could be a game changer for treating various kidney disorders. Mesenchymal stem cells, therefore, by virtue of their renotropic property and tubular regenerative potential are currently being tested for their potential use in cell and gene therapy for several human debilitating diseases and genetic disorders.

16.6 Concluding Remarks and Future Perspectives

Chronic kidney disease is acknowledged as a considerable medical problem globally and is a crucial issue of public health concern. Kidney possesses a complex architectural structure having an intricate cellular composition which poses challenges to mitigate kidney diseases. Use of stem cells as a curative therapy has become a much-wanted choice now for various types of acute as well as chronic kidney pathologies is gaining ground. MSC form a population of cells that are well distinguished and are easily isolated from a wide variety of human as well animal sources. There are several mechanisms through which MSC exert their therapeutic effect but there are strong evidences which demonstrate that the most promising and effective mechanism of stem cells consists fundamentally in their paracrine and immunomodulatory action. An extensive survey of research supports the restorative efficacy of stem cells in numerous experimental studies of renal disorders and has evidenced outstanding results. Although long-term use of MSCs still remains debatable, therefore researchers are speeding up the pace of this area of research so as to address the flaws associated with stem cell therapy such as mechanism of homing, in vivo tissue differentiation, and tissue-specific delivery of MSCs. Although much has been learned about the therapeutic applications of stem cells, there is still a great deal that has to be achieved before using them clinically and the day is not far when the use of stem cells will speed up exponentially over time, thus paving our way to the most exciting and interesting new frontiers the domain is likely to take in the upcoming future.