Resident Stem Cells and Regenerative Therapy

Brazil

Chapter 1

Resident Stem Cells in Kidney Tissue

Milene R. Costa∗,†,‡,§,∗∗, Karine S. Verdoorn∗,†,‡,§,∗∗, Rafael S. Lindoso∗,†,‡,§,∗∗, Marcelo Einicker-Lamas∗,†,‡,§,∗∗, Hellen J. Vieira-Beiral∗,†,‡,§,∗∗, Oliver Wessely†† and Adalberto Vieyra∗,†,‡,§,∗∗

∗Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil

†Programa de Terapia Celular e Bioengenharia, Rio de Janeiro, Brazil

‡Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Rio de Janeiro, Brazil

§Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem -INBEB, Rio de Janeiro, Brazil

∗∗Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

††Cleveland Clinic, Lerner Research Institute, Department of Cell Biology, Cleveland, Ohio, USA

Introductory Remarks

The mammalian kidney is recognized for its highly specialized structures and terminally differentiated cells arranged in an elaborate form to accomplish its function. Although these specialized cells have a low rate of renewal, the mammalian kidney possesses an inherent ability to regenerate or repair after ischemic or toxic injury, but the regenerative process is not fully understood. In the past few decades, investigations have concentrated on three main issues: (1) contribution of extrarenal stem cells (e.g., hematopoietic and mesenchymal bone marrow cells), (2) intrarenal regeneration by dedifferentiation of surviving tubular cells, and (3) intrarenal regeneration by recruitment of resident renal stem cells. The very low rate of extrarenal stem cell transdifferentiation into specialized kidney cells reinforces the intrinsic renal regenerative capacity. In parallel, the discovery of adult stem cells in many different tissues encouraged the search for renal resident stem/progenitor cells. This issue challenged well-established concepts and raised a new paradigm: renal resident stem/progenitor cells exist and may contribute to tissue regeneration. Identification and characterization of these stem cells has presented an enormous challenge, given the complexity of the kidney structure. In this chapter, we will discuss the main findings to date and debate some of the open questions. The structural organization of the kidney and its multiple specialized cell types are important details when considering the susceptibility of kidney cells to injury and their ability to regenerate. We therefore present a brief overview in the following section of the structural characteristics of the kidney to give a better understanding of the issues that will be considered later in the chapter.

Brief Description of the Structural Organization of the Mammalian Adult Kidney

A longitudinal section of a mammalian kidney shows three clearly delimitated regions: cortex, external medulla, and internal medulla (Fig. 1.1). A close-up of the cortex moreover allows the recognition of two subregions, the more superficial cortex corticis (CC) and the internal cortex (IC) (Fig. 1.1A). The cortex corticis is particularly important in the context of this chapter because its cell population comprises >90% of the proximal tubule cells that are important targets in acute renal injury (AKI). The subregion called papilla (P) is found in the tip of the internal medulla (Fig. 1.1A and B), where a niche of stem/progenitor cells was described some years ago [1] (discussed later). The functional unit of the kidney, the nephron (more than 1 million in a human kidney; Fig. 1.1B and C), is a tubular structure, in which different segments can be distinguished by their specialized cell morphology and by their different permeability and transport properties. The nephron starts in the cortex with the glomerular or Bowman’s capsule (BC) that receives the ultrafiltrate from a capillary network that originates from the afferent arteriole (AA), a vessel of the interlobular artery (IA) (Fig. 1.1B). As shown in Figure 1.2 the endothelial cells (EC) of the intertwined vessels are organized in a globular structure by two additional cell types, the podocytes (PD) and the mesangial cells (MC) [2]. Although the podocytes are critical structures for ultrafiltration—and are also especially sensitive to injuries—the mesangial cells are the more important targets for the action of stem cells in any potential regenerative process (Fig. 1.2).

Figure 1.1 Structural organization of an adult kidney. (A) Longitudinal section of a mammalian kidney. CC, cortex corticis ; IC, internal cortex; CX, calyx; P, papilla. To complete the description of the morphologic organization of an adult kidney, panel A also shows the renal artery (RA), the renal vein (RV), the renal pelvis (RP), and the ureter (U). (B) Enlargement of the area indicated in A shows the location of cortical and juxtamedullary nephrons. (C) Amplified picture of a nephron and its association with the vascular network. AA, afferent arteriole; BC, Bowman’s capsule; EA, efferent arteriole; PTA, peritubular arteriole; PCT, proximal convoluted tubule; TDL, thin descending limb; TAL, thin ascending limb; TMAL, thick medullary ascending limb; DT, distal tubule; CD, collecting duct; IA, interlobular artery; IV, interlobular vein; VR, vasa reta .

(© Kari C. Toverud CMI.)

Figure 1.2 Detailed view of the glomerular compartment. AA, afferent arteriole; EA, efferent arteriole. The lowercase letters indicate possible pathways for cell replacement; a-c : podocyte replacement from the capsular epithelium, by self-renewal or by circulating stem cells; d,e : endothelial cell replacement from resident stem/progenitor cells or circulating cells; f,g : replacement of mesangial cells from macula densa or circulating cells; h : potential route of regeneration with the use of embryonic stem cells (ESC) or induced pluripotent stem (iPS) cells; i : possible regeneration of proximal tubule cells from parietal capsular epithelial cells.

(From [2], with permission; see this reference for details.)

After the capsular space, the next segment is the proximal convoluted tubule (PCT)¹ (Fig. 1.1B and C). Its terminal portion, pars recta (PR) dips into the outer medullary region. From here the tubule forms a hairpin structure (Henle’s loop) (Fig. 1.1B and C), which is subdivided in three regions based on their differentiated structure and transport properties: the thin descending limb (TDL), the thin ascending limb (TAL), and the thick medullary ascending limb (TMAL). Reaching again the cortex, the distal tubule (DT) is the last nephron segment.² It fuses with the collecting duct (CD), which merges into the medulla and ends in the calyx (CX) (Fig. 1.1A). From a functional point of view, it is remarkable that specialized tubular cells in the region of the terminal part of the ascending limb and the early portion of the distal tubule (macula densa cells) (MD) (Fig. 1.2) form a complex (the juxtaglomerular apparatus) with cells of the afferent arteriole. This organized structure is key in the most important biochemical pathway of the kidney, the renin/angiotensin system (RAS), which plays a pivotal role in kidney disease and regeneration [5].

In respect to the vasculature, the glomerular capillary network described earlier converges to form the efferent arteriole (EA) (Fig. 1.1B and C; Fig. 1.2). This vessel exits the glomerulus and ramifies around the proximal and distal convoluted tubules, forming the peritubular arteriolar network (PTA) (Fig. 1.1C); it then fuses into small venules that drain into the interlobular veins (IV) (Fig. 1.1C). In the case of the juxtamedullary nephrons (i.e., the deeper nephrons below the cortex corticis) the efferent arteriole forms a straight-bore vessel that merges into the medulla (the descending arm of the vasa reta) (VR) in close proximity with the ascending limb of Henle’s loop up to the papillary tip. Here it turns to ascend toward the external medulla as a venule (the ascending arm of the vasa reta) that finally ends into an interlobular vein [6]. Along their trajectories, the descending and ascending vasa reta anastomose with one another several times.

The special organization of the renal vasculature, with its intense blood flow in the cortex, and a lower oxygen and substrate supply in the medulla results in different metabolic characteristics to the three main kidney areas. Although in the cortex, intense oxidative metabolism occurs (~10% of the oxygen consumption of the body at rest), the metabolism in the inner medulla is almost purely glycolytic [7]. The cells of the outer medulla show mixed metabolic characteristics depending on the blood flow and the local oxygen pressure. Interestingly, these differences in local oxygen pressure are important in AKI episodes and, possibly, in kidney regeneration.

Stem Cells in Kidney Organogenesis

The nephron epithelia and collecting system of the mammalian kidney derive from two precursor tissues, both of the intermediate mesoderm (Fig. 1.3) [8]. Early in the mouse embryogenesis at approximately day 9.5 (E9.5), the nephric duct (ND), or Wolffian duct, a simple epithelial tube, originates from a solid cord of cells from the dorsal intermediate mesoderm (IM). Interactions between the nephric duct and the undifferentiated ventral intermediate mesoderm, the nephrogenic cord, give rise to the pronephric and mesonephric kidneys. In mammals, these more primitive kidney forms are replaced soon thereafter by the adult metanephric kidney. At E10.5, mesenchymal-epithelial interactions between the nephric duct and the metanephric mesenchyme (MM)—a more specialized region of the intermediate mesoderm—will form the ureteric bud (UB) near its caudal end. The UB is an epithelial protrusion from the nephric duct into the MM. Reciprocal inductive signals from the UB and the MM induce growth and specialization of these two components. The UB will undergo elongation and repeated branching into the MM, giving rise to the collecting system, whereas the cells of the MM aggregate around the UB tips and undergo mesenchymal-epithelial transition to form a polarized renal vesicle (Fig. 1.4) [9]. This renal vesicle will contact the tip epithelium of the UB, generating the comma-shaped body, after which elongation of this structure forms a second cleft generating the S-shaped body. The distal end of the S-shaped body will subsequently fuse with the UB epithelium, while the proximal end will be invaded by blood vessels and stromal cells forming the glomerular tuft.

Figure 1.3 Renal organogenesis: origins of metanephric mesenchyme and ureteric bud. Mammalian metanephric kidneys develop from the intermediate mesoderm (IM) located bilaterally to the neural tube (NT), between the lateral plate mesoderm (LPM) and the somites (S) in the caudal region of the embryo (A) . The nephric duct (green, ND)—a simple epithelial tube originated from a solid cord of cells at the dorsal intermediate mesoderm (C-E) —interacts with the nephrogenic cord (blue, NC), the undifferentiated ventral IM. The nephric duct undergoes elongation and epithelialization (D and E) while the caudal end of the NC specializes into metanephric mesenchyme (pink, MM) (F) . The pseudo-stratified epithelium in the caudal ND gives rise to the ureteric bud (UB) (G) . Interactions between the UB and the MM promote the UB outgrowth (H) . and repeated branching within the MM (I-K) . The elongation of the collecting ducts forms the medulla (L) and papilla (M) . E, embryogenesis circa day, and P, postnatal day.

(From [8], with permission.) See Plate 1.

Figure 1.4 Renal organogenesis: specification of the metanephric mesenchyme and ureteric bud. The ureteric bud progenitor population is characterized by Pax2, Lim1, Gata3, and β-catenin expression. The metanephric mesenchyme is settled by different precursor populations: those that give rise to the stromal cells, which are Foxd1 + , and those that form the condensed mesenchyme, which are Six2 + . In the condensed mesenchyme, two subpopulations can be distinguished, those of the capping mesenchyme, which are Six2 + , Cited1 + , and Wnt4 − , and those of the induced mesenchyme, which are Six2 + , Cited1 − , and Wnt4 + . The cells of the induced mesenchyme form the renal vesicle via mesenchymal-epithelial transition (MET); they continue to express Wnt4 and give rise to all tubular epithelial cells. Differently, the cells of the capping mesenchyme stay undifferentiated and proliferate, providing nephron epithelial precursors until completion of renal development.

(From [9], with permission.)

This sequence of events is coordinated by reciprocal signaling between the UB and MM cells and accompanied by multiple steps of differential gene expression and stem/progenitor cell maintenance or differentiation. The first precursor cell population that will give rise to all kidney structures is the intermediate mesoderm. Differentiation of these cells from the surrounding tissues begins with the expression of the genes that encode the two transcription factors Lhx1/Lim1 (LIM homeobox 1) and Osr1/Odd1 (odd-skipped related 1) [8, 9]. Lhx1 activity is required for the intermediate mesoderm differentiation, but its expression, like the expression of Osr1, also extends to the lateral plate mesoderm