Stem Cells and Generation of New Cells in the Adult Kidney

Epithelial Cells

All epithelial cells of the adult kidney are believed to derive from the intermediate mesoderm, from which both the ureteric bud (a branch of the Wolffian duct) and the metanephric mesenchyme originate. Renal morphogenesis starts when the ureteric bud invades the metanephric mesenchyme and starts branching. The cells in the tip of each ureteric bud branch give rise to the collecting duct cells and the metanephric mesenchyme cells in contact with each ureteric bud tip give rise, after a series of morphogenic steps, to the cells of the remaining nephron segments spanning from the connecting tubule to the glomerulus. The ureteric bud, like the Wolffian duct, expresses the homeobox gene HoxB7 , and transgenic mice expressing HoxB7- GFP or HoxB7- Cre recombinase have been used to label most, if not all, of the cells in the ureteric bud branches of the embryonic collecting duct, and their progeny in the adult kidney.

The metanephric mesenchymal cells undergo simultaneous differentiation (to generate a nephron for each ureteric bud tip) and growth, so that the appropriate number of nephrons will be generated for the branches of the ureteric bud. It was recently found that the metanephric mesenchymal cells that are in contact with the tips of the ureteric bud, referred to as the cap mesenchyme, are the progenitor cells of all nephron epithelia (except the collecting duct). These cells were found to express the transcription factors Cited1 and Six2 , thereby allowing generation of transgenic mice that label all nephron epithelial cells except those of the collecting ducts. More relevant to the present discussion is that these mice can be used to permanently identify the progeny of adult nephron epithelial cells, thus providing an invaluable tool for analysis of epithelial cell regeneration after kidney injury and/or disease, as discussed below.

Mesenchymal/Stromal Cells

Like renal epithelial cells, the vast majority of the stroma cells in the adult kidney derive from the intermediate mesoderm that in the kidney gives rise to a cell population that expresses the forkhead transcription factor Foxd1 . These cells generate many renal interstitial cells, as well as mesangial cells, vascular smooth muscle, pericytes, and renal capsule, and likely mesenchymal stem cells. Foxd1 -expressing cells are absolutely required for normal kidney development, and their adult progeny is of extreme interest because it likely contains pluripotent MSC and pericytes, although a clear-cut distinction between these two cell types is not yet possible. In addition, identification of Foxd1 as marker of these cells has made it possible to develop transgenic mice that can be used to label the stroma cell progeny in the adult kidney.

Another population of renal stromal cells derives from the paraxial mesoderm, but the precise contribution of these cells to the interstitial and mesenchymal cell populations of the adult kidney remains to be defined. Finally, an area of the intermediate mesoderm located ventro–lateral to the dorsal aorta generates renal interstitial cells that express the stem cell factor receptor ( c-kit ). During embryogenesis, these cells appear to be involved in the maintenance of the metanephric mesenchyme-derived cells, but identification of their progeny in the adult kidney remains to be established.

Endothelial Cells

The renal circulation is both anatomically and functionally complex, and likely contains many types of endothelial cells. Their exact origin is still poorly-understood. It is clear that once kidney development starts, the renal anlage contains angioblats that give rise to endothelial cells. It is currently unknown whether these endothelial precursors migrate into the developing kidney or differentiated from cells that reside in the metanephric mesenchyme. The latter appears more likely, as we found that many cells in the renal anlage express tyrosine kinases that are characteristic of adult endothelial cells. Interestingly, in addition to being endothelial precursors, angioblasts in the renal anlage appear to provide signals important for development and differentiation of the metanephric mesenchyme. It is currently unknown if there exists interaction in the adult kidney between endothelial cells and either interstitial or epithelial cells that might be involved in maintaining kidney homeostasis or repair from injury. Recent work by Lin et al. suggests that endothelial-to-pericyte cross-talk is involved in the generation of kidney myofibroblats, as detailed below.

Normal Conditions

As assayed by a variety of methods, the normal adult kidney has a low rate of cellular proliferation. Using antibodies to Ki67, a nuclear protein expressed in cycling cells during G0 and G1, between 0.4–1% of all cells were cycling in the adult rat kidney. Interestingly, age has a profound effect in the abundance of proliferating cells found in the kidney. Vogesterder et al. found that while only ~0.4% of all renal cells were positive for Ki67 in the kidneys of 16- to 20-week-old rats, in animals that were only 4 weeks old the number of cycling cells was ~5%. This suggests that in the kidney there is an age-dependent progressive decline in the number of cycling cells, and that workers examining renal cell proliferation should take into account the age of the animal as an important variable.

Proliferating cells in the renal cortex of 4-week-old rats were found preferentially located to the S3 segment of the proximal tubule, when compared to the S1 and S2 segments. This interesting result might have important implications in designing strategies to identify renal stem cells, a subject to which we will return below. In contrast, in 1-year-old rats, we found that most of the kidney parenchyma had a homogeneous fraction of ~1% of Ki67 positive cells Figure 29.1 . There were two exceptions to this, however, the body of the papilla where there were extremely low numbers of cycling cells (<0.1% of the cells were positive for Ki67), and the upper part of the papilla (at the papilla–medullary junction), where we found the highest frequency of cycling cells (~2.5%), indicating that it is an area of privileged cellular proliferation in the adult rat. Detailed morphological observations indicate that terminally differentiated tubular epithelia cells can generate new cells, but these observations don’t exclude the possibility that there are epithelial stem cells.

Organ Repair from Injury

In contrast to normal conditions, the kidney displays a remarkable proliferation capacity shortly after transient injury. For example, injury induced by 30–45 minutes of complete renal artery occlusion in rodents causes functional failure, and widespread cellular apoptosis and necrosis that are followed by diffuse cellular proliferation and functional recovery. What is the origin of these new cells? While the kidney has multiple cell types, studies on the generation of new cells after injury have fundamentally focused on the epithelial cells, likely because of their better-understood functional importance and easier identification. It is now established that the new epithelial cells after kidney injury develop from within the parenchyma, rather than being derived from extrarenal sources such as the bone marrow, and thus three possibilities appear likely: (1) any surviving terminally differentiated epithelial cell can generate identical cells; (2) there exist kidney epithelial stem cells capable of generating any epithelial cell type, similar to what was found in the interfollicular epidermis; and (3) pluripotent renal stem cells generate epithelial as well as other cell types, as in the case of the stem cells in the bulge of the skin.

Morphological observations and functional studies with nucleotide analogs have provided strong evidence that terminally-differentiated epithelial cells generate new epithelial cells after injury. More recent elegant genetic cell fate-mapping studies have confirmed this suspicion; Humpreys et al. used reporter mice in which the renal epithelial cell compartment was labeled by a Cre recombinase driven by the promoter of Six2 , a gene that is specifically expressed in embryonic epithelial precursors (see above), and examined their response to acute kidney injury. They found that injury induced massive cellular proliferation, and that all new epithelial cells expressed the reporter gene ( Figure 29.2 ). Since RT-PCR of adult kidney tissue could neither detect Six2 nor Cre , it is apparent that the labeled cells originated from epithelial cells labeled previously during kidney development, thus excluding the interstitial/stroma cell compartment as the origin of new epithelial cells. Needless to say, this experiment does not address whether there exists a group of restricted epithelial cells that are responsible for all the new epithelia generated after injury; these cells could function as adult renal epithelial stem cells. In a recent study, Humphreys et al. examined this possibility by labeling cycling cells after transient kidney injury with two different thymidine analogs administered sequentially. Since the number of epithelial cells that were positive for both nucleotides was very low, these workers concluded that surviving epithelial cells repopulate the nephron epithelium in a stochastic manner, suggesting that the nephron epithelia has no stem cells. However, while control experiments clearly showed appropriate specificity of both antibodies, detection of closely related thymidine analogs during conditions that, unlike in the control experiments, probably result in incorporation of very different amounts of the two analogs in a given cell are fraught with potential problems. More importantly, their conclusion is based on the implicit assumption that a putative population of epithelial stem cells would be a small fraction of the total number of cells. Under these conditions, to repopulate the damaged epithelia the stem cell progeny would need to divide rapidly, and would thus incorporate both nucleotide analogs. However, as detailed below, stem cells in Drosophila Malpighian tubules are a very large fraction of the total cells, and there is no reason why this may not also be the case in mammalian kidneys. Similarly, in organs other than the kidney such as the adult airway epithelia, stem cells account for about one-third of the total number of cells.

Identification of the site where cellular proliferation first starts after transient injury could potentially facilitate identification of precursor/stem cells. Unfortunately, for most insults that cause acute kidney injury with functional failure, cellular proliferation has most often been examined one or a few days afterwards, at which time cell proliferation, while very prominent in the S3 segment of the proximal tubule, is also widespread in other parts of the kidney parenchyma, particularly the medulla.

Following acute kidney injury by renal artery occlusion we could not detect proliferating cells until ~24 hours later when we examined kidney sections of ~5 μm thickness, as is done routinely. However, with 100 μm vibrotome sections, one hour after injury we found that the upper part of the papilla had more proliferative cells than other parts of the kidney (see Figure 29.3 ), suggesting that this is the site of initial cellular proliferation after kidney injury, which we previously found to be the site of enhanced cell cycling under normal conditions (see above). Interestingly, Vinsonneau et al. reported that the first cells that they found cycling after ischemia reperfusion injury to the kidney were uro-epithelial cells in the upper part of the urinary (intrarenal) space and neighboring interstitial cells; detailed analysis with Ki67 and BrdU incorporation showed that these cells were proliferating ~16 hours after ischemic injury and ~4 hours before proliferation could be detected elsewhere in the kidney. The site identified by Vinsonneau et al. is where the base of the papilla attaches to the medulla, it is in close proximity to the cortex, and appears to be the same proliferating site we identified in the upper papilla. Needless to say, identification of these “early” proliferating cells and of their progeny would be of extreme interest.

An additional observation merits mention. In the few studies that identified the cells that first started proliferating after injury, either after transient ischemic injury or aminoglycoside toxicity, it is remarkable that in all instances they were interstitial cells, perhaps suggesting that some interstitial cell plays a critical role in initiating epithelial regeneration. Indeed, the likelihood of renal functional recovery after injury was found to correlate with increases in the number of interstitial cells, many of which were likely myofibroblasts. Although these results raise the possibility that myofibroblasts might be involved in epithelial regeneration, other interstitial cells such as macrophages are known to be involved in kidney repair, and a detailed characterization of the interstitial cell compartment following transient kidney injury is lacking.

Effect of Age

For many organs, including the kidney, the capacity to recover from injury decreases with age, an observation familiar to most practicing nephrologists. In most organs, it remains to be determined whether this is due to a decrease in the number of organ-specific stem cells or to the inability of the stem cells to be activated, but recent work suggest that the latter is more likely (reviewed by Liu and Rando ). For example, it was recently found that aging muscle had a normal number of satellite cells, but the cells failed to activate in response to exercise. Interestingly, progenitor/stem cells in advanced age can display a “young” response by exposure to a young systemic environment, indicating that the changes responsible for the functional decrease of precursor/stem cells are potentially reversible.

While the effect of age on the renal proliferative capacity after injury has not been studied in detail, it appears that cell proliferation after acute kidney injury decreases with age, in agreement with the poor prognosis of recovery from acute kidney injury in elderly patients. Mechanisms that could account for these observations include telomere shortening and increased expression by renal epithelial cells of zinc-α ² -glycoprotein (Zag), an adipokine associated with cachexia. To examine whether the poor regenerative capacity of the aging kidney is due to a decrease in the numbers or in the functionality of putative renal stem/progenitor cells awaits definitive identification of these cells.

In sum, both during normal conditions and particularly after transient kidney injury, the S3 segment of the proximal tubule is a site of intense cell proliferation, suggesting the presence of progenitor cells in this part of the nephron. In addition, the upper part of the papilla and close to the urinary space is also a site of robust cell cycling, both under normal conditions and after transient kidney injury.