The Drosophila MTs provide an excellent model organ for studying cell fate specification, organization of epithelial sheets, and physiology of renal systems, and serve as an excellent model for studying human kidney diseases (Dow and Davies, 2006; Chintapalli et al., 2007). MTs develop from two sources: the ectodermal epithelial bud (hindgut primordium) and the mesenchymal mesoderm. The MTs consist of monolayer epithelial cells with a distinct apical-basal polarity. The development of Drosophila MTs, which is largely completed during embryogenesis, includes an allocation of tubule primordia and tube budding, followed by growth and elongation via cell rearrangements, and, eventually, cell differentiation (Ainsworth et al., 2000; Delholm et al., 2003; Jung et al., 2005; Dow, 2007a; Dow, 2007b). In the early stages of MT development, interaction between the midgut and hindgut anlagen redefines the expression of the two transcription factors, zinc-finger transcription factor, Krüppel (Kr) and homeodomain-containing protein, Cut in primordial cells (Gaul and Weigel, 1990, Liu and Jack, 1992). Each gene is expressed in the tubule primordia independently of the other and they act together to specify tubule cell fate (see Hatton-Ellis et al. 2007). Following tubule evagination, extension of the cylindrical buds into crescent-shaped tubes requires ribbon (rib), which encodes a BTB/POZ-type nuclear protein, faint sausage (fas), which encodes an extracellular, immunoglobulin-like protein, and Drosophila ret (Halm and Bishop, 2001). The tubule cell shape changes, and the rearrangement of the cycloskeleton leads to tube elongation, which is regulated by the walrus, zipper, hibris, rib, rolling pebbles, myoblast city, and rac genes (Liu et al., 1999). Signaling from the tip cell is needed for the proliferation of the distal cells of the tubule. The four MTs are derived from two cell populations: ectodermal epithelial buds and the surrounding mesenchymal mesoderm (Sozen et al., 1997). The tubules derived from the initial ectodermal epithelium buds consist of principal cells (PC). Mesenchymal cells from the visceral mesoderm migrate and polarize along the epithelial tubules, where they undergo a mesenchymal-to-epithelial transition (MET) and incorporate into tubules as stellate cells (SC) that require hibris, an ortholog of the vertebrate NEPHRIN (Denholm et al., 2003). Several genes and signaling pathways are known to play a crucial role in the early and later developmental events underlying the formation of functional MTs (Sozen et al., 1997; Wan et al., 2000; Ainsworth et al., 2000; Sudarsan et al., 2002; Denholm et al., 2003; Dow and Davies, 2006; Hatton-Ellis et al., 2007; Evans et al., 2008).

In mammals, two kinds of stem cells, embryonic and adult, have been identified, and each has distinct functions and characteristics. Embryonic stem cells are considered as true stem cells, as they generate mature progeny of all cell types; in contrast, the adult stem cells are thought to have limited proliferation potential and can only differentiate into the mature tissue in which they reside. However, the accumulative evidence suggests that adult stem cells may have greater plasticity than previously recognized. Despite their extensive proliferative capacities, stem cells may be quiescent in vivo until injury or tissue degradation stimulates the regenerative signal. The adult stem cells may repair the damaged tissues by differentiating into appropriate cell phenotypes, by providing cytokines and other factors to enhance recovery of endogenous cells, or by undergoing cell fusion (Morris and Spradling, 2008). Adult stem cells play an important role in cell turnover and regeneration. However, several obstacles exist in the use of adult stem cells as disease therapy. First, the ability to identify most adult stem cells is hampered by the lack of stem cell markers. Second, in vitro systems for manipulating adult stem cell populations are often not well defined. Finally, our understanding of how adult stem cells are regulated within their niche is in its immature stage. Multipotent adult stem cells have been reported in many organs, such as bone marrow, skin, intestine, prostate, pancreas, lung, and testes, with unrestricted potential to form different cell types after tissue injury (Blanpain et al., 2007; Barker et al., 2008).

Adult stem cells play an important role in cell turnover and regeneration. The kidney, on the other hand, has a low rate of cellular turnover but has a great potential for tissue regeneration following an ischemic or toxic injury. Ischemic injury to the kidney causes acute renal failure, loss of tubular polarity, necrosis, and cell death, followed by tubular regeneration and recovery of renal function (Anglani et al., 2008; Gupta and Rosenberg, 2008; Yokoo et al., 2008; Vaidya et al., 2008). In humans, two broad categories of kidney disease have been characterized: acute and chronic. Acute renal failure is a leading cause of morbidity and mortality. Developmental studies reveal that the kidney has more than 26 terminally differentiated cells types, suggesting that the differentiation potential would be helpful in renal regeneration after injury. Many animal models provide evidence of regenerating completely degenerated renal tissues after injury (Elger et al., 2003; Haller et al., 2005), which can help us understand the cellular and molecular mechanisms of ischemic injury and tubular regeneration (Imai et al., 2007, Gupta and Rosenberg, 2008, Yokoo et al., 2008, Vaidya et al., 2008). There is an ongoing debate about the source of proliferating cells (stem cells) in the adult kidney that repopulate injured nephrons or regenerate the lost renal tissues. Whether the adult stem cells that repopulate the renal tubule following injury come from within the renal tubule or from extra-renal cells remain controversial. Kidney regeneration and repair occur through three possible sources of stem cells: dedifferentiation of surviving tubule cells, bone marrow–derived stem cells, or resident kidney stem cells (Fig. 1; Oliver et al., 2004; Cantley, 2005; Duffield et al., 2005; Gupta et al., 2006; Imai et al., 2007; Imberti et al. 2007; Bussolati et al., 2008; Gupta and Rosenberg, 2008; Yokoo et al., 2008; Vaidya et al., 2008). Stem cell–based renal regeneration would be critical in reducing the incidence and severity of acute renal failure and treatment of several other kidney diseases and cancer.

Several studies in preclinical models of acute and chronic kidney injury have demonstrated that cells from bone marrow (bone marrow–derived stem cells, mesenchymal stem cells) may migrate to the kidney and participate in the generation of new epithelial cells following injury (for details, see reviews, Gupta and Rosenberg, 2008; Sagrinati et al., 2008). Mesenchymal stem cells have also been found to produce several growth factors such as VEGF, HGF, IGF-1, BMP-7, and TGF-α, suggesting that, after renal injury, a paracrine effect of renal vasculature may provide regeneration and repair. Further studies have examined the role of bone marrow–derived stem cells in renal regeneration, with different results (Gupta and Rosenberg, 2008; Sagrinati et al., 2008). Moreover, several recent studies have shown that tubular cell replacement with bone marrow–derived stem cells occurs less frequently than previously recognized, suggesting that these cells may not represent a major tool in cell therapy after tubular injury and that regenerative cells originate from intra-renal cells (Lin et al., 2005). A recent study even suggests that treatment of renal failure with bone marrow stem cells can be offset by a partial maldifferentiation of bone marrow stem cells into adipocytes, resulting in glomerular sclerosis (Kunter et al., 2007). Thus, kidney-specific stem cells may be better for tissue replacement because of their inherent organ-specific identity, which can reduce the risk of maldifferentiation. Several other studies suggest that renal tubular injury may be repaired by the less damaged cells, which can migrate, proliferate, and ultimately repopulate into normal renal tubules by the dedifferentiation (Lin et al., 2005; Humpherys et al., 2008).

Accumulative evidence suggests that the repair of kidney injuries is predominately regulated by different endogenous renal stem cells (Table 1). However, the location and behavior of renal stem cells remain controversial. Adult kidney stem cells have been isolated using four different selection strategies that have been used to successfully isolate stem cells from other organs (Gupta and Rosenberg, 2008). First, because stem cells are slow-cycling cells and they can retain the 5-bromo-2-deoxyuridine (BrdU) for a long period of time. By BrdU labeling it has been shown that label-retaining cells function as a source of regenerating cells in adult kidney (Maeshima et al., 2003). These cells retain for long period of time at the interstitial cells of renal papilla, and are multipotent in vitro (Oliver et al., 2004). However, a recent study used the genetic fate mapping technique in mice to exclude the presence of stem cells in the interstitial renal cells, and the results indicate that surviving tubule epithelial cells are the predominant mechanism of adult mammalian kidney repair after ischemic injury (Humphreys et al., 2008). Second, isolating side-population (SP) cells because these cells extrude Hoechst dye through the activity of multidrug resistance proteins of ATP-binding cassette transporter superfamily (Gupta and Rosenberg, 2008). SP cells (Hoechst low cells) isolated from many different organs are multipotent stem cells (Challen and Littlle, 2006) Stem cells are also found in the side population (SP) cells of the adult kidney with multilineage differential potential (Iwatani et al., 2004; Challen et al., 2006). Third, identify and isolate kidney stem cells using specific cell surface markers that have been used to identify stem cells in other organs including kidney. Gupta et al. (2006) identified multipotent renal stem cells in the proximal tubules of the rat kidney and showed that these cells express several markers, including CD90, Oct4, and Pax2. Recently, multipotent renal stem cells have been identified in the adult human kidney in a subset of parietal epithelial cells located in Bowman’s capsule (Bussolati et al., 2005; Sagrinati et al., 2006). These progenitor cells have the ability to self-renew and differentiate into several cell types in the kidney, and are characterized by the expression of stem cell markers, such as CD24, CD133, SDF-1, CXCR4, and CXCR7 (Sagrinati et al., 2006; Mazzinghi et al., 2008). These findings in the adult human kidney suggest that CD24, CD133, SDF-1, CXCR4, and CXCR7 play an essential role in the therapeutic homing of human renal progenitor cells in acute renal failure, with important implications for the development of stem cell–based therapies for renal injury (Mazzinghi et al., 2008).

Cancer is a leading cause of death worldwide. Cancer begins when a group of cells display uncontrolled growth, invasion and sometimes metastasis. The malignant tumors develop through the accumulation of genetic changes in proliferating cells, such as the activation of oncogenes, dysfunction of stability genes, or inactivation of tumor-suppressor genes. Kidney cancer is a combination of different types of cancer, each with a different histology, requiring different clinical courses and responding to different forms of therapy, and each associated with the alteration of a different gene. Renal cell carcinoma (RCC) is the most common type of kidney cancer, accounting for more than 90% of malignant kidney tumors. RCC originates primarily in proximal renal tubules and, rarely, in collecting ducts (Fig. 4). Like most cancers, RCC is difficult to treat once it has metastasized. The five human genes associated with predisposition to RCC are von Hippel-Lindau (VHL; clear cell RCC); met proto-oncogene (c-MET; papillary RCC); fumarate hydratase (FH; papillary RCC); Birt-Hogg-Dubé (BHD/FLCN-chromophore, oncocytomas, clear cell); and hyperparathyroidism 2 (HRPT2; papillary RCC) (Fig. 4). RCC could develop following chronic renal regeneration and repair in individuals with polycystic kidney disease or in renal allograft. A detailed investigation of kidney neoplasms suggests that some RCCs, such as Wilms’ tumor (WT) and hereditary papillary renal carcinoma, are caused by mutations in the genes involved in normal nephrogenesis. WT is a pediatric kidney cancer that arises from multipotent embryonic renal precursors of the metanepric blastema that fail to differentiate. Some of the neoplasms are caused by mutations in genes expressed during normal development; for example, RCC is associated with the tuberous sclerosis complex (TSC) gene, and clear cell renal carcinoma with the VHL gene. The majority of RCCs develop in abnormalities of renal epithelial cells, and only a mutation in TSC causes abnormalities in both mesenchymal cells and epithelial cells (Fig. 4; Henske, 2005; Pfaffenroth and Linehan, 2008)

Studies of the genetic conditions associated with RCC, such as VHL, BHD, and MET, as well as genetic analyses of the tumors have provided considerable insight into the pathogenesis of these lesions. Although RCC is resistant to chemotherapy, kinase inhibitors and interleukin-2 are used to treat advanced RCC. However, because of the side effects, these therapies are not effective. Cancer is considered a stem cell disease because of both its propagation by a minority of cells with stem-cell-like properties (termed cancer stem cells, or CSCs) and its possible derivation from normal-tissue stem cells (Sneddon and Werb, 2008). Furthermore, overlapping sets of molecules and pathways regulate both stem cell migration and cancer metastasis. Normally, most adult stem cells reside in a quiescent state. However, the effect of repeated injury (as in chronic kidney injury, for example) would, over time, increase the pool size of stem cells in an active state of renewal and increase cancer incidence. The presence of more stem cells in a tissue may enhance the possibility of a stem cell being trapped in the activated state by an oncogenic event (Beachy et al., 2004).

Abnormal functioning of signaling pathways is believed to contribute to the pathogenesis of many malignancies and is particularly relevant to renal cancers. To understand the cancer stem cells in tumors, we need to know first what signaling pathways and pathway interactions regulate cancer formation. It has been shown that TSC complex (hamartin/tuberin [TSC1/TSC2]) regulates early renal progenitor cells because individuals with TSC mutations have an increased incidence of RCC. TSC mutations activate the mammalian target of rapamycin (mTOR) by inhibiting the Rheb (ras homolog enriched in brain) and biochemically resemble VHL phenotypes. In addition, mTOR has been shown to regulate hypoxia inducible factor (HIF) activity, and much evidence implicates mTOR as a valid target for treatment of renal cell carcinoma. Epidermal growth factor receptor (EGFR) activates pI3K and downstream targets including AKT (protein kinase B) and mTOR, resulting in increased HIF-1 expression leading to tumor metastasis. The c-MET proto-oncogene, expressed in both stem and cancer cells, is a key regulator of invasive growth in normal conditions because it binds with HGF to induce receptor dimerization and phosphorylation, and interacts with several other intracellular factors, including the ras oncogene–mitogen-activated protein kinase (RAS-MAPK) and protein kinase B (AKT) pathways. VHL, a tumor-suppressor gene, plays a role in the regulation of tumor angiogenesis by targeting the hypoxia-inducible growth factor-1 (HIF-1) for ubiquitin-mediated degradation. HIF-1 induces the dedifferentiation of cancer cells, maintains stem cell identity, and increases the metastatic potential (Kondo et al., 2003). VHL interacts with HIF-1, resulting in the loss of VHL and overproduction of the HIF-1, which contributes to the development RCC. These findings suggest that HIF-1 is downstream of VHL and are able to treat RCC caused by VHL. Further, genetic inactivation of VHL prevents HIF-1 down-regulation, leading to the expression of the c-MET proto-oncogene, an important regulator of invasion and metastasis. VHL and glycogen synthase kinase 3 (GSK3) function together in a ciliary-maintenance signaling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation. Recently it has been reported that PTEN (phosphatase and tensin homolog) tumor-suppressor protein cooperates with VHL to regulate kidney tumorigenesis (Frew et al., 2008). STRA13 is a cancer-associated protein regulated by VHL and HIF-1 pathway and is overexpressed in many common malignancies. VHL deficiency or HIF-1 activation results in the repression of endogenous STAT1, which possesses tumor-suppressor properties and is mediated by STRA13 (Ivanov et al., 2007). BHD is another dominantly inherited hamartoma syndrome (Schmidt et al., 2005) that shares several features with TSC; mutation in both genes causes renal carcinoma, which suggests that the BHD and TSC proteins may function within a common pathway. However, clinical phenotypes and the risk of malignancy are higher in BHD than in TSC. Recently, we demonstrated that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and differentiate into RCs, which results in tumorous overgrowth in the MT (Singh et al., 2007). Previously, we have reported that BHD interacts with JAK-STAT signaling and regulates germline stem cell in the Drosophila testis and functions downstream of JAK-STAT signaling pathways (Singh et al., 2006). Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study cancer stem cell regulation in renal tubules. The genetic interactions of different oncogenes and tumor-suppressor genes regulating renal carcinoma and other kidney cancers are summarized in Fig. 5. A key goal in clinical oncology is the development of medical therapies specific to pathways that are misregulated in cancer. Understanding the biological pathways involving VHL, MET, FH, BHD, and HRPT2 will provide new therapeutic approaches for kidney cancer.

Using genetic-labeling techniques and molecular markers, we have identified many multipotent stem cells in the MTs of the adult Drosophila. These renal stem cells have the ability to generate all cell types of the adult. Further, we found that autocrine JAK-STAT signaling regulates self-renewal or differentiation of renal stem cells. However, several important questions remain regarding the biological properties of multipotent stem cells in Drosophila renal tubules, such as how the self-renewal or differentiation is balanced in the MTs to avoid tissue overgrowth; whether their self-renewal is unlimited; what are the molecular mechanisms controlling asymmetric stem cell division: whether cell polarity, centrosomal asymmetry, and cell cycle regulators, determines asymmetric division of RNSCs, as in other stem cells system, and finally, how the tumor suppressor genes regulate stem cell proliferation and self-renewal in MTs. Answering these questions is crucial and will provide a better understanding of the mechanisms behind self-renewal, proliferation and differentiation of stem cells in general.