Adult Mammalian Kidney Research Articles

The Role of the Epidermal Growth Factor Receptor in Diabetic Kidney Disease.

The epidermal growth factor receptor (EGFR) is expressed in numerous cell types in the adult mammalian kidney and is activated by a family of EGF-like ligands. EGFR activation has been implicated in a variety of physiologic and pathophysiologic functions. There is increasing evidence that aberrant EGFR activation is a mediator of progressive kidney injury in diabetic kidney disease. This review will highlight recent studies indicating its potential role and mechanisms of injury of both glomerular and tubular cells in development and progression of diabetic kidney disease.

Aqp2+ Progenitor Cells Maintain and Repair Distal Renal Segments.

Adult progenitor cells presumably demonstrate clonogenicity, self-renewal, and multipotentiality, and can regenerate cells under various conditions. Definitive evidence demonstrating the existence of such progenitor cells in adult mammalian kidneys is lacking. We performed in vivo lineage tracing and thymidine analogue labeling using adult tamoxifen-inducible (Aqp2ECE/+ RFP/+, Aqp2ECE/+ Brainbow/+, and Aqp2ECE/+ Brainbow/Brainbow) and WT mice. The tamoxifen-inducible mice were analyzed between 1 and 300 days postinduction. Alternatively, WT and tamoxifen-induced mice were subjected to unilateral ureteral obstruction and thymidine analogue labeling and analyzed 2-14 days post-surgery. Multiple cell-specific markers were used for high-resolution immunofluorescence confocal microscopy to identify the cell types derived from Aqp2+ cells. Like their embryonic counterparts, adult cells expressing Aqp2 and V-ATPase subunits B1 and B2 (Aqp2+ B1B2+) are the potential Aqp2+ progenitor cells (APs). Adult APs rarely divide to generate daughter cells, either maintaining the property of the AP (self-renewal) or differentiating into DCT2/CNT/CD cells (multipotentiality), forming single cell-derived, multiple-cell clones (clonogenicity) during tissue maintenance. APs selectively and continuously regenerate DCT2/CNT/CD cells in response to injury resulting from ureteral ligation. AP proliferation demonstrated direct correlation with Notch activation and was inversely correlated with development of kidney fibrosis. Derivation of both intercalated and DCT2 cells was found to be cell division-dependent and -independent, most likely through AP differentiation which requires cell division and through direct conversion of APs and/or regular principal cells without cell division, respectively. Our study demonstrates that Aqp2+ B1B2+ cells behave as adult APs to maintain and repair DCT2/CNT1/CNT2/CD segments.

Adult mouse kidney stem cells orchestrate the de novo assembly of a nephron via Sirt2‐ modulated canonical Wnt/β‐catenin signaling

End‐stage renal disease affects an estimated 2.4 million people annually worldwide. With cases increasing 8% every year, regenerative strategies are needed to help alleviate, and potentially replace, the increasing demand for kidney transplants. An important question remains whether stem cells reside in the adult mammalian kidney. Here we developed a methodology for isolation of kidney stem cells from adult mouse kidney by using Sca1 and Oct4 stem cell markers. Using these cells, we generated three types of kidney‐like structures. First, we observed the assembly of self‐organizing three‐dimensional kidney organoids. These kidney organoids show characteristic of kidney structures and contain podocytes and endothelial cells that form networks of capillary loop‐like structures. We also observed the de novogeneration of a self‐organizing nephron (and its associated collecting duct) from monolayer cultures. Comma‐ and S‐shaped bodies that expressed activated Notch1 were present during this formation, suggesting that Notch signaling played a key role in driving nephrogenesis. This is consistent with our current knowledge of nephron formation in the mouse embryo. Furthermore, we observed the differentiation of monolayer cells into functionally mature tubules and self‐organizing kidney‐shaped structures that selectively endocytose dextran. Finally, we demonstrated that expression of Sirt2 was upregulated, which modulated the dynamics of canonical Wnt/β‐catenin signaling during kidney organoid development. Altogether, our observations provide the first evidence for the existence of stem cells in the adult mouse kidney. These findings offer powerful tools for future applications, including drug screening, disease modelling, cell therapy, and kidney regeneration.

Transcription factors AP-2\u03b1 and AP-2\u03b2 regulate distinct segments of the distal nephron in the mammalian kidney

Transcription factors AP-2α and AP-2β have been suggested to regulate the differentiation of nephron precursor populations towards distal nephron segments. Here, we show that in the adult mammalian kidney AP-2α is found in medullary collecting ducts, whereas AP-2β is found in distal nephron segments except for medullary collecting ducts. Inactivation of AP-2α in nephron progenitor cells does not affect mammalian nephrogenesis, whereas its inactivation in collecting ducts leads to defects in medullary collecting ducts in the adult. Heterozygosity for AP-2β in nephron progenitor cells leads to progressive distal convoluted tubule abnormalities and β-catenin/mTOR hyperactivation that is associated with renal fibrosis and cysts. Complete loss of AP-2β in nephron progenitor cells caused an absence of distal convoluted tubules, renal cysts, and fibrosis with β-catenin/mTOR hyperactivation, and early postnatal death. Thus, AP-2α and AP-2β have non-redundant distinct spatiotemporal functions in separate segments of the distal nephron in the mammalian kidney.

Epidermal growth factor deficiency predisposes to progressive renal disease.

Epidermal growth factor (EGF) is produced in the kidney by thick ascending limbs of the loop of Henle and by distal convoluted tubules (DCTs). Reduced urinary EGF levels have been associated with chronic kidney disease but it is not known whether physiological levels of EGF protect the kidney from progressive renal disease. Here, we show that EGF-deficient mice on a mixed genetic background had increased urinary microalbumin, and a subset of these mice developed severe progressive renal disease with azotemia that was not seen in WT or TGFα-deficient littermates with this mixed genetic background. These azotemic EGF-deficient mice developed crescentic glomerulonephritis linked to HB-EGF/EGFR hyperactivation in glomeruli, as well as attenuation of the proximal tubule brush border, distal convoluted tubule (DCT) dilatation, and kidney fibrosis associated with renal β-catenin/mTOR hyperactivation. The observation of these severe renal pathologies only in a subset of EGF-deficient mice suggests that independent segregation of strain-specific modifier alleles contributes to the severity of the renal abnormalities that only manifest when EGF is lacking. These findings link the lack of EGF to renal pathologies in the adult mammalian kidney, in support of a role of physiological levels of EGF for maintaining the function of glomeruli, proximal tubules, and DCTs. These observations suggest that diminished EGF levels predispose kidneys to progressive renal disease.

Adult Mouse Kidney Stem Cells Orchestrate the De Novo Assembly of a Nephron via Sirt2-Modulated Canonical Wnt/β-Catenin Signaling.

Generation of kidney organoids using autologous kidney stem cells represents an attractive strategy for treating and potentially replacing the failing kidneys. However, whether adult mammalian kidney stem cells have regenerative capacity remains unknown. Here, previously unidentified adult kidney Sca1+ Oct4+ stem/progenitor cells are isolated. Interestingly, culturing these cells leads to generation of kidney‐like structures. First, the assembly of self‐organizing 3D kidney‐like structures is observed. These kidney organoids contain podocytes, proximal tubules, and endothelial cells that form networks of capillary loop‐like structures. Second, the differentiation of kidney stem cells into functionally mature tubules and self‐organizing kidney‐shaped structures in monolayer culture that selectively endocytoses dextran, is shown. Finally, the de novo generation of an entire self‐organizing nephron from monolayer cultures is observed. Mechanistically, it is demonstrated that Sirt2‐mediated canonical Wnt/β‐catenin signaling is critical for the development of kidney organoids. Thus, the first evidence is provided that the adult mouse kidney stem cells are capable of de novo generating kidney organoids.

Embryonic Kidney Development, Stem Cells and the Origin of Wilms Tumor.

The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature kidney, the embryonic kidney hosts at least three types of lineage-specific stem cells that give rise to (a) a ureter and collecting duct system, (b) nephrons, and (c) mesangial cells together with connective tissue of the stroma. Extensive interest has been raised towards these embryonic progenitor cells, which are normally lost before birth in humans but remain part of the undifferentiated nephrogenic rests in the pediatric renal cancer Wilms tumor. Here, we discuss the current understanding of kidney-specific embryonic progenitor regulation in the innate environment of the developing kidney and the types of disruptions in their balanced regulation that lead to the formation of Wilms tumor.

Differential role of planar cell polarity gene Vangl2 in embryonic and adult mammalian kidneys.

Planar cell polarity (PCP) pathway is crucial for tissue morphogenesis. Mutations in PCP genes cause multi-organ anomalies including dysplastic kidneys. Defective PCP signaling was postulated to contribute to cystogenesis in polycystic kidney disease. This work was undertaken to elucidate the role of the key PCP gene, Vangl2, in embryonic and postnatal renal tubules and ascertain whether its loss contributes to cyst formation and defective tubular function in mature animals. We generated mice with ubiquitous and collecting duct-restricted excision of Vangl2. We analyzed renal tubules in mutant and control mice at embryonic day E17.5 and postnatal days P1, P7, P30, P90, 6- and 9-month old animals. The collecting duct functions were analyzed in young and adult mutant and control mice. Loss of Vangl2 leads to profound tubular dilatation and microcysts in embryonic kidneys. Mechanistically, these abnormalities are caused by defective convergent extension (larger tubular cross-sectional area) and apical constriction (cuboidal cell shape and a reduction of activated actomyosin at the luminal surface). However, the embryonic tubule defects were rapidly resolved by Vangl2-independent mechanisms after birth. Normal collecting duct architecture and functions were found in young and mature animals. During embryogenesis, Vangl2 controls tubular size via convergent extension and apical constriction. However, rapidly after birth, PCP-dependent control of tubular size is switched to a PCP-independent regulatory mechanism. We conclude that loss of the Vangl2 gene is dispensable for tubular elongation and maintenance postnatally. It does not lead to cyst formation and is unlikely to contribute to polycystic kidney disease.

Graft immaturity and safety concerns in transplanted human kidney organoids

For chronic kidney disease, regeneration of lost nephrons with human kidney organoids derived from induced pluripotent stem (iPS) cells is proposed to be an attractive potential therapeutic option. It remains unclear, however, whether organoids transplanted into kidneys in vivo would be safe or functional. Here, we purified kidney organoids and transplanted them beneath the kidney capsules of immunodeficient mice to test their safety and maturity. Kidney organoid grafts survived for months after transplantation and became vascularized from host mouse endothelial cells. Nephron-like structures in grafts appeared more mature than kidney organoids in vitro, but remained immature compared with the neighboring mouse kidney tissue. Ultrastructural analysis revealed filtration barrier-like structures, capillary lumens, and tubules with brush border in the transplanted kidney organoids, which were more mature than those of the kidney organoids in vitro but not as organized as adult mammalian kidneys. Immaturity was a common feature of three separate differentiation protocols by immunofluorescence analysis and single cell RNA sequencing. Stroma of transplanted kidney organoid grafts were filled with vimentin-positive mesenchymal cells, and chondrogenesis, cystogenesis, and stromal expansion were observed in the long term. Transcription profiles showed that long-term maintenance after kidney organoid transplantation induced transcriptomic reprogramming with prominent suppression of cell-cycle-related genes and upregulation of extracellular matrix organization. Our data suggest that kidney organoids derived from iPS cells may be transplantable but strategies to improve nephron differentiation and purity are required before they can be applied in humans as a therapeutic option.

Bayesian inference of agent-based models: a tool for studying kidney branching morphogenesis

The adult mammalian kidney has a complex, highly-branched collecting duct epithelium that arises as a ureteric bud sidebranch from an epithelial tube known as the nephric duct. Subsequent branching of the ureteric bud to form the collecting duct tree is regulated by subcellular interactions between the epithelium and a population of mesenchymal cells that surround the tips of outgrowing branches. The mesenchymal cells produce glial cell-line derived neurotrophic factor (GDNF), that binds with RET receptors on the surface of the epithelial cells to stimulate several subcellular pathways in the epithelium. Such interactions are known to be a prerequisite for normal branching development, although competing theories exist for their role in morphogenesis. Here we introduce the first agent-based model of ex vivo kidney uretic branching. Through comparison with experimental data, we show that growth factor-regulated growth mechanisms can explain early epithelial cell branching, but only if epithelial cell division depends in a switch-like way on the local growth factor concentration; cell division occurring only if the driving growth factor level exceeds a threshold. We also show how a recently-developed method, “Approximate Approximate Bayesian Computation”, can be used to infer key model parameters, and reveal the dependency between the parameters controlling a growth factor-dependent growth switch. These results are consistent with a requirement for signals controlling proliferation and chemotaxis, both of which are previously identified roles for GDNF.

A Subpopulation of Label-Retaining Cells of the Kidney Papilla Regenerates Injured Kidney Medullary Tubules.

SummaryTo determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools.

In Vivo Clonal Analysis Reveals Lineage-Restricted Progenitor Characteristics in Mammalian Kidney Development, Maintenance, and Regeneration

The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

Of mice and men: the riddle of tubular regeneration

Regeneration can occur through multiple distinct mechanisms, such as pluripotent stem cells, lineage-committed progenitors or dedifferentiation. The respective contribution of each of these regenerative strategies in every organ or tissue may be different. Recent results indicate that dedifferentiation contributes less than previously thought, and that stem or progenitor cells seem to be the main drivers of regenerative processes. Our views of regeneration in the kidney are undergoing the same process of revision. Indeed, studies in humans have established the existence of a scattered population of tubular progenitors in the adult kidney. Renal progenitors have been discovered also in other animal classes such as fish and insects. In contrast, in rodents a tubular progenitor phenotype seems to be induced only after tubular injury, suggesting some differences may exist. Is this difference really related to a distinct regenerative strategy or is it simply a matter of the type and modality of cellular markers expressed? It may also be possible that progenitor cells, as well as tubular cell dedifferentiation, act in concert to allow regeneration of a complex organ like the adult mammalian kidney, as recently proposed also for the liver. Further studies are needed to resolve the riddle of tubular regeneration. However, beyond the controversial results obtained from humans and rodents, identification of tubular progenitors in humans can move the field forward and provide a novel perspective for understanding tubular regeneration.

Assessing cardio‐renal function in zebrafish larvae

The larva of the Zebrafish (Danio rerio) has become a leading model in various fields of physiology (e.g. Panula, 2010; Leong et al. 2010) and in developmental physiology (Grunwald & Eisen, 2002; Zetterberg, 2011). It is now possible to track gene expression with imaging methods and thereby gain detailed understanding of organ function in an intact vertebrate. In a recent issue of The Journal of Physiology, Rider et al. (2012) demonstrate this physiological application to better assess renal function. Previous measures of renal clearance in the zebrafish larvae have been made in the absence of flow changes and disregarding trapped fluorescence (Hentschel et al. 2007). In these new studies assays are established to determine renal clearance based on cardiovascular erythrocyte flow and extinction of injected fluorescein isothiocyanate (FITC)-labelled inulin. The method was evaluated in two models of kidney damage: using the nephrotoxic gentamicin or by giving a salt challenge. No matter how well we are acquainted with graphed or tabulated data, imaging is often more convincing. Seeing is believing. However, invasive visualization perturbs the steady-state and it is often not possible to image physiological processes over extended time periods. The zebrafish model provides a way around these limitations because the transparency of the embryo allows long-term monitoring. The development of the zebrafish embryo is established and the specific development of the kidney and the vasculature are well known (Kimmel et al. 1995; Ackermann & Paw, 2003). The zebrafish kidney is genetically and morphologically related to that of mammals, although the pronephric kidney of zebrafish larvae consist of only a fused glomerulus with two nephrons (Drummond & Davidson, 2010). The metanephros is the most complex kind of kidney, and is found only in birds and mammals; it develops out of the simpler pronephric and mesonephric kidneys. Although zebrafish lack the metanephros, they have pronephric (embryonic) and mesonephric (adult) kidneys. Three and half days after fertilization, the pronephros is functional and exhibits many features of the metanephros. On the other hand, important features of adult mammalian kidney function are not seen in the zebrafish pronephros. For instance, as described in the highlighted article by Rider et al., renal blood flow is not uncoupled from systemic blood flow. Thus, renal autoregulation, a key feature of renal haemodynamics, is not functional. Renal filtration is the surrogate parameter used for overall renal function. The gold standard for assessing glomerular filtration rate (GFR) is the inulin method. Inulin is a polysaccharide which is freely filtered through the glomerular filtration barrier into the tubules, where it cannot be reabsorbed or secreted. Therefore, provided the urine flow rate can be assessed, the clearance of inulin can be taken as a measure for GFR. Unfortunately, zebrafish urine flow rate is not yet assessable, but two measures were used by Rider et al. for determining renal clearance in the mature pronephric kidney: FITC intensity changes in the caudal region of the larvae and, reciprocally, the excretion of FITC inulin in water, determined over a 4 h period. The values of FITC intensity change and venous erythrocyte velocity were compared to the development of oedema in the yolk sac and pericardium. In this study it was also shown that renal clearance does not adjust in response to salt loading. This is different from the mature human kidney, where high salt decreases GFR via tubuloglomerular feedback (Schnermann et al. 1976). These new techniques for measuring renal clearance in the zebrafish are milestones in the study of the development of kidney haemodynamics. Before these techniques became available, the zebrafish renal model was restricted to more morphological studies, of, for example, cilia and the filtration barrier (Hentschel et al. 2007; Swanhart et al. 2011). Even determining GFR may become possible, once a method is established to determine zebrafish urine flow rate.

Hox genes and kidney development

The adult mammalian kidney is generated by the differentiation and integration of several distinct cell types, including the nephrogenic mesenchyme, ureteric epithelium, stromal and endothelial cells. How and where these cell types are generated and what signals lead to their differentiation and integration into a functional organ system is a main focus of current studies. Herein, we review the formation of distinct cell types within the adult mammalian kidney; what is understood regarding their origin and the signaling pathways that lead to their formation and integration; morphogenetic changes the metanephric kidney undergoes during development; and what is known regarding the role of Hox genes in these processes.

Uncharted waters: nephrogenesis and renal regeneration in fish and mammals

The major functions of the vertebrate kidney are the removal of metabolic waste and the balance of salt and water. These roles are fulfilled by nephrons, which generally comprise a blood filter (glomerulus) attached to an epithelial tubule. The number of nephrons in the mammalian kidney is set at the end of kidney organogenesis in the late fetal or neonatal period. Subsequent increases in nephron size and functionality then occur during postnatal growth to match increases in body mass/fluid. Because of this strategy of renal development, injuries or birth defects that reduce nephron number lead to a permanent nephron deficit and increase the risk of kidney disease. In contrast to mammals, fish kidneys continue to add nephrons throughout their lifespan. In response to renal injury, fish increase the rate of nephrogenesis, effectively replacing lost nephrons and maintaining their nephron endowment. A better understanding of the remarkable nephrogenic abilities of fish kidneys may lead to innovative ways to restore nephrogenesis in the adult mammalian kidney. This review examines our current understanding of nephrogenesis in mammals and fish and explores possible explanations for why fish, but not mammals, utilize a perpetual nephrogenesis strategy to grow and maintain their kidneys.

Impaired Wnt–β-catenin signaling disrupts adult renal homeostasis and leads to cystic kidney ciliopathy

Cystic kidney disease represents a major cause of end-stage renal disease, yet the molecular mechanisms of pathogenesis remain largely unclear. Recent emphasis has been placed on a potential role for canonical Wnt signaling, but investigation of this pathway in adult renal homeostasis is lacking. Here we provide evidence of a previously unidentified canonical Wnt activity in adult mammalian kidney homeostasis, the loss of which leads to cystic kidney disease. Loss of the Jouberin (Jbn) protein in mouse leads to the cystic kidney disease nephronophthisis, owing to an unexpected decrease in endogenous Wnt activity. Jbn interacts with and facilitates beta-catenin nuclear accumulation, resulting in positive modulation of downstream transcription. Finally, we show that Jbn is required in vivo for a Wnt response to injury and renal tubule repair, the absence of which triggers cystogenesis.

CBP and p300 in renin homeostasis: can they drive the fate?

CBP and p300 in renin homeostasis: can they drive the fate?

BMP-7 protects mesangial cells from injury by polymeric IgA

Bone morphogenetic protein-7 (BMP-7) is a potential therapeutic agent for acute and chronic renal diseases. Here we found that addition of polymeric IgA, isolated from patients with IgA nephropathy, increased the synthesis of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and fibronectin in cultured human mesangial cells, effects blunted by BMP-7. When mesangial cells were cultured with both polymeric IgA and BMP-7 there was an increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The activation of NF kappaB and TNF-alpha synthesis induced by polymeric IgA or TNF-alpha were downregulated by BMP-7 or rosiglitazone. BMP-7 inhibited TNF-alpha release from polymeric IgA-stimulated mesangial cells by activation of PPAR-gamma but suppressed TGF-beta release by mechanisms independent of PPAR-gamma. The expression of inhibitory Smad6 and 7 was increased whereas the expression of active Smad2 and 3 was reduced in these mesangial cells by BMP-7. Our study shows that BMP-7 ameliorates IgA nephropathy-derived polymeric IgA-induced TNF-alpha and TGF-beta synthesis in human mesangial cells through multiple mechanisms involving inhibitory Smads and PPAR-gamma.

Intrinsic Epithelial Cells Repair the Kidney after Injury

Understanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%-95% of tubular epithelial cells, but no interstitial cells, were labeled with either beta-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney.