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During the past decades, remarkable progress has been made in our understanding of the molecular basis of kidney diseases, as well as in the ability to pinpoint disease-causing genetic changes. Congenital anomalies of the kidney and urinary tract (CAKUT) are remarkably diverse, and may be either isolated to the kidney or involve other systems, and are notorious in their variable genotype-phenotype correlations. Genetic conditions underlying CAKUT are individually rare, but collectively contribute to disease etiology in ~ 16% of children with CAKUT. In this review, we will discuss basic concepts of kidney development and genetics, common causes of monogenic CAKUT, and the approach to diagnosing and managing a patient with suspected monogenic CAKUT. Altogether, the concepts presented herein represent an introduction to the emergence of nephrogenetics, a fast-growing multi-disciplinary field that is focused on deciphering the causes and manifestations of genetic kidney diseases as well as providing the framework for managing patients with genetic forms of CAKUT.

We aimed to describe the experience of a large single-center cohort for the clinical, radiological, and genetic characteristics, as well as to determine the efficacy of different anti-epileptic strategies in children and adults with tuberous sclerosis complex (TSC). We carried out a historical cohort study on 91 TSC patients treated in a single center between 2008 and 2018. Our cohort comprised 46 males and 45 females, with a median age of 15.6 years at the last follow-up. Mean follow-up time was 2.5 ± 0.75–5.5 years (range 0–9.5 years). Of those tested, a disease-causing mutation was identified in 90% of patients, 53% in TSC2, and 37% in TSC1. Epilepsy prevalence was similar among TSC1 and TSC2 mutated patients. The most common radiological finding were cortical tubers in 95% of patients, while subependymal giant cell astrocytoma (SEGA) were detected in 36% of patients. Notably, infantile spasms (IS) were diagnosed in 29%, with SEGA representing the only finding significantly different in prevalence between those with and without IS (62% vs. 28%, respectively, p = 0.009). Lastly, we did not find any difference in efficacy between three anti-epileptic treatments: Vagus nerve stimulation (VNS), CBD-based products, and the ketogenic diet, all showing approximately 30%–40% response rates. Altogether, we provide a comprehensive description of our experience in treating TSC, which could serve to expand current knowledge of the disease and its treatments.

OBJECTIVE The Klotho protein family plays important roles in several metabolic pathways. Soluble Klotho has been recently put forward as an antiaging protein, demonstrating renal and cardiovascular protective traits. Cardiopulmonary bypass (CPB) support during cardiac surgery has been implicated in several adverse outcomes in pediatric and adult patients. Our goal was to assess whether serum Klotho levels can be used to predict outcomes in children undergoing cardiac surgery with CPB due to congenital heart defects (CHDs). METHODS This prospective study was conducted on pediatric patients admitted to two Pediatric Cardiac Intensive Care Units, between 2012 and 2018. All patients were born with CHD and underwent corrective surgery with CPB. Sequential blood samples were analyzed by enzyme-linked immunosorbent assay for soluble Klotho levels at baseline, 2, 6, and 24 h after surgery. The association between Klotho levels and several demographic, intraoperative, and postoperative clinical and laboratory parameters was studied. RESULTS Twenty-nine children undergoing cardiac surgery with CPB support were included. Serum Klotho levels were shown to significantly decrease 2 h after surgery and increase to baseline levels after 6 h (p

Chronological and biological age correlate with DNA methylation levels at specific sites in the genome. Linear combinations of multiple methylation sites, termed epigenetic clocks, can inform us of the chronological age and predict multiple health-related outcomes. However, why some sites correlate with lifespan, healthspan, or specific medical conditions remains poorly understood. Kidney fibrosis is the common pathway for Chronic Kidney Disease, which affects 10% of Europe and USA population. Here we identify epigenetic clocks and methylation sites that correlate with kidney function. Moreover, we identify methylation sites that have a unique methylation signature in the kidney. Methylation levels in the majority of these sites correlates with kidney state and function. When kidney function deteriorates, all of these sites regress towards the common methylation pattern observed in other tissues. Interestingly, while the majority of sites are less methylated in the kidney and become more methylated with loss of function, a fraction of the sites are highly methylated in the kidney and become less methylated when kidney function declines. These methylation sites are enriched for specific transcription-factor binding sites. In a large subset of sites, changes in methylation pattern are accompanied by changes in gene expression in kidneys of chronic kidney disease patients. These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. However, this information loss is not random, but guided towards a baseline that is dependent on the genomic loci.

We investigated the risk of kidney injury among adolescents with and without a congenital single functioning kidney (SFK). This retrospective study is based on a medical evaluation database of 17-year-old Israeli conscripts, born during 1989–1999. Those with congenital SFK diagnosis, verified by a pediatric nephrologist’s review of the original military medical committee classifications, were compared to the rest of the cohort. Kidney injury (KI) was defined as proteinuria, high blood pressure (BP), or estimated glomerular filtration rate (eGFR)

In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 106 cells, and analyzed their characteristics using transcriptomics. A comparison between clonal cultures revealed restriction to either proximal or distal kidney sub-lineages with distinct cellular and molecular characteristics; rapidly amplifying de-differentiated clones and a stably proliferating cuboidal epithelial-appearing clones, respectively. Furthermore, each showed distinct molecular features including cell-cycle, epithelial-mesenchymal transition, oxidative phosphorylation, BMP signaling pathway and cell surface markers. In addition, analysis of clonal versus bulk cultures show early clones to be more quiescent, with elevated expression of renal developmental genes and overall reduction in renal identity markers, but with an overlapping expression of nephron segment identifiers and multiple identity. Thus, ex-vivo clonal growth mimics the in-vivo situation displaying lineage-restricted precursor characteristics of mature renal cells. These data suggest that for reconstruction of varying renal lineages with human adult kidney based organoid technology and kidney regeneration ex-vivo, use of multiple heterogeneous precursors is warranted.

Background Increasing cancer incidence among children alongside improved treatments has resulted in a growing number of pediatric cancer survivors. Despite childhood cancer survivors' exposure to various factors that compromise kidney function, few studies have investigated the association between childhood cancer and future kidney disease. Methods To assess the risk of ESKD among childhood cancer survivors, we conducted a nationwide, population-based, retrospective cohort study that encompassed all Israeli adolescents evaluated for mandatory military service from 1967 to 1997. After obtaining detailed histories, we divided the cohort into three groups: participants without a history of tumors, those with a history of a benign tumor (nonmalignant tumor with functional impairment), and those with a history of malignancy (excluding kidney cancer). This database was linked to the Israeli ESKD registry to identify incident ESKD cases. We used Cox proportional hazards models to estimate the hazard ratio (HR) of ESKD. Results Of the 1,468,600 participants in the cohort, 1,444,345 had no history of tumors, 23,282 had a history of a benign tumor, and 973 had a history of malignancy. During a mean follow-up of 30.3 years, 2416 (0.2%) participants without a history of tumors developed ESKD. Although a history of benign tumors was not associated with an increased ESKD risk, participants with a history of malignancy exhibited a substantially elevated risk for ESKD compared with participants lacking a history of tumors, after controlling for age, sex, enrollment period, and paternal origin (adjusted HR, 3.2; 95% confidence interval, 1.3 to 7.7). Conclusions Childhood cancer is associated with an increased risk for ESKD, suggesting the need for tighter and longer nephrological follow-up.

Pyelonephritis is the most common serious bacterial infection during childhood. The long-term importance of kidney scarring is unclear. To assess the risk of end-stage kidney disease (ESKD) in adolescents and young adults with history of pyelonephritis. A nationwide, population-based, historical cohort study, including 1,509,902 persons (62% male) examined for military service between 1967 and 1997. Participants with a history of pyelonephritis were sub-grouped according to presence of kidney scarring and baseline kidney function. Data were linked to the Israeli ESKD registry to identify incident ESKD cases. Cox proportional hazards models were used to estimate the hazard ratio (HR) of treated ESKD (dialysis or kidney transplant). Pyelonephritis was diagnosed in 6979 participants (0.46%). 6479 had normal kidney function and no evidence of kidney scarring, 400 had normal kidney function with evidence of scarring, and 100 demonstrated reduced baseline kidney function. Treated ESKD developed in 2352 individuals (0.2%) without history of pyelonephritis, 58 individuals (0.9%) with normal kidney function, history of pyelonephritis and no kidney scarring, 14 individuals (3.5%) with normal kidney function, history of pyelonephritis and kidney scarring, and 23 individuals (23.0%) with history of pyelonephritis and reduced baseline kidney function, yielding HR of 3.3, 34.8 and 43.2, respectively, controlling for age, gender, paternal origin, enrollment year, body mass index, and blood pressure, and accounting for death as a competing risk. History of pyelonephritis was associated with significantly increased risk of treated ESKD, particularly when associated with kidney scarring or reduced baseline kidney function.

Diabetic kidney disease (DKD) is becoming increasingly common among children. We aimed to estimate the risk of end-stage renal disease (ESKD) and mortality among adolescents with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) and normal renal function compared with non-diabetics. We hypothesized that childhood onset T1DM vs. T2DM would be associated with a different risk profile for developing ESKD and its complications. A nationwide, population-based, retrospective cohort study, including 1,500,522 adolescents examined for military service between 1967 and 1997, which were classified according to the presence and type of diabetes. Data were linked to the Israeli ESKD registry. Cox proportional-hazards models were used to estimate the hazard ratio (HR) for ESKD. At study enrolment, 1183 adolescents had T1DM and 196 had T2DM. ESKD developed in 2386 non-diabetic individuals (0.2%) compared with 72 individuals (6.1%) with T1DM and 8 individuals (4.1%) with T2DM. Participants with T1DM were younger at ESKD onset than participants with T2DM (median age, 36.0 vs. 40.5 years, P < 0.05). In a multivariate model adjusted for age, sex, paternal origin, enrollment year, BMI, and blood pressure, T1DM and T2DM were associated with HR of 36.4 (95% CI 28.3–46.9) and 19.3 (95% CI 9.6–38.8) for ESKD, respectively. Stratification according to sex, ethnicity, immigration, and socioeconomic status did not materially change the HR. During the follow-up period, mortality rates were higher in T2DM as compared with T1DM and controls (8.7 %, 2.2%, and 2.7% respectively). T1DM and T2DM in adolescents with normal renal function confer a significantly increased risk for ESKD. T1DM is associated with younger age at ESKD onset while T2DM is associated with higher mortality rate.

Background Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks. Methods We administered renal tubule-forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of "renovascular units" comprising both vessels and tubules with potential interaction. Results Directly injecting vessel-forming cells and renal tubule-forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule-forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal-epithelial transition in renal tubule-forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule-forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo. Conclusions Combined cell therapy of vessel-forming cells and renal tubule-forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.

Over the past few decades, there have been tremendous advancements in the field of nephrology due to developments in genetics and molecular biology, such as the ability to pinpoint the causative mutations in congenital syndromes involving the kidneys, animal models of kidney disease and an array of tools for manipulating nucleic acids. However, despite these achievements, in most cases, these sophisticated technologies have yet to translate into improved outcomes. Thus, there are still several important challenges in the field of pediatric nephrology, the most important of which are reviewed herein. These include: 1. Better understanding of the association between a specific genotype and disease phenotype in congenital anomalies of the kidney and urinary tract, and development of effective treatments for these anomalies. 2. Deeper understanding of the pathophysiology of genetic kidney diseases. 3. Application of the available molecular tools for the purpose of genetic treatments of congenital kidney disease. 4. Uncovering the underlying mechanisms of renal fibrosis and establishment of effective means of halting/preventing it. Advancements in any of these areas have a great potential to influence the prognosis of children with kidney diseases, and considering the fast pace in which new knowledge is acquired and technologies are developed, it is expected that at least some of these challenges will be met in the foreseeable future.

Background Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. Methods Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. Results We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. Conclusions SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

We investigated the risk of kidney injury among adolescents with and without a congenital single functioning kidney (SFK).This retrospective study is based on a medical evaluation database of 17-year-old Israeli conscripts, born during 1989-1999. Those with congenital SFK diagnosis, verified by a pediatric nephrologist's review of the original military medical committee classifications, were compared to the rest of the cohort. Kidney injury (KI) was defined as proteinuria, high blood pressure (BP), or estimated glomerular filtration rate (eGFR) 90 ml/min/1.73 mOf 979,630 screened candidates, 353 were diagnosed with SFK. The yearly incidence of SFK gradually increased in the first years of the study, reaching a plateau in 1995 (5.5 ± 1.2/10,000 births/year). The male to female ratio was 2.7:1. Concomitant genital malformations were documented in 5.5% of those with SFK. KI was more prevalent in the SFK than the control group (42.2% vs. 23.5%, p 0.001). All three components of KI were more common in the SFK than the control group: high BP (31.7% vs. 23.1%, p 0.001), proteinuria (18.2% vs. 0.4%, p 0.001), and eGFR90 ml/min/1.73mThis large population-based study documents a significant risk for KI among adolescents with SFK. Obesity represents a major modifiable risk factor for KI, implicating the need for closer follow-up in this group during childhood.

Diabetic kidney disease (DKD) is becoming increasingly common among children. We aimed to estimate the risk of end-stage renal disease (ESKD) and mortality among adolescents with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) and normal renal function compared with non-diabetics. We hypothesized that childhood onset T1DM vs. T2DM would be associated with a different risk profile for developing ESKD and its complications.A nationwide, population-based, retrospective cohort study, including 1,500,522 adolescents examined for military service between 1967 and 1997, which were classified according to the presence and type of diabetes. Data were linked to the Israeli ESKD registry. Cox proportional-hazards models were used to estimate the hazard ratio (HR) for ESKD.At study enrolment, 1183 adolescents had T1DM and 196 had T2DM. ESKD developed in 2386 non-diabetic individuals (0.2%) compared with 72 individuals (6.1%) with T1DM and 8 individuals (4.1%) with T2DM. Participants with T1DM were younger at ESKD onset than participants with T2DM (median age, 36.0 vs. 40.5 years, P0.05). In a multivariate model adjusted for age, sex, paternal origin, enrollment year, BMI, and blood pressure, T1DM and T2DM were associated with HR of 36.4 (95% CI 28.3-46.9) and 19.3 (95% CI 9.6-38.8) for ESKD, respectively. Stratification according to sex, ethnicity, immigration, and socioeconomic status did not materially change the HR. During the follow-up period, mortality rates were higher in T2DM as compared with T1DM and controls (8.7 %, 2.2%, and 2.7% respectively).T1DM and T2DM in adolescents with normal renal function confer a significantly increased risk for ESKD. T1DM is associated with younger age at ESKD onset while T2DM is associated with higher mortality rate.

Pleuropulmonary blastoma (PPB) is a rare pediatric lung neoplasm that recapitulates developmental pathways of early embryonic lungs. As lung development proceeds with highly regulated mesenchymal-epithelial interactions, a DICER1 mutation in PPB generates a faulty lung differentiation program with resultant biphasic tumors composed of a primitive epithelial and mesenchymal stroma with early progenitor blastomatous cells. Deciphering of PPB progression has been hampered by the difficulty of culturing PPB cells, and specifically progenitor blastomatous cells. Here, we show that in contrast with in-vitro culture, establishment of PPB patient-derived xenograft (PDX) in NOD-SCID mice selects for highly proliferating progenitor blastoma overexpressing critical regulators of lung development and multiple imprinted genes. These stem-like tumors were sequentially interrogated by gene profiling to show a FGF module that is activated alongside Neural cell adhesion molecule 1 (NCAM1). Targeting the progenitor blastoma and these transitions with an anti-NCAM1 immunoconjugate (Lorvotuzumab mertansine) inhibited tumor growth and progression providing new paradigms for PPB therapeutics. Altogether, our novel in-vivo PPB xenograft model allowed us to enrich for highly proliferating stem-like cells and to identify FGFR and NCAM1 as two key players that can serve as therapeutic targets in this poorly understood and aggressive disease.

In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 10

Epidemiological studies demonstrate an association of increased body mass index and risk of kidney stone formation in adults. We conducted a population based pediatric study to examine the epidemiology of nephrolithiasis in Israeli children during a 30-year period, and to determine body mass index distribution during the same period.We accessed data from the compulsory medical evaluations of 17-year-old military service candidates in Israel before their enlistment during 1980 to 2013. Candidates for the army with a history of stone disease were compared to those without such a history.Of 1,908,893 candidates 1,691 reported a history of nephrolithiasis, yielding an average prevalence rate of 88.6 per 100,000. During 1980 to 1995 the average reported prevalence of nephrolithiasis was 69 cases per 100,000. From 1995 onward the reported prevalence increased by an average of 6% yearly, reaching 120 per 100,000 during 2010 to 2012. This increased prevalence was observed for males and females but was more prominent among males. Mean ± SD body mass index of stone formers was higher than that of controls (22.7 ± 3.5 vs 22.1 ± 3.9 kg/mThis large, population based study documents an increasing prevalence of nephrolithiasis in children. The possible association of this finding with the increase in body mass index during the same period warrants further investigation.

The mammalian kidney is a highly complex organ, composed of various cell types within a unique structural framework. Nonetheless, in recent years, giant leaps in our understanding of nephrogenesis and the origin of new cells in the adult kidney have resulted in novel routes to regenerate damaged nephrons. While several strategies can be envisioned to achieve this aim, one common theme is the reliance on renal lineage cells, as extrarenal cells, such as bone marrow-derived cells, have been shown to be devoid of renal differentiation capacity. Herein, we will present the main motivation for the pursuit for cell-based therapies, which is the ever growing problem of chronic kidney disease (CKD), and discuss different strategies toward replenishing the damaged renal parenchyma. These include transplantation of fetal kidney grafts or fetal kidney stem cells, directed differentiation of pluripotent stem cells into kidney epithelia, establishment of renal progenitors from the adult kidney, and genetic reprogramming of mature kidney cells into a progenitor state. Taken together with novel techniques recapitulating the three-dimensional developmental environment, these advances are expected to take the field into a new era, bringing us closer than ever to the day when kidney stem cell-based therapy becomes a viable therapeutic option.

Isolated familial hypoparathyroidism is an extremely rare disorder, which to date has been linked to several loci including mutations in CASR, GCM2, and PTH, as well as a rare condition defined as X-linked recessive hypoparathyroidism, previously associated with a 1.5 Mb region on Xq26-q27. Here, we report a patient with hypocalcemia-induced seizures leading to the diagnosis of primary hypoparathyroidism. Mutations in CASR, GCM2, and PTH were ruled out, while whole exome sequencing of the family suggested FHL1, located on chromosome Xq26, as the most likely causative gene variant (FHL1, exon 4, c.C283T, p.R95W). Since FHL1 has not been linked to calcium regulation before, we provide evidence for its functional role in hypoparathyroidism by: (i) bioinformatics analysis coupling its action to known modulators of PTH function; (ii) observing strong expression of fhl1b in Corpuscles of Stannius, gland-like aggregates in zebrafish that function in calcium regulation similar to mammalian PTH; and (iii) implicating fhl1b and FHL1 as regulators of calcium homeostasis in zebrafish and human cells, respectively. Altogether, our data suggest that FHL1 is a novel regulator of calcium homeostasis and implicate it as the causative gene for X-linked recessive hypoparathyroidism. Electronic supplementary material The online version of this article (doi:10.1007/s00439-017-1804-9) contains supplementary material, which is available to authorized users.

When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms’ tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1+CD133− marks SIX2+ multipotent renal stem cells transiting to NCAM1+CD133+ differentiating segment-specific SIX2− epithelial progenitors and NCAM1−CD133+ differentiated nephron cells. In tumorigenesis, NCAM1+CD133− marks SIX2+ blastema that includes the ALDH1+ WT cancer stem/initiating cells, while NCAM1+CD133+ and NCAM1−CD133+ specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1+ nephron stem cells in normal and malignant nephrogenesis.

Nearly 26 million Americans, one in every nine, harbor kidney disease. Despite recent medical advances, current treatment options for patients with renal failure are limited. Alternatives available to patients who succumb to terminal renal disease are either supportive treatment in the form of dialysis or whole-organ replacement by kidney transplantation. Dialysis is associated with long-term morbidity, mortality, and poor quality of life. The shortage of donor organs and the long wait time on the recipient list hamper renal transplantation. The number of patients with end-stage renal disease has increased significantly in recent decades, and the treatment costs for these patients now exceed the cumulative costs of treating cancer patients. Against this background of the growing number of patients with kidney disease and limited treatment options, alternative treatments are needed.

End-stage renal failure is a major cause of death with currently only dialysis and transplantation available as therapeutic options, each with its own limitations and drawbacks. To allow regenerative medicine-based kidney replacement therapies and due to the fact that neither haematopoietic stem cells nor mesenchymal stem cells, the most accessible human stem cells, can be used to derive genuine nephron progenitors, much attention has been given to finding adult renal stem cells. Several candidates for this have been described, but their true identity as stem or progenitor cells and their potential use in therapy has not yet been shown. However, the analysis of embryonic renal stem cells, specifically stem/progenitor cells that are induced into the nephrogenic pathway to form nephrons until the 34th week of gestation, has been much more conclusive.

The generation of nephrons during development depends on differentiation via a mesenchymal to epithelial transition (MET) of self-renewing, tissue-specific stem cells confined to a specific anatomic niche of the nephrogenic cortex. These cells may transform to generate oncogenic stem cells and drive pediatric renal cancer. Once nephron epithelia are formed the view of post-MET tissue renal growth and maintenance by adult tissue-specific epithelial stem cells becomes controversial. Recently, genetic lineage tracing that followed clonal evolution of single kidney cells showed that the need for new cells is constantly driven by fate-restricted unipotent clonal expansions in varying kidney segments arguing against a multipotent adult stem cell model. Lineage-restriction was similarly maintained in kidney organoids grown in culture. Importantly, kidney cells in which Wnt was activated were traced to give significant clonal progeny indicating a clonogenic hierarchy. In vivo nephron epithelia may be endowed with the capacity akin to that of unipotent epithelial stem/progenitor such that under specific stimuli can clonally expand/self renew by local proliferation of mature differentiated cells. Finding ways to ex vivo preserve and expand the observed in vivo kidney-forming capacity inherent to both the fetal and adult kidneys is crucial for taking renal regenerative medicine forward. Some of the strategies used to achieve this are sorting human fetal nephron stem/progenitor cells, growing adult nephrospheres or reprogramming differentiated kidney cells toward expandable renal progenitors.

Recent studies have suggested that epigenetic modulation with chromatin-modifying agents can induce stemness and dedifferentiation and increase developmental plasticity. For instance, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been shown to promote self-renewal/expansion of hematopoietic stem cells and facilitate the generation of induced pluripotent stem cells (iPSCs). Previously, we observed that downregulation of embryonic renal stem/progenitor genes in the adult kidney was associated, at least in part, with epigenetic silencing. Therefore, we hypothesized that VPA may alter the expression of these genes and reprogram mature human adult kidney epithelial cells (hKEpCs) to a stem/progenitor-like state. Here, using quantitative RT-PCR and flow cytometry [fluorescence-activated cell sorting (FACS)] analysis, we show in VPA-treated primary cultures of human adult and fetal kidney significant reinduction of the renal stem/progenitor markers SIX2, OSR1, SALL1, NCAM, and PSA-NCAM. Robust SIX2 mRNA re-expression was confirmed at the protein level by western blot and was associated with epigenetic changes of the histones at multiple sites of the SIX2 promoter leading to gene activation, significantly increased acetylation of histones H4, and methylation of lysine 4 on H3. Furthermore, we could demonstrate synergistic effects of VPA and Wnt antagonists on SIX2 and also OSR1 reinduction. Nevertheless, VPA resulted in upregulation of E-CADHERIN and reduction in VIMENTIN, preventing the skewing of hKEpCs towards a more replicative mesenchymal state required for clonogenic expansion and acquisition of stem cell characters, altogether inducing cell senescence at early passages. These results demonstrating that chromatin-modifying agents prevent dedifferentiation of hKEpCs have important clinical implications as they may limit ex-vivo self-renewal/expansion and possibly the in vivo renal regenerative capacity initiated by dedifferentiation.

SummaryThe 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.

The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1 + cells overexpressed early nephron progenitor markers ( PAX2 , SALL1 , SIX2 , WT1 ) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial–mesenchymal transition. NCAM1 + cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1 + cells from hKEpCs abrogated stemness traits in vitro . Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo . Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases.

During the process of development, a single cell, the fertilized ovum, becomes an entire functional organism that is made up of a vast number of cell types, each acting within the context of a specific organ or tissue, usually in cooperation with other types of cells.

Chronic kidney disease (CKD) is a progressive loss in renal function over a period of months or years. End-stage renal disease (ESRD) or stage 5 CKD ensues when renal function deteriorates to under 15% of the normal range. ESRD requires either dialysis or, preferentially, a kidney organ allograft, which is severely limited due to organ shortage for transplantation. To combat this situation, one needs to either increase supply of organs or decrease their demand. Two strategies therefore exist: for those that have completely lost their kidney function (ESRD), we will need to supply new kidneys. Taking into account the kidneys' extremely complex structure, this may prove to be impossible in the near future. In contrast, for those patients that are in the slow progression route from CKD to ESRD but still have functional kidneys, we might be able to halt progression by introducing stem cell therapy to diseased kidneys to rejuvenate or regenerate individual cell types. Multiple cell compartments that fall into three categories are likely to be worthy targets for cell repair: vessels, stroma (interstitium) and nephron epithelia. Different stem/progenitor cells can be linked to regeneration of specific cell types; hematopoietic progenitors and hemangioblastic cell types have specific effects on the vascular niche (vasculogenesis and angiogenesis). Multipotent stromal cells (MSC), whether derived from the bone marrow or isolated from the kidney's non-tubular compartment, may, in turn, heal nephron epithelia via paracrine mechanisms. Nevertheless, as we now know that all of the above lack nephrogenic potential, we should continue our quest to derive genuine nephron (epithelial) progenitors from differentiated pluripotent stem cells, from fetal and adult kidneys and from directly reprogrammed somatic cells.

End-stage renal disease (ESRD) is defined as the inability of the kidneys to remove waste products and excess fluid from the blood. ESRD progresses from earlier stages of chronic kidney disease (CKD) and occurs when the glomerular filtration rate (GFR) is below 15 ml/minute/1.73 m2. CKD and ESRD are dramatically rising due to increasing aging population, population demographics, and the growing rate of diabetes and hypertension. Identification of multipotential stem/progenitor populations in mammalian tissues is important for therapeutic applications and for understanding developmental processes and tissue homeostasis. Progenitor populations are ideal targets for gene therapy, cell transplantation, and tissue engineering. The demand for kidney progenitors is increasing due to severe shortage of donor organs. Because dialysis and transplantation are currently the only successful therapies for ESRD, cell therapy offers an alternative approach for kidney diseases. However, this approach may be relevant only in earlier stages of CKD, when kidney function and histology are still preserved, allowing for the integration of cells and/or for their paracrine effects, but not when small and fibrotic end-stage kidneys develop. Although blood- and bone marrow-derived stem cells hold a therapeutic promise, they are devoid of nephrogenic potential, emphasizing the need to seek kidney stem cells beyond known extrarenal sources. Moreover, controversies regarding the existence of a true adult kidney stem cell highlight the importance of studying cell-based therapies using pluripotent cells, progenitor cells from fetal kidney, or dedifferentiated/reprogrammed adult kidney cells.