Epigenetic regulation of kidney progenitor cells

Abstract The reciprocal interactions among the different embryonic kidney progenitor populations lay the basis for proper kidney organogenesis. During kidney development, three types of progenitor cells, including nephron progenitor cells, ureteric bud progenitor cells, and interstitial progenitor cells, generate the three major kidney structures—the nephrons, the collecting duct network, and the stroma, respectively. Epigenetic mechanisms are well recognized for playing important roles in organism development, in fine‐tuned control of physiological activities, and in responses to environment stimuli. Recently, evidence supporting the importance of epigenetic mechanisms underlying kidney organogenesis has emerged. In this perspective, we summarize the research progress and discuss the potential contribution of novel stem cell, organoid, and next‐generation sequencing tools in advancing this field in the future.


| INTRODUCTION
The kidney is responsible for maintaining homeostasis. It is involved in removing metabolic waste products and adjusting water, salt, and pH to maintain the homeostatic balance of fluids in mammals. 1 In addition, the kidney also participates in the control of blood pressure through the renin-angiotensin-aldosterone system and secretes erythropoietin to promote erythrocyte production. 1 During embryogenesis, the native kidney progenitor cells that give rise to the kidney include nephron progenitor cells (NPCs), ureteric bud (UB) progenitor cells (UPCs), and interstitial progenitor cells (IPCs). NPCs form nephrons, the functional units of the kidney; UPCs form the collecting duct network and the ureter that drain urine; and IPCs form various stromal cell types. Additionally, vascular progenitor cells are also present in the developing kidney to form the blood vessels. [1][2][3][4] Epigenetic mechanisms regulate heritable phenotype changes that do not involve alterations in the DNA sequence. In this manner, finetuning of biological processes is usually achieved in response to environmental stimuli. Epigenetic mechanisms mainly include DNA methylation, histone modifications, and regulatory noncoding RNAs. To carry out these epigenetic changes, associated functional proteins serve as mediators to add or remove related epigenetic markers. 5 Epigenetics are involved in regulating several physiological processes, such as cell differentiation, 6 organogenesis, 7 immune response, 8 and organism aging. 9 In this perspective, we focus on discussing the recent findings of epigenetic regulation of kidney progenitor cell fates during kidney development ( Figure 1). Biao Huang and Zhenqing Liu are co-first authors.

| EPIGENETIC REGULATION OF NPCs
The functional unit of the kidney is the nephron. A typical mouse kidney consists of 12 000 to 16 000 nephrons, while the human kidney has 1 000 000 nephrons on average, with significant variations among individuals. 1 Six2+/Cited1+ NPCs have been identified as the selfrenewing progenitor cell population that generates the main body of the nephron, including glomerulus, podocytes, proximal tubule, the loop of Henle, and distal tubule. 10 12  deletion resulted in a remarkable reduction in nephron numbers, as well as renal hypoplasia at birth, suggesting that Dnmt1 regulates NPC self-renewal. 13 Further analyses indicated that global DNA hypomethylation promotes ectopic expression of germline-related genes F I G U R E 1 Representative scheme of epigenetic regulation of kidney progenitors within a nephrogenic niche during kidney development. Epigenetic mechanisms involved in kidney organogenesis include DNA methylation, histone acetylation, chromatin remodeling complexes, and versatile noncoding RNAs. These mechanisms are mediated by special epigenetic modifiers and play important roles in the regulation of selfrenewal maintenance and differentiation of three types of kidney progenitors during kidney development and Cited1, was significantly decreased and NPC proliferation capacity was reduced. 17 The genetic interaction between Mi2b with Sall1, a key transcription factor for the specification and self-renewal of NPCs, was revealed through the generation of heterozygous double mutants, suggesting a potential NuRD function through Sall1.
However, the detailed mechanisms remain to be addressed to explain the strong phenotype of Mi2b knockout.
In view of these studies, it appears that NPC self-renewal maintenance and proper differentiation of NPCs require a relatively repressed chromatin landscape considering that DNA hypomethylation 13,14 and histone hyperacetylation 16

| EPIGENETIC REGULATION OF UB
During mouse kidney development, UB starts to branch to form a Tshaped structure by E11.5. The UB-derived epithelium then undergoes 12 continuous branching steps before ceasing around 2 days after birth, finally generating the entire collecting duct system for draining of urine. Wnt11+ UB progenitor cells (UPCs) are positioned on the UB tips. They self-renew to generate new UPCs at branching UB tips, while also differentiating into mature collecting duct cells, including principal cells and intercalated cells. 1 Moreover, p63 was identified to specifically define a subpopulation of UB tip cells, responsible for the formation of intercalated cells. 24 The genetic determinants of UB branching have been studied extensively, 25 while epigenetic regulations in this process are relatively less studied.
Similar to the function of HDACs in maintaining NPC selfrenewal, HDACs are also involved in UB branching morphogenesis. Hdac1 fl/fl ;Hdac2 fl/fl mice were crossed with Hoxb7-CreEGFP transgenic mice to enable the conditional knockout in the ureteric epithelial of the kidney. UB-specific deletion of both Hdac1 and Hdac2 resulted in bilateral renal hypodysplasia due to the impairment of canonical Wnt signaling pathway and the hyperacetylation of the tumor suppressor protein p53, which might inhibit UB cell growth and survival 26 In addition, HDACs were also reported to participate in the renin-angiotensin system (RAS)-associated UB branching.
HDACs are involved in regulating not only UB morphogenetic program genes, but also expression of RAS genes. 27 Additionally, the Yu group demonstrated the significance of miRNAs in UB branching morphogenesis via UB-specific Dicer deletion (Dicer fl/fl ; Hoxb7-CreEGFP). 19 Loss of Dicer in the UB results in a premature termination of branching morphogenesis. Let-7 family miRNAs are expressed higher in the later-stage UB than in the early-stage UB, and computational analysis revealed the presence of let-7 family miRNA binding sites for many early UB genes. These types of evidence led to the hypothesis that let-7 family miRNAs might be involved in promoting differentiation of collecting duct via inhibiting expression of early UB genes at later developmental stages. 28 In all these studies, Hoxb7-Cre mouse strain was used. However, since Hoxb7 is expressed in both UB tip (UPCs) and UB trunk (maturating into collecting duct), it remains unknown whether the phenotypes observed in the above studies were due to changes in the UPCs or the UPC progenies. Future studies using Wnt11-Cre to mediate a specific gene manipulation in the UPCs were able to address these questions.

| EPIGENETIC REGULATION OF INTERSTITIAL PROGENITOR CELLS
IPCs generate most stromal cell types in the kidney during development, including pericytes, mesangial cells, and interstitium, with Foxd1 serving as a critical marker gene of IPCs. 29  Most of our current knowledge in this field is obtained from studies in mouse models. Considering the differences in kidney development between mouse and human, 32 it is imperative to investigate how the epigenetic mechanisms govern the cell fates in the human kidney progenitor cell types. Directed differentiation from human pluripotent stem cells (hPSCs) into kidney progenitor cells 33 and kidney organoids 34,35 has emerged as a novel platform for modeling human kidney development and diseases. [36][37][38][39] In addition, complementing the directed differentiation from hPSCs, our group and others have developed culture conditions to expand primary mouse and human nephron progenitor cells, which could further generate nephron organoids. [40][41][42] With these in vitro culture systems and gene-editing tools, such as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) 9, 36,43-45 the simultaneous manipulation of multiple genes can be achieved with high efficiency, which is technically challenging and expensive to do using the traditional mouse models. In this manner, these in vitro systems can supplement the traditional mouse models in addressing the detailed epigenetic mechanisms involved in both mouse and human kidney development. The availability of large quantities of relatively pure progenitor cells will also enable novel applications that were previously impossible to do with the small number of cells isolated from the mice.
For example, the genetic screening using the CRISPR-Cas9 system 46,47 can now be done with the availability of a large number of NPCs produced in vitro. Moreover, single cell sequencing technologies have proved to be a powerful research tool recently. With a higher resolution gene expression profiling of kidney development at the single cell level, novel epigenetic mechanisms will potentially be discovered. Taken together, in the future, a better understanding of the epigenetic regulatory mechanisms will contribute to our understanding of kidney development, and the knowledge will potentially help address the pathogenesis of congenital kidney diseases that occur frequently in newborns ( Figure 2).

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
B.H.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Z.L.: conception and design, data analysis, and interpretation; A.V.: data analysis and interpretation; Z.Z.: collection and/or assembly of data, data analysis and interpretation; Z.L.: conception and design, manuscript writing, data analysis and interpretation, final approval of manuscript.