MicroRNAs in Kidney Development And Disease Ⅱ

Kidney development 

The mammalian kidney (or metanephros) is a vital organ that plays a critical role in the excretion of metabolic wastes, regulation of extracellular fluid volume, and maintenance of electrolyte and acid-base homeostasis. Moreover, the kidney produces important hormones, such as erythropoietin, calcitriol, renin, and prostaglandins (48). The functional capacity of the kidney correlates with the number of functioning nephrons that are formed during kidney development before birth, also termed nephron endowment. Each human kidney contains on average 1,000,000 nephrons, although this number varies considerably, with estimates ranging from 200,000 to 2,000,000 nephrons (49, 50). With aging, loss of functional nephron reserve occurs over time (51, 52); therefore, low nephron endowment at birth is associated with an increased risk of developing hypertension and chronic kidney disease (CKD) later in life (53–55). Moreover, CAKUT, which leads to decreased nephron endowment and nephron function, is the leading cause of renal failure in children, resulting in significant morbidity and mortality associated with transplant and dialysis (56, 57). Thus, a better understanding of the cellular and molecular mechanisms underlying the establishment of nephron number and normal nephron formation provides insights into novel avenues to predict, prevent, and treat childhood kidney disease.

HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK FOR KIDNEY DISEASE PATIENTS?

Metanephric kidney development starts around embryonic day 10.5 (E10.5) in mice and around the fifth week of gestation in humans (58). In response to inductive signals from the metanephric mesenchyme, the ureteric bud extends from the caudal end of the Wolffian duct and invades into the adjacent mesenchyme (Figure 2). Simultaneously, morphogens emanating from the ureteric bud induce condensation of the metanephric mesenchyme to form the cap mesenchyme (also termed nephron progenitors) around the tips of the ureteric bud. As nephrogenesis progresses, the ureteric bud undergoes successive rounds of branching, elongation, and differentiation to generate the collecting ducts of the kidney. A subpopulation of nephron progenitors undergoes mesenchymal-epithelial transition to form renal vesicles, which after polarization and elongation become comma- and S-shaped body structures. Finally, the distal portion of the S-shaped body fuses with the collecting duct to form a functional nephron (59–61). The S-shaped body undergoes further differentiation to form the mature cell types of the nephron, apart from the collecting duct. Foxd1+ stromal progenitor cells are adjacent to nephron progenitors in the outer cortical or nephrogenic zone of the developing kidney (Figure 2) (62). Signals from the cortical stroma are thought to inhibit nephron progenitor cell expansion and stimulate its differentiation, as ablation of the renal stroma results in impaired nephron progenitor differentiation (63). The Foxd1+ progenitor cells give rise to all stromal cells in the metanephric kidney, including renal cortical and medullary interstitial cells, pericytes, perivascular fibroblasts, mesangial cells, and vascular smooth muscle cells (64, 65). Perturbations in any step of this process can lead to CAKUT, the major cause of childhood CKD (66, 67).

The mature nephron is composed of a glomerulus that acts as the filtration unit and a tubular rearsorption compartment that is subdivided into a proximal convoluted tubule, a loop of Henle, a distal convoluted tubule, and a collecting duct (Figure 2). The filtration barrier of the glomerulus (which comprises the fenestrated endothelium, glomerular basement membrane, foot processes, and slit diaphragms of podocytes) allows the filtration of plasma and small solutes, while selectively retaining proteins such as albumin and immunoglobulins in the blood (68, 69). Meanwhile, the tubular reabsorption compartment is responsible for the maintenance of water homeostasis, reabsorption of solutes (including sodium, potassium, calcium, phosphorous, magnesium, glucose, and many others), and excretion of acid and other wastes.

MiRNAs in the developing kidney

In studies in conditional transgenic mice, miRNAs have emerged as critical regulators of kidney morphogenesis in multiple cell lineages. The initial studies evaluating a functional role for miRNAs in kidney development utilized conditional deletion of Dicer (70) in different renal lineages. However, Dicer is also known to have miRNA-independent roles (71), which has complicated the interpretation of these models. Conconditional deletion of Dicer in early metanephric mesenchyme or nephron progenitors results in augmented apoptosis of nephron progenitors, elevated levels of the proapoptotic protein Bim, and premature cessation of nephrogenesis (72–75) (Table 1). Interestingly, the loss of Bim expression in Dicer-deficient nephron progenitors decreases apoptosis and partially restores nephron formation. Two miRNAs expressed in nephron progenitors, miR-17 and miR-106, were identified as suppressors of BIM expression (76). Together, these findings indicate that miRNAs control the balance between survival and apoptosis in nephron progenitors to ensure the formation of a correct number of nephrons throughout nephrogenesis.

Figure 2. Schematic illustration of the stages of metanephric kidney development. Signals from the ureteric bud trigger condensation of the metanephric mesenchyme to form a cap of nephron progenitors (cap mesenchyme) around the ureteric bud tips. The cap mesenchyme undergoes a mesenchymal-epithelial transition to form renal vesicles, which develop sequentially into comma- and S-shaped bodies. These structures connect to the ureteric bud stalk, which gives rise to the collecting duct. Cells in the proximal domain of the S-shaped body differentiate into specialized epithelial cells of the mature renal corpuscle (i.e., podocytes and Bowman's capsule cells), while cells in the mid-and distal portions differentiate into the tubular segments of nephron (proximal tubules, loops of Henle, and distal tubules). Created with BioRender.com.

Conditional deletion of Dicer in the ureteric bud lineage results in a spectrum of abnormalities that strongly resemble CAKUT, including renal dysplasia and the development of collecting duct cysts (73, 77, 78). Premature termination of branching morphogenesis (in response to decreased expression of Wnt11 and c-Ret from the ureteric bud) is likely the major contributing factor for renal dysplasia (73). The onset of cyst formation occurs at around E15.5 and is associated with defects in primary cilia length, increased apoptotic cell death, and excessive cell proliferation (73). As Dicer has important miRNA-independent roles in the cell, conditional deletion of Dgcr8 has been used to confirm that the phenotypes observed in conditional Dicer-knockout models are indeed the result of loss of miRNAs. Animals with Dgcr8 deletion in the distal nephron and derivatives of the collecting duct develop hydronephrosis and collecting duct cysts (79), a CAKUT-like phenotype that resembles the loss of Dicer activity in the ureteric bud lineage.

Studies from two independent groups demonstrated that ablation of Dicer from the Foxd1+ renal stroma lineage and its derivatives results in a spectrum of renal anomalies, with consistent findings regarding hypoplastic kidneys, reduced glomerular numbers, abnormal glomerular maturation, and defective vascular patterning (80, 81). Though both groups described largely concordant phenotypes using similar mouse models, two distinct differences were noted. Nakagawa et al. observed a lack of the inner medulla and papilla, as well as a decrease in the nephrogenic zone (80). In contrast, Phua et al. showed an expansion of the nephron progenitor population and preserved renal papilla (81). Nakagawa et al. proposed that these defects are related to the disruption of Wnt pathway signaling, resulting in changes in stromal cell migration and proliferation, due to downregulation of the stromal cell miRNAs, miR-214, miR-199a-5p, and miR-199a-3p (80). The study by Phua et al. suggested that changes in apoptotic programs (including augmented expression of Bim and p53 effector genes) contribute to phenotypic defects (81). It is conceivable that genetic background differences and/or the efficiency of Cre-mediated recombination may be responsible for the differences these studies describe. Nevertheless, the described phenotypes are consistent with the known multifaceted roles of renal stroma in kidney development, and the mechanisms underlying these phenotypes are likely complex given the nature of a Dicer deletion. Further studies examining specific miRNAs in various stromal subpopulations are needed to better define the regulatory mechanisms at play.

More recent work has addressed the question of the function of specific miRNAs in both the developing kidney and nephron progenitors. Using a human embryonic stem cell model, Bantounas et al. showed that inhibition of the miR-199a~214 cluster results in dysmorphic glomeruli, aberrant proximal tubules, decreased WT1 expression, and increased interstitial capillaries in kidney-like organoids (82). Interestingly, global deletion of hypoxia-responsive miR-210 results in a male-specific nephron deficit (83). For example, conditional deletion of the miR-17~92 cluster in nephron progenitors and their derivatives in mice impairs progenitor cell proliferation and reduces the number of developing nephrons. As a result, mutant mice develop proteinuria, renal fibrosis, and impaired renal function (84). Dysregulated levels of the miR-17~92 target gene, CFTR, are implicated in the defective proliferation of progenitor cells and reduced nephron endowment in this mouse model (85).

Small RNA sequencing (smRNA-Seq) has been increasingly used to profile miRNA expression patterns and for the discovery of novel miRNA species. smRNA-Seq of E15.5 nephrogenic mesenchymal cells identified 162 annotated miRNAs that are differentially expressed in this cell population compared with whole kidneys and 49 novel miRNA species (86). Interestingly, levels of miR-200 family miRNAs were significantly reduced in nephron progenitors. Given that members of the miR-200 family are key regulators of mesenchymal-epithelial transition in the collecting duct (87), we speculate that their expression might be tightly regulated to ensure normal epithelial differentiation of nephron progenitors during kidney development.

MiRNA function in the mature nephron

In addition to their requirement during kidney development, miRNAs regulate numerous biological processes in the major cell lineages that form the mature nephron (69, 88–91). In keeping with this, segment-specific expression of miRNAs along the nephron has been described, including miR-143 and miR-195a in the glomerulus, miR-107 and miR-34a in the proximal tubule, miR-193 and miR-378a in the thick ascending limb, miR-874 and miR-155 in the distal convoluted tubule, and miR-200c in the collecting duct (87). Moreover, functional studies in compartments of the mature nephron support distinct roles for miRNAs.

Mice lacking either Dicer or Drosha in podocytes exhibit marked proteinuria, glomerulosclerosis, and rapid progression to kidney failure, secondary to disruption of the glomerular filtration barrier (90–93). In silico analyses revealed that various upregulated transcripts in mutant glomeruli contain target sequences for miR-30 family members. As all four miR-30 family members (miR-30c-1, miR-30b, miR-30d, and miR-30c-2) are normally highly expressed in podocytes, these miRNAs may be responsible for the podocyte abnormalities and disruption of the glomerular filtration barrier in mutant mice (91).

Somewhat surprisingly, deletion of Dicer from postnatal mammalian proximal tubules does not affect kidney development, histology, or function but does protect against renal ischemia/reperfusion injury. Mutant mice exhibit better kidney function, reduced kidney injury, lower tubular apoptosis, and improved survival compared with their WT littermates (94). This likely reflects the "sum" of the effect of deletion of multiple miRNAs in the proximal tubule, as other work has since demonstrated that the expression of specific miRNAs is protective in renal ischemia/reperfusion injury (e.g., miR-16 and miR-21; refs. 95, 96); whereas others are injurious (e.g., miR-182; ref. 97).

Although miRNAs seem to be dispensable for proximal tubule function, they are essential for distal nephrons and collecting duct homeostasis (79, 88, 98). Collecting duct-specific inactivation of Dicer and other critical miRNA biogenesis-associated genes (including Dgcr8, Ago1, 2, 3, and 4) causes renal failure in adult mice because of progressive tubulointerstitial fibrosis and interstitial inflammation (88). This is preceded by a partial epithelial-mesenchymal transition (EMT) of collecting duct cells, and downregulation of miR-200 family members, which inhibit EMT (88). Likewise, ablation of either Dicer or Dgcr8 from distal nephron and ureteric bud derivatives, respectively, results in renal abnormalities and kidney failure (78, 98), which are ultimately associated with downregulation of miR-200 family members (98). Increased expression of miR-200 target gene Pkd1 in these mutant mice disrupts tubulogenesis and produces cyst-like structures (98). These differences in the requirement for functional miRNAs in proximal tubules and distal nephron/collecting duct might be explained by the segmental distribution of miRNAs along the length of the nephron and collecting duct in WT kidneys (88).

Deletion of Dicer in renin-secreting cells in the juxtaglomerular apparatus results in a deficit of juxtaglomerular cells, reduced circulating renin levels with consequent reduction in arterial blood pressure, reduced kidney function, striped pattern of interstitial fibrosis, and vascular abnormalities (89). The reduction in juxtaglomerular cells suggests a requirement for mature miRNAs in the maintenance of their phenotype. Later, miR-330 and miR-125b-5p were identified as potential candidates that either inhibit or promote, respectively, the smooth muscle phenotype of juxtaglomerular cells (99).

Other active areas of research on miRNAs in acute kidney injury (8–10), polycystic kidney disease (11), and kidney transplant (10), have been comprehensively addressed in other recent reviews.

MiRNAs in pediatric kidney diseases In this section, we provide an overview of the role of miRNAs in developmental kidney diseases, including CAKUT and Wilms tumors. CAKUT are among the most frequent forms of malformations at birth, affecting approximately 3–7 out of 1000 live births (100). Disruption of kidney and lower urinary tract development leads to a wide spectrum of clinical manifestations observed in CAKUT, including kidney anomalies (i.e., renal agenesis, renal hypoplasia and dysplasia, and multicystic dysplastic kidneys), ureteropelvic anomalies (i.e., ureteropelvic junction obstruction), duplex collecting system, and anomalies of the bladder and urethra (101–103). This phenotypic heterogeneity is likely due to complex interactions between genetic, epigenetic, and/or prenatal environmental factors that affect kidney and lower urinary tract development, resulting in CAKUT (101). Most of our current knowledge of CAKUT pathogenesis has arisen from mouse models and syndromic forms of CAKUT. These studies have led to the identification of several CAKUT genes, many of which are implicated in early kidney development, including PAX2, SALL1, HNF1B, EYA1, GATA3, RET, WNT4, GDNF, SIX1, SIX2, and others (101, 104, 105). However, single mutations or copy number variants in protein-coding genes do not explain the majority of CAKUT cases (~80%) (101, 106).

As mentioned above, depletion of mature miRNAs from different cell lineages of the developing kidney in mouse models results in renal abnormalities that mimic human CAKUT (73, 74, 78, 79). In addition, germline deletions of MIR17HG, which encodes the miR-17~92 cluster, cause type 2 Feingold syndrome in humans (107). Although a renal phenotype in type 2 Feingold syndrome patients with MIR17HG mutations remains undefined, an 18% incidence of CAKUT has been reported in Feingold syndrome cases associated with MYCN mutations (108, 109). Together, these observations suggest that mutations in miRNAs expressed during kidney development might cause CAKUT in humans, particularly as many miRNAs are highly conserved between mice and humans.

To test this hypothesis, one study investigated 1248 patients with nonsyndromic CAKUT from 980 families and looked for mutations in 96 stem-loop regions of 73 renal developmental miRNA genes (106). Within this cohort, 31 individuals with 17 different single nucleotide variants affecting 16 different miRNA genes were identified. Among these, two novel variants in miRNAs were found to be potentially pathogenic. MIR19B1 (a member of the miR-17~92 cluster) was associated with the presence of right renal agenesis, and MIR99A was associated with severe vesicoureteral reflux and kidney ptosis. This surprisingly low number of candidate pathogenic variants is partly due to the limitations of this study, as the analysis only accounted for mutations in miRNA genes that were included in the candidate gene approach and did not detect copy number variations and large DNA rearrangements (106).

In an alternative approach, ureter segments from patients with a variety of CAKUT were analyzed for differential transcript expression via microarray, for the presence of bioinformatically predicted miRNA targets, and mature miRNAs via qPCR (110). Using this multipronged approach, seven miRNAs were identified with potential roles in CAKUT, and among these, has-miR-144 was significantly increased in patients with CAKUT. Gene ontology analysis indicated that predicted has-miR-144 target genes contribute to biological processes involved in CAKUT development, including tube development (22 target genes), urogenital system development (18 target genes), kidney development (14 target genes), and embryonic organ development (18 target genes) (110).

Further studies are needed to define the molecular mechanisms underlying the pathogenic roles of miRNAs in CAKUT. Findings from such studies will be critical in improving the care of patients with CAKUT and preventing their progression to CKD, providing appropriate genetic counseling for patients and their families, and developing novel therapeutic strategies.