Suppression of ureteric bud apoptosis rescues nephron endowment and adult renal function in Pax2 mutant mice

The molecular mechanisms that set congenital nephron number are unknown. However, humans with modest suboptimal nephron number may be at increased risk for essential hypertension, and those with more severe nephron deficits at birth may develop progressive renal insufficiency. A model of branching morphogenesis during fetal kidney development in which the extent of ureteric bud arborization is dependent on suppression of programmed cell death has been proposed. This study shows that the increased apoptosis and reduced ureteric bud branching of heterozygous Pax2 mutant mice is associated with 40% decrease in nephron number at birth. This leads to postnatal glomerular hypertrophy and long-term renal insufficiency in the absence of glomerulosclerosis. To determine whether restoration of antiapoptotic factors alone is sufficient to rescue the nephron deficit in these mice, a BCL2 transgene that is under the control of the PAX2 promoter was targeted to the ureteric bud. The transgene suppressed programmed cell death in the ureteric bud lineage, increased nephron number to 90% of that of wild-type littermates at birth, and normalized renal function at 1 yr. These observations lend strong support to the hypothesis that factors that control ureteric bud apoptosis are powerful determinants of congenital nephron endowment.     

BMP signaling and its modifiers in kidney development

The kidney develops through mutual interactions between the metanephric mesenchyme and the ureteric bud, the former of which contains nephron progenitors that give rise to glomeruli and renal tubules. Bone morphogenetic protein (BMP) signaling and its modifiers play important roles in many steps of kidney development. BMP4 inhibits ureteric bud attraction, and the BMP antagonist Gremlin is essential for the initial stage of ureteric budding. During mid-gestation, BMP7 maintains the nephron progenitors and, at the same time, sensitizes them to the ureteric bud-derived differentiation signal. Crossveinless2 is a pro-BMP factor, and its absence leads to kidney hypoplasia. After birth, when nephron progenitors have disappeared, Dullard, a phosphatase that inactivates BMP receptors, keeps BMP signaling at an appropriate level. Deletion of Dullard results in excessive BMP signaling and apoptosis of the postnatal nephrons. In this review I discuss the similarities and differences of BMP functions in kidney development, as well as in diseases.     

miRNAs in mammalian ureteric bud development

The collecting duct network and the urothelium of the ureter of the metanephric kidney are derived from the ureteric bud epithelium, initially an outgrowth from the caudal end of the Wolffian duct at the onset of the metanephric kidney development. The tips of the ureteric bud epithelium undergo reiterative branching morphogenesis, which generates more tips and trunks, whereas the ureteric trunks grow and differentiate into principal cells and intercalated cells of the collecting ducts that regulate body water and acid–base homeostasis. microRNAs (miRNAs) are a family of small non-coding RNAs that regulate a diversity of biological processes including organogenesis, mostly by negatively regulating their target gene expression. In this review, I will summarize the current knowledge on the critical roles of miRNAs expressed in the ureteric bud epithelium in ureteric bud morphogenesis and differentiation, including ureteric bud branching morphogenesis, collecting duct terminal differentiation, cystogenesis of the collecting ducts, and ureter development.     

Stem cells in the kidney

The kidney is derived from the ureteric bud and the metanephrogenic mesenchyme, and these two progenitor cells differentiate into more than 26 different cell types in the adult kidney. The ureteric bud contains the precursor of the epithelial cells of the collecting duct and the renal mesenchyme contains precursors of all the epithelia of the rest of the nephron, endothelial cell precursors and stroma cells, but the relatedness among these cells is unclear. A single metanephric mesenchymal cell can generate all the epithelial cells of the nephron (except the collecting duct), indicating that the kidney contains epithelial stem cells. It is currently unknown whether these stem cells also are present in the adult kidney but experience in other organs makes this likely. It also is unclear whether embryonic renal epithelial stem cells can generate other cell types, but preliminary studies in our laboratory suggest that they can differentiate into myofibroblasts, smooth muscle, and perhaps endothelial cells, indicating that they are pluripotent renal stem cells. The important problem to be solved now is the identification and location of adult renal stem cells. This article discusses work done in other organs and in renal development that we believe may be useful for the resolution of this problem.     

Retinoids and renal development

Although it has long been appreciated that retinoids play an essential role in kidney organogenesis, it has only recently been recognized that even mild fetal vitamin A deficiency syndromes can result in a reduction in nephron number. Recent studies have also begun to define the cellular and molecular events associated with retinoid actions in the fetal kidney and have demonstrated the essential function of retinoids in branching growth of the ureteric bud. Importantly, characterization of the renal developmental effects of RAR alpha/beta 2 double homozygous mice combined with metanephric organ culture studies have together shown that one essential function of retinoid action in the developing kidney is the maintenance of c-ret expression in the tips of the ureteric bud. However, many other potential retinoid target genes including midkine, sonic hedgehog, Hox d-11, matrix metalloproteinases, and tissue inhibitors of metalloproteinases appear to play important roles in renal development and might be important downstream mediators of retinoid effects in the developing kidney. It can, therefore, be anticipated that important new insights into fetal kidney development will be forthcoming in the near future, as the essential target genes affected by retinoid signal transduction are progressively elucidated.     

Ureteric bud apoptosis and renal hypoplasia in transgenic PAX2-Bax fetal mice mimics the renal-coloboma syndrome

In humans, PAX2 haploinsufficiency causes renal-coloboma syndrome (RCS) involving eye abnormalities, renal hypoplasia, and renal failure in early life. The authors previously showed that heterozygous mutant Pax2 mice have smaller kidneys with fewer nephrons, associated with elevated apoptosis in the ureteric bud (UB). However, PAX2 may have a variety of developmental functions such as effects on cell fate and differentiation. To determine whether apoptosis alone is sufficient to cause a UB branching deficit, the authors targeted a pro-apoptotic gene (Baxalpha) to the embryonic kidney under the control of human PAX2 regulatory elements. The exogenous PAX2 promoter directed Baxalpha gene expression specifically to the developing kidney UB, eye, and mid/hindbrain. At E15.5 PAX2Promoter-Baxalpha fetal mice exhibited renal hypoplasia, elevated UB apoptosis, and retinal defects, mimicking the phenotype observed in RCS. The kidneys of E15.5 PAX2Promoter-Baxalpha fetal mice were 55% smaller than those of wild-type fetal mice, and they contained 70% of the normal level of UB branching. The data indicate that loss of Pax2 anti-apoptotic activity is sufficient to account for the reduced UB branching observed in RCS and suggest that elevated UB apoptosis may be a key process responsible for renal hypoplasia. The authors propose a morphogenic unit model in which cell survival influences the rate of UB branching and determines final nephron endowment.     

Review: The role of microRNAs in kidney disease

MicroRNAs (miRNAs) are short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression via blockade of protein translation or by inducing mRNA degradation. These miRNAs potentially regulate the expression of thousands of proteins. As a result, miRNAs have emerged rapidly as a major new area of biomedical research with relevance to kidney disease. MiRNA expression has been shown to differ between the kidney and other organs as well as between different kidney regions. Furthermore, miRNAs have been found to be functionally important in models of podocyte development, diabetic nephropathy and polycystic kidney disease. Of particular interest, podocyte-specific deletion of Dicer, a key enzyme in the biogenesis of miRNA, results in proteinuria and severe renal impairment in mice. One miRNA (miR-192) can also act as an effector of transforming growth factor-β activity in the high-glucose environment of diabetic nephropathy. Differential expression of miRNAs has been reported in kidney allograft rejection. It is anticipated that future studies involving miRNAs will generate new insights into the complex pathophysiology underlying various kidney diseases, generate diagnostic biomarkers and might be of value as therapeutic targets for progressive kidney diseases. The purpose of this review is to highlight key miRNA developments in kidney diseases and how this might influence the diagnosis and management of patients with kidney disease in the future.     

Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) are expressed in the ureteric bud and metanephric mesenchyme of the developing kidney. Furthermore, in vitro and in vivo studies have shown that exogenous fibroblast growth factors (Fgfs) increase growth and maturation of the metanephric mesenchyme and ureteric bud. Deletion of fgf7, fgf10, and fgfr2IIIb (the receptor isoform that binds Fgf7 and Fgf10) in mice lead to smaller kidneys with fewer collecting ducts and nephrons. Overexpression of a dominant negative receptor isoform in transgenic mice has revealed more striking defects including renal aplasia or severe dysplasia. Moreover, deletion of many fgf ligands and receptors in mice results in early embryonic lethality, making it difficult to determine their roles in kidney development. Recently, conditional targeting approaches revealed that deletion of fgf8 from the metanephric mesenchyme interrupts nephron formation. Furthermore, deletion of fgfr2 from the ureteric bud resulted in both ureteric bud branching and stromal mesenchymal patterning defects. Deletion of both fgfr1 and fgfr2 in the metanephric mesenchyme resulted in renal aplasia, characterized by defects in metanephric mesenchyme formation and initial ureteric bud elongation and branching. Thus, Fgfr signaling is critical for growth and patterning of all renal lineages at early and later stages of kidney development.     

Nephron progenitors in the metanephric mesenchyme

The kidney is formed by a reciprocally inductive interaction between two precursor tissues, the metanephric mesenchyme and the ureteric bud. This interaction can be divided into three processes: attraction of the ureteric bud toward the mesenchyme, maintenance of the mesenchyme in an undifferentiated state versus transition to an epithelial state, and further differentiation into multiple epithelial lineages, such as glomeruli and renal tubules. In this review we describe our recent findings related to each process. A mesenchymal nuclear zinc finger protein, Sall1, controls ureteric bud attraction by regulating a novel kinesin, Kif26b. The Sall1 gene is highly expressed in multipotent nephron progenitors in the mesenchyme, and these cells can partially reconstitute a three-dimensional structure in organ cultures following Wnt4 stimulation. While Notch2 is required for further differentiation of proximal nephron structures, ectopic Notch2 activation in the embryonic kidney depletes nephron progenitors, suggesting that Notch2 stabilizes—rather than dictates—nephron fate by shutting down the maintenance of undifferentiated progenitor cells.     

Biochemical and histochemical characterization of a murine tubular antigen.

Cellular components of the developing fetal nephron express certain cell surface antigens at discrete stages of nephron development. To further evaluate the expression of antigens by tubular epithelium, a rat monoclonal antibody (BL26) was developed against murine fetal kidney. BL26 identified a differentiation antigen expressed by epithelial cells of the ureteric bud and late S-body in developing nephrons and by endothelium, mesangium, and distal tubular epithelium in mature nephrons. The polypeptide identified by BL26 was shown to be synthesized by fetal kidney and renal adenocarcinoma cells. The polypeptide contained no detectable carbohydrate modifications but was found in two isoforms, an acylated (26,000-Da) form and a nonacylated (24,000-Da) form. The murine antigen recognized by BL26 and the human CD9 moiety, an acylated polypeptide with a tissue distribution similar to that of the BL26 antigen, comigrated in polyacrylamide gels. We speculate that expression of the BL26 antigen and CD9 reflects processes relating to the activation of epithelial cells.     

The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy

Micro-RNAs (miRNAs) are short (average 22 nucleotides) noncoding regulatory RNAs that inhibit gene expression by targeting complementary 3'-untranslated regions of protein-encoding mRNAs for translational repression or degradation. miRNAs play key roles in both the function and differentiation of many cell types. Drosha and Dicer, two RNAase III enzymes, function in a stepwise manner to generate a mature miRNA. Previous studies have shown that podocyte-specific deletion of Dicer during development results in proteinuric renal disease and collapsing glomerulopathy (CG); however, Dicer has functions other than the generation of miRNAs. Here we found that the podocyte-specific deletion of Drosha results in a similar phenotype to Dicer mutants, confirming that the Dicer mutant phenotype is due to the loss of miRNAs. Moreover, the inducible deletion of Drosha in 2- to 3-month-old mice (Tet-On system) resulted in CG. Thus, continuous generation of miRNAs are required for the normal function of mature podocytes and their loss leads to CG. Identifying these miRNAs may provide new insight into disease pathogenesis and novel therapeutic targets in various podocytopathies.     

Renal hypoplasia: lessons from Pax2

The functions of Pax2 during renal development are many. It organizes caudal descent of the nephric duct, emergence of the ureteric bud, branching morphogenesis, and sustained arborization of the collecting system. In this review, we use lessons from the study of Pax2 as organizing principles to focus on the developmental processes which, if disrupted, might lead to renal hypoplasia in humans. We consider the problem of renal hypoplasia as a continuum, ranging from renal agenesis to subtle congenital nephron deficits. Early failure in the first two developmental stages (e.g. homozygous inactivation of Pax2) should preclude formation of metanephric kidneys and cause bilateral renal agenesis, incompatible with life. Interference with the later stages affects the extent of branching morphogenesis (e.g. heterozygous Pax2 mutations). Although the resulting nephron deficits are compatible with life, they may be moderately severe and account for up to 40% of the children in dialysis and transplant units around the world. Finally, the effect of Pax2 on apoptosis in the branching ureteric bud seems to imply a quantitative process which is finely tuned. Modest changes in this program could account for subtle nephron deficits in normal humans and increased risk of hypertension or susceptibility to acquired renal disease later in life.     

Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells

Endothelial progenitor cells are known to reverse acute kidney injury by paracrine mechanisms. We previously found that microvesicles released from these progenitor cells activate an angiogenic program in endothelial cells by horizontal mRNA transfer. Here, we tested whether these microvesicles prevent acute kidney injury in a rat model of ischemia-reperfusion injury. The RNA content of microvesicles was enriched in microRNAs (miRNAs) that modulate proliferation, angiogenesis, and apoptosis. After intravenous injection following ischemia-reperfusion, the microvesicles were localized within peritubular capillaries and tubular cells. This conferred functional and morphologic protection from acute kidney injury by enhanced tubular cell proliferation, reduced apoptosis, and leukocyte infiltration. Microvesicles also protected against progression of chronic kidney damage by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis. The renoprotective effect of microvesicles was lost after treatment with RNase, nonspecific miRNA depletion of microvesicles by Dicer knock-down in the progenitor cells, or depletion of pro-angiogenic miR-126 and miR-296 by transfection with specific miR-antagomirs. Thus, microvesicles derived from endothelial progenitor cells protect the kidney from ischemic acute injury by delivering their RNA content, the miRNA cargo of which contributes to reprogramming hypoxic resident renal cells to a regenerative program.     

Advances in renal development

Branching morphogenesis, mesenchymal cell condensation and mesenchymal-to-epithelial conversion are key steps in kidney development and morphogenesis of several other organs. Review articles describing our current knowledge of the genetic and molecular regulation of these processes have been published. Therefore, this review focuses on the important question of cell lineage specification during kidney development. We describe how new insights are being made into the specification of kidney cells in the intermediate mesoderm, as well as specification of cells that will form the renal mesenchyme, ureteric duct, nephron tubule epithelium and renal vasculature.     

Stem cells in the embryonic kidney

The mammalian kidney, the metanephros, is formed by a reciprocally inductive interaction between two precursor tissues, the metanephric mesenchyme and the ureteric bud. The ureteric bud induces the metanephric mesenchyme to differentiate into the epithelia of glomeruli and renal tubules. Multipotent renal progenitors that form colonies upon Wnt4 stimulation and strongly express Sall1 exist in the metanephric mesenchyme; these cells can partially reconstitute a three-dimensional structure in an organ culture setting. Six2 maintains this mesenchymal progenitor population by opposing Wnt4-mediated epithelialization. Upon epithelial tube formation, Notch2 is required for the differentiation of proximal nephron structures (podocyte and proximal tubules). In addition, the induction methods of the intermediate mesoderm, the precursor of the metanephric mesenchyme, begin to be elucidated. If derivation of metanephric mesenchyme becomes possible, we will be closer to the generation and manipulation of multiple cell lineages in the kidney.     

Role of extracellular matrix in kidney development and repair

Extracellular matrix (ECM) molecules and their receptors exert a dynamic role in cell-matrix interactions during kidney development and repair processes. They provide a physical substratum for the spatial organization of the cells, but also regulate cell growth and proliferation by interacting with growth factors. In addition, they can regulate signal transduction pathways by binding to integrins or by modulating the activity of signaling molecules such as Wnts. ECM and ECM-related molecules control multiple (if not all) steps of kidney development, including ureteric bud branching morphogenesis, mesenchymal condensation, nephron formation, terminal differentiation of renal tubules, and glomerular basement membrane assembly. Their role still needs to be better documented in renal repair. The emergence of conditionally mutated mice for basement membrane components will provide a useful tool to demonstrate further the involvement of ECM and ECM-related proteins in development and repair.