Dicer Cuts the Kidney
Authors: J.J. David Ho, Philip A. Marsden
Journal: Journal of the American Society of Nephrology (2008)

We should occasionally stop, take a breath, and reflect about what is happening at the interface between medicine and science. Collectively, we are uncovering paradigms that are relevant to understanding disease at a frantic pace. One can only turn to wonder, in this moment of reflection, when trying to imagine what it must feel like as a treating physician trying to filter all of these new concepts. Which advances are relevant? Am I filtering out those that are merely incremental? Is this concept transformative? Does this article have the potential to affect my understanding of the cause, prevention, and treatment of kidney disease? Recognizing an article that reports a landmark discovery is a challenge. An excellent example is Fire and Mello's1 discovery that double-stranded RNA (dsRNA) could potently and specifically silence the function of an endogenous gene in nematode worms. This obscure report from a decade ago, effectively teaching us about a new pathway for how dsRNA is "cut" by RNases within a cell to generate a silencing signal, heralded the beginning of what has become the RNA interference (RNAi) revolution. The 2006 Nobel Prize in Physiology and Medicine was awarded to Fire and Mello for this discovery. In this issue of JASN, three articles provide compelling and concordant evidence that RNAi is critically important for the kidney.2–4 Kidneys fail when the RNAi pathway is disrupted in podocytes of the glomerulus. These articles underscore that the newer concept of RNAi is relevant to our understanding of the cause of kidney disease. RNAi has evolved from a newly appreciated, erudite pathway to an essential technique in the molecular biologist's toolbox in the past 10 yr. As first described, RNAi is mediated by short dsRNA and involves the sequence-specific endonucleolytic cleavage and degradation of messenger RNA (mRNA) that is elicited by the base pairing of two short complementary RNA strands, each approximately 22 nucleotides (nt) in length. These are termed small interfering RNA (siRNA) duplexes and are generated in the cell cytoplasm from the cleavage of endogenous long dsRNA or from short "hairpin" RNA molecules (shRNA). The introduction of exogenous siRNA into cells is an efficient way of silencing genes within cells and, we suspect, is how most scientists first became aware of the promise offered by this approach. After its initial discovery, it was quickly revealed that RNAi is an ancient, widely conserved genomic defense mechanism that involves several classes of small RNA.5–11 The microRNA (miRNA) class is one group of these endogenous small RNA. They are especially important because, like siRNA, they also silence gene function.12–14 In brief, RNAi and miRNA both result in decreased levels of functional protein within cells, but RNAi tends to decrease steady-state mRNA levels, whereas miRNA usually impairs the efficiency with which mRNA is translated into protein on the ribosomes. Where do miRNA come from? Hundreds of discrete regions within the genome are transcribed by RNA polymerase II as pri-miRNA species (Figure 1). Most of these are evolutionarily conserved across the 65 million yr that separate humans from mice. Pri-miRNA get trimmed in the nucleus by a microprocessor complex containing the Drosha RNase into smaller approximately 60- to 70-nt precursor molecules, termed pre-miRNA. After being transported into the cytoplasm by an exportin-5–dependent process, the pre-miRNA get a final key "cut" by an RNase known as Dicer.15–20 This enzyme is a key step in this biosynthetic pathway. Both siRNA and miRNA are "cut" or processed into approximately 22-nt RNA duplexes with characteristic 2-nt 3′ overhangs. In addition, Dicer is involved in the immediate downstream effector steps of this pathway, whereby it serves as an essential component of miRNA-containing catalytic enzyme complexes, such as the RNA-induced silencing complex.21–24 Thus, Dicer functions both as a physical platform and a functional bridge that couples miRNA biogenesis to miRNA-mediated gene silencing. A thorough understanding of the functional impact of Dicer is critical because of its vital role in RNAi. In contrast to lower species, such as plants, mammals have only one Dicer protein. Importantly, Dicer knockout in every cell of a mouse results in early embryonic lethality25; therefore, at least during development, Dicer is a key enzyme. Then came the task of sorting out the tissue and cell specificity of Dicer function. To date, 22 articles have addressed this question, although none has been done in the kidney. In this issue of JASN, three independent groups—Ho et al., Shi et al., and Harvey et al.—report the phenotypes of mice with podocyte-specific genetic ablation of Dicer.2–4 The overall goal of these three studies was to examine the impact of ablating Dicer activity, because it is key to miRNA production, on the phenotype of podocytes and, ultimately, glomerular and kidney function. Most endogenous miRNA function as a sophisticated conductor of genetic pathways by modifying the translational regulation of a number of genes at the same time. The specificity of this manipulation is relatively low, in part because target mRNA species contain sequences that are only partially related to the complementary miRNA sequence. Instead of looking at the specific function of one unique miRNA, targeting the enzyme that produces the bulk of miRNA allows for a more global view of their collective action. The groups reporting in this issue of JASN used independent, wild-type floxed Dicer alleles as a target26,27 and expressed Cre recombinase only in podocytes (NPSH2 promoter). This strategy allows for podocyte-specific gene rearrangements of the Dicer gene. When done correctly, the gene rearrangement disrupts gene function. Key to interpreting these tissue-specific studies is that every other cell in the germline has functional forms of the Dicer gene. Severe physiologic morbidity and mortality have previously been reported for other tissue-specific Dicer knockouts. For example, seven of the 22 tissue-specific Dicer ablations and especially those in key organs result in lethality. The combined findings of the three groups that have focused on the kidney reinforce concepts derived from Dicer-knockout studies in other tissues and provide unique insight into the essential role of Dicer in the glomerulus. Given the similarities between the knockout strategies, it is not surprising that all three groups produced largely consistent observations. Briefly, the major phenotypes were proteinuria, foot process effacement, and glomerular basement membrane abnormalities by approximately 2 to 4 wk. Notably, all groups reported the rapid progression of disease phenotypes, with accompanying pathologic features of glomerulosclerosis and tubulointerstitial fibrosis, such that advanced kidney failure and death was evident at approximately 6 to 8 wk. Importantly, it is argued that phenotypes are attributable to the global loss of pre-miRNA processing because of Dicer ablation. It is refreshing for readers that the three groups used different approaches that, when the data are taken together, allow a unique window on the role of RNAi on the glomerulus. Although each article can be criticized for missing a key piece, each one also adds a wonderful new perspective. For instance, Ho et al.3 provide key evidence that Dicer is disrupted. Using elegant approaches, they demonstrated that pre-miRNA accumulate when Dicer function is lost. Assessments of global patterns of miRNA expression by microarray analyses by Ho et al. give valuable baseline information with regard to miRNA that are abundant in the kidney, miRNA that are restricted in expression to the specific cell types of the kidney, and miRNA that are regulated in kidney disease. All three groups identify members of the miR-30 miRNA family as kidney-specific and important players in the development of the observed phenotypes.2–4 Shi et al.4 provide evidence that miRNA are involved in the regulation of apoptosis and cytoskeletal formation. Future studies should focus on identifying miRNA that are selectively expressed in specific kidney cell types, such as glomerular endothelial cells, podocytes, or proximal tubular renal epithelial cells. The findings of these studies bring up several key concepts that are broadly relevant to Dicer function and miRNA biogenesis in living organisms. First, all three studies provided clear evidence that Dicer is qualitatively essential for normal glomerular and kidney function. It would be interesting, then, to ask whether the quantity of functional Dicer protein present in the cell is important. Heterozygous mutant Dicer mice are, in general, phenotypically indistinguishable from wild-type counterparts under baseline conditions; however, Harvey et al. note that mice carrying a disrupted Dicer gene in every cell of the body and one podocyte-disrupted allele (DicerΔ/fl) exhibit a more severe phenotype than mice that carry two podocyte-disrupted alleles (Dicerfl/fl).2 All three studies suggest that neighboring cell types become activated secondary to Dicer-dependent podocyte injury.2–4 Indeed, such secondary responses may be functionally important in mediating the progression of kidney disease and therefore warrant further investigation. Dicer is an enzyme. Perhaps there is a threshold for Dicer activity below which Dicer-dependent cellular processes are impaired. With an injured podocyte, half as much Dicer in a glomerular endothelial cell may not be enough. To date, most studies have focused on miRNA levels, mRNA, or protein regulation, all of which are downstream of Dicer activity; however, not much attention has been given to how Dicer itself may be regulated under physiologic conditions and, more important, whether and how Dicer regulation is affected in disease conditions such as glomerular injury. Multiple Dicer mRNA variants exist, some of which are expressed in a tissue-specific manner, and they themselves are regulated by translation.28 Thus, it is plausible that Dicer is activated, at least in part, through translational enhancement under cellular stress. New evidence highlights that abnormalities in the regulation of the RNAi/miRNA biogenesis pathway may be relevant to disease.29–31 Clearly, disruption of this pathway causes glomerular injury. Which genes in the glomerulus are targets for Dicer? Because 30% of human genes may be regulated by miRNA,32 we can only speculate. As an example, analyses of miRNA (Ho et al.),3 mRNA (Shi et al.),4 and protein levels (Harvey et al.)2 all point to disruption of the normal cytoskeleton as an early consequence of impaired miRNA biogenesis and function; however, global analyses of mRNA and protein expression, performed by Shi et al. and Harvey et al., respectively, remind us the ablation or reduction of Dicer activity does not simply lead to loss of inhibition of miRNA-mediated gene silencing.2,4 Such a response would mean more protein. Indeed, both groups observed that levels of some mRNA and proteins go down. At first glance, this seems to be a paradox, but as Harvey et al. correctly point out, such decreased expression could reflect downstream effects of ablating Dicer, especially if inhibitors of gene expression are regulated by a specific miRNA.2 Finally, we should also bear in mind that miRNA are not the only targets of Dicer. Others have suggested that RNAi first evolved as an antiviral defense mechanism directed against dsRNA viruses. In summary, Fire and Mello discovered that dsRNA could potently and specifically silence the function of endogenous gene. Now we know that this fresh paradigm is relevant to the kidney. Our challenge is to consider how this is important in our understanding of the cause, prevention, and treatment of kidney disease. We are offered hopes for advances in therapy. For instance, the rapid development of RNAi-based therapeutics in hepatitis C (anti–miR-122) affirms the promise of the method in patients with chronic viral hepatitis, a sad burden for many patients with ESRD, yet setbacks in exploiting RNAi as a potential therapy are to be expected and serve as sobering reminders that the design of safe and effective RNA-based therapies will depend on a comprehensive understanding of the endogenous mechanisms involved.33 These three groups are to be commended for teaching us that "Dicer cuts the kidney" and that miRNA are critical in the maintenance of glomerular homeostasis. DISCLOSURES None.Figure 1: Tissue-specific Dicer knockout in the mouse kidney. Pri- and pre-miRNA is an endogenous product of the transcription of host genomic DNA. Exportin-5 is a key component of the machinery used to export the pre-miRNA to the cytoplasm, where it is processed by Dicer. Dicer is a key enzyme that catalyzes the final maturation step of miRNA biogenesis, specifically the production of miRNA from pre-miRNA. In the cytoplasm, mature miRNA are incorporated into Dicer- and Argonaute (Ago)-containing RNA-induced silencing complexes (RISC), which mediate the degradation of mRNA targets by endonucleolytic cleavage and/or translational inhibition of specific target mRNA. In this issue of JASN, three independent groups report that podocyte-specific ablation of Dicer function injures the glomerulus at approximately 2 to 4 wk with ensuing glomerulosclerosis and tubulointerstitial fibrosis leading to end-stage kidneys at approximately 6 to 8 wk.P.A.M. is a Heart and Stroke Foundation of Canada Career Investigator and is supported by a grant from the Heart and Stroke Foundation of Canada (T-5658).