Biology of Renal Disease: Laboratory to Clinic

MicroRNAs and Their Role in Progressive Kidney Diseases

Mitsuo Kato, Laura Arce and Rama Natarajan
CJASN July 2009, 4 (7) 1255-1266; DOI: https://doi.org/10.2215/CJN.00520109

Abstract

MicroRNAs (miRs) are a family of short non-coding RNAs. These endogenously produced factors have been shown to play important roles in gene regulation. The discovery of miRs has greatly expanded our knowledge of gene regulation at the posttranscriptional level. miRs inhibit target gene expression by blocking protein translation or by inducing mRNA degradation and therefore have the potential to modulate physiologic and pathologic processes. The imperative need to determine their cellular targets and disease relevance has sparked an unprecedented explosion of research in the miR field. Recent findings have revealed critical functions for specific miRs in cellular events such as proliferation, differentiation, development, and immune responses and in the regulation of genes relevant to human diseases. Of particular interest to renal researchers are recent reports that key miRs are highly expressed in the kidney and can act as effectors of TGF-β actions and high glucose in diabetic kidney disease. Moreover, podocyte-specific deletion of Dicer, a key enzyme involved in miR biogenesis, led to proteinuria and severe renal dysfunction in mice. Hence, studies aimed at determining the in vitro and in vivo functions of miRs in the kidney could determine their value as therapeutic targets for progressive renal glomerular and tubular diseases. Translational approaches could be facilitated by the development of effective inhibitors of specific miRs and methods for optimal delivery of anti-miRs to the kidney. The major goal of this review is to highlight key functions of these miRs and their relationships to human diseases, with special emphasis on diabetic kidney disease.

MicroRNAs (miRs) are a family of short, non-coding RNAs that are approximately 22 nucleotides long. These endogenously produced transcripts have been shown to play important roles in gene regulation (1–12). The discovery of miRs, only approximately 15 yr ago, has greatly expanded our knowledge of gene regulation and provided a new perspective on the mechanisms of gene expression under disease states. In the early 1990s, the first miR, lin-4, was reported as a small, non-coding RNA controlling a specific step in developmental timing in Caenorhabditis elegans by downregulating a conventional protein-coding gene (lin-14) (13,14). The lin-14 3′ untranslated region (3′UTR) harbors multiple sites of imperfect complementarity to the lin-4 small RNA. lin-4 binds to these sites and blocks lin-14 translation. In 2000, the second miR (let-7) and its target, lin-41, were discovered and shown to be conserved across species (1,2,15). These important discoveries provided the foundation for our current understanding of mammalian miR function. Estimates indicate that more than 1000 human miRs target and downregulate at least 60% of human protein-coding genes expressed in the genome (4,8,16–20). It is therefore not surprising that there is an unprecedented explosion in miR research to determine their biologic functions and disease relevance.

Recent findings have revealed critical functions for specific miRs in several cellular and biologic processes, including proliferation, differentiation, and development, and in the regulation of genes relevant to cancer, insulin secretion (5,11,21,22), modulation of immune responses in macrophages (23), and cardiac and muscle differentiation (24–26). Because miRs are important regulators of gene expression, misregulation or mutations of miRs are expected to play key roles in several diseases (5,11,27,28). Cancer was one of the first diseases to be related to abnormal expression or actions of miRs (5,11,27). Several miR genes have been detected at chromosomal breakpoints associated with cancer (29,30). miRs with both tumor suppressor and oncogenic properties have been reported (31–38), and miR research is very active in the cancer field. In addition, miRs are highly expressed in the cardiovascular system and play important roles in cardiovascular development, biology, and pathology (39). Key miRs have been implicated in cardiomyocyte differentiation, growth, and hypertrophy (24,25,40–42). In addition, miR-21 was implicated in vascular smooth muscle cell proliferation and neointimal thickening (43) and myocardial disease (44). Thus, several groups are actively evaluating miRs as potential therapeutic targets for various vascular and cardiac diseases.

Of particular interest to the renal community are reports showing that a cluster of key miRs are highly expressed in the kidney and that there are differences in the miR expression profile in renal cortex versus medulla (45,46); however, it was not until recently that specific roles of miRs in renal function were investigated. These studies revealed that key miRs can play roles in TGF-β1 actions and diabetic kidney disease (47,48) and that podocyte-specific deletion of Dicer led to progressive glomerular and tubular damage along with proteinuria and other podocyte defects in mice (49–51). As such, there is increasing interest in evaluating the in vitro and in vivo functions of miRs in the kidney and thereby determine their value as therapeutic targets for renal glomerular and tubular diseases. In this review, we highlight key functions of miRs and their relationships to human diseases, with emphasis on diabetic nephropathy.

Biogenesis and Mechanism of Action of miRs

miR transcripts initially originate as long primary miRs (Figure 1). Primary miRs are processed to a stem-loop (hairpin) structure fragment termed precursor miR in the nucleus by an RNase III enzyme, Drosha, in complex with the double-strand RNA–binding protein DGCR8 (10,12). The approximately 70-nucleotide hairpin structure precursor miRs are then exported to the cytoplasm by Exportin-5 and further cleaved to RNA duplexes by a second RNase III family enzyme, Dicer (10,12). The miR duplexes are then unwound, and one strand, termed “mature miR” guide strand, which contains complementarity to mRNA targets, is loaded into the RNA-induced silencing complex (RISC) (6,10,12,52), which contains Argonaute 2, Dicer, and transactivating response RNA-binding proteins. miRs in the RISC complex then guide the recognition of target genes. If the complementarity is perfect, then RISC usually cleaves the target mRNA (classical RNA interference); however, if the complementarity is not perfect, then RISC induces translational repression of target genes by targeting their 3′UTR (Figure 1) (6,8,10,11). Although complete complementarity is not required for miR-mediated regulation of a target transcript, the “seed sequence,” namely seven key nucleotides of the miR 5′ sequence, is critical for target recognition and inhibition (18,20), whereas other factors, such as a bulge in the central region and reasonable complementarity even at miR 3′ sequence, have also been suggested (53,54). miRs inhibit the initiation and elongation steps of translation to reduce protein expression (55–58). They can also repress gene expression by sequestering targeted mRNAs to cytoplasmic mRNA processing bodies for degradation (59–61). In addition to their role in such posttranscriptional repression, miRs are now implicated in transcriptional gene silencing by targeting the promoter region (62). Thus, miRs can inhibit gene expression via translational repression, target mRNA degradation, or transcriptional inhibition.

Figure 1.
Figure 1.

Biogenesis and mechanism of action of microRNAs (miRs). miR transcripts initially originate as primary miRs that are then processed into precursor miRs, which are further cleaved to result in miR–miR duplexes. The miR duplexes are then unwound, and the mature miR guide strand is loaded into the RNA-induced silencing complex (RISC) complex. miRs in the RISC complex then guide the recognition of target RNAs to induce their downregulation depending on the type of complementarity.

Mouse models of Dicer, Dgcr8 or Argonaute 2 knockout, displayed embryonic lethality (63–66). Tissue-specific conditional Dicer knockout mice showed defects in various organs, suggesting that miRs are essential for specific tissue and organ development (3,24,67–69). Cardiac development defects were observed in miR-1–to -2–deficient mice (24) and cardiac hypertrophy in miR-208–deficient mice (25), whereas miR-133 was shown to control cardiac hypertrophy (67). Myeloid-specific miR-223 could regulate progenitor cell proliferation and granulocyte function by targeting Mef2c (68). Thus, miR function seems essential not only for embryonic development but also for adult tissue/organ development; therefore, miRs can be expected to play important roles in disease.

miRs in Diabetes and Metabolism

Whereas several studies have evaluated the role of miRs in cancer, much less is known in the field of diabetes. Overexpression of miR-375, an islet-specific miR, could suppress glucose-induced insulin secretion and, conversely, inhibition of endogenous miR-375–enhanced insulin secretion (21). Myotrophin, a protein that inhibits insulin secretion, was a predicted target of miR-375. Recent reports showed that miR-375 also targets phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biologic response in pancreatic β-cells (70), whereas miR-9 could control insulin secretion (71). A correlation between miR-204/211 expression and insulinoma also suggests that these miRs may be related to insulin secretion (72). miR-122, a liver-expressed miR, was implicated in cholesterol and lipid metabolism and hence a potential target for the treatment of hypercholesterolemia (73,74). miR-124a was shown to play a role in glucose homeostasis (28,75) by targeting Rab27 and FOXA2 and MTPN. FOXA2 may regulate several targets that are relevant to diabetes, including insulin and potassium channel subunits, and also affect glucose homeostasis and pancreas development (28,75). It is interesting that 27 miR genes were reported to be located in nine of 19 insulin-dependent diabetes (IDDM) susceptibility loci (76). Their potential targets were autoimmune- and β cell–related genes. miR-192 and miR-375 were located in IDDM4 and IDDM13, respectively. A single-nucleotide polymorphism (SNP) identified in the miR-192 precursor sequence might alter its secondary structure to play a role in diabetes (77). These results suggest that specific miRs may serve as novel targets for the treatment of diabetes.

Diabetic Nephropathy and TGF-β Actions

Diabetic nephropathy (DN) is a progressive kidney disease and a major debilitating complication of both type 1 and type 2 diabetes that can lead to ESRD and related cardiovascular disorders. The cellular mechanisms underlying diabetes-induced dysfunction of key renal cells have been studied extensively, and several therapies for DN are available; however, patients with diabetes are still reaching ESRD at alarming proportions, and it therefore is imperative to evaluate newer molecular mechanisms and therapeutic targets.

Histologically, DN is characterized by glomerular basement membrane thickening, mesangial expansion, and extracellular matrix (ECM) accumulation. This ECM accumulation is due to coordinate alterations in ECM proteins such as types I and IV collagen, laminin, and fibronectin (78–80); ECM regulatory enzymes such as matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (81,82); and growth factors such as PDGF, TGF-β, and angiotensin II (83–86). Podocyte effacement and albuminuria are also major features of DN (87,88). Factors that are relevant to the pathogenesis of DN can increase TGF-β expression in glomerular mesangial cells (MCs) in vitro and in vivo (84,89–93). TGF-β is a profibrotic agent with several effects in renal cells, including the production of ECM proteins such as type I and II collagens, laminin, fibronectin, and plasminogen activator inhibitor-1 (84,90,91,94–96). Thus, TGF-β has been studied as a major target for DN treatment (97).

Smad transcription factors have been studied extensively as the major effectors of TGF-β signaling (98,99). Evidence shows that ECM genes including collagen type I-α1 and 2 (Col1a1/a2) are regulated by TGF-β via Smads in MCs (96,100). Interaction of TGF-β with its receptors induces the phosphorylation and nuclear translocation of the receptor-regulated Smad2/3 transcription factors (98,99,101) to regulate gene expression (95,96,100,102). TGF-β also activates the phosphoinositide-3-kinase/Akt kinase pathway in MCs (103–110), and this has been implicated in its fibrotic responses, such as expression of collagen and fibronectin (105,106). Activated Akt kinase phosphorylates several downstream proteins, including GSK3-β and Forkhead (FoxO) transcription factors, to control cell growth, survival, and protein synthesis (111,112). Akt activated by TGF-β phosphorylates and inactivates FoxO3a and thereby downregulates key FoxO3a targets, such as the proapoptotic Bim and antioxidant manganese superoxide dismutase genes (103,109). The combination of these events in response to TGF-β can result in enhanced MC survival, oxidative stress, and hypertrophy and thereby accelerate kidney diseases such as DN.

Although several studies showed that TGF-β signaling events are crucial in regulating its fibrotic effects in MCs and other renal cells, the subtle molecular mechanisms are not fully clear. Using cDNA microarray profiling of genes regulated by TGF-β in mouse MCs (47), we discovered that TGF-β decreases the expression of Zeb1, also known as δEF1 (Figure 2). ZEB1 is a repressor of E-cadherin (113), osteocalcin (114), and E2 box (115). Notably, it is also a repressor of collagen type I and type II genes in various cells (116–118). E-box elements are located in the far-upstream enhancer region of the collagen gene (116,118). It is interesting that TGF-β also decreased the expression of another E-box repressor belonging to the Zeb1 family, namely Zeb2 (also called Smad-interacting protein-1) (119). These results suggested that TGF-β can increase Col1a2 gene expression in MCs by downregulating the E-box repressors ZEB1 and ZEB2 (Figure 2) (47).

Figure 2.
Figure 2.

miR-dependent mechanisms for TGF-β–induced expression of extracellular matrix (ECM) genes related to the pathogenesis of diabetic nephropathy (DN). The expression of miR-192 is increased in diabetic kidney glomeruli in mouse models along with that of collagen type I-α 2 (Col1a2) and TGF-β expression (47). Col1a2 expression is increased by TGF-β or diabetic conditions via decrease in ZEB2 targeted by miR-192 upregulated by TGF-β in mesangial cells (MCs) (47). Because E-boxes are also present in the upstream genomic regions of the miR-200 family, miR-200 family members might themselves be regulated by ZEB1 and ZEB2 (136,138). miR-192 may initiate signaling from TGF-β also to upregulate the miR-200 family, and then the miR-200 family might amplify or accelerate the signaling by further upregulating themselves via downregulation of E-box repressors (Zeb1/Zeb2). The miR-192–regulated circuit may amplify TGF-β signaling under diabetic conditions. miR-377 has also been shown to induce fibronectin (ECM protein) expression via downregulation of manganese superoxide dismutase (MnSOD) and p21-activated kinase (PAK1) in MCs (48).

miRs in the Kidney: Role in TGF-β Actions, ECM Production, and the Diabetic Kidney

Sun et al. (45) identified five miRs (192, 194, 204, 215, and 216) that were highly expressed in human and mouse kidney using miR microarray. Tian et al. (46) combined miR microarray and proteomics to identify a differential profile of miR expression in rat renal cortex versus medulla, as well as several miR-target protein pairs. Recent reports using new proteomic approaches to profile and identify miR targets demonstrated that miRs repress their targets at both the mRNA and translational levels and that the effects are mostly relatively mild (120–122). Such complementary methods are expected to identify key miRs more accurately as well as their targets under normal versus disease states.

Reports have implicated miRs in TGF-β signaling and actions in various systems. Zebrafish miR-430 was found to affect the expression of the Nodal (TGF-β) agonist and antagonist (123). Xenopus laevis miR-15 and miR-16 could alter development processes by targeting the Nodal type II receptor (124). miR-24 was implicated in the inhibition of skeletal muscle differentiation by TGF-β (26). Certain gastric cancer cells were resistant to TGF-β because the miR-106b-25 cluster was upregulated and inhibited the expression of p21 (cell-cycle arrest gene) and Bim (apoptotic gene) that are downstream of TGF-β (125). It is interesting that the processing of miR-21 was reported to be enhanced by Smad proteins phosphorylated by TGF-β (126).

We demonstrated that the expression miR-192, one of the highly expressed miRs in human and mouse kidney (45) and rat kidney cortex (46), is increased in renal glomeruli obtained from mouse models of type 1 diabetes (streptozotocin injected) and type 2 diabetes (db/db mice) relative to the corresponding control mice (47). It is interesting that the expression of miR-192 was increased by TGF-β in mouse MCs, whereas, conversely, the expression of its target, Zeb2, was decreased (47). This also paralleled the increased Col1a2 and TGF-β expression (47). These results suggested that the increase in TGF-β in vivo in diabetic glomeruli and in vitro in MCs can induce miR-192 expression, which can target and downregulate Zeb2 thereby to increase Col1a2. This is supported by the report showing that miR-192 is upregulated in human MCs treated with high glucose (48). Recent computational analysis of miRs and mRNA in colon cancer cells also revealed ZEB2 as a target of miR-192 (127). The molecular mechanisms by which miR-192 is induced by TGF-β needs further investigation. It is interesting that the human miR-192 promoter is regulated by hepatocyte nuclear factor 1α (128), a factor whose mutations have been associated with maturity-onset diabetes of the young (129).

TGF-β induced downregulation of Zeb2 (via miR-192), and Zeb1 (via potentially another miR) can cooperate to enhance Col1a2 expression via de-repression at E-box elements (47) (Figure 2). A recent report also showed that bone morphogenic protein 6–induced miR-192 decreases the expression of ZEB1 in breast cancer cells (130). Enhanced expression of miR-192 and collagen genes were reported in nasopharyngeal carcinomas (131). Thus, miR-192 might regulate collagen expression by targeting E-box repressors not only in kidney disease but also in various cancers.

Several articles have shown that the miR-200 family targets ZEB1 and ZEB2 (132–138). We also observed that Zeb1 is a target of certain miR-200 family members that are also upregulated by TGF-β in MCs (M.K. and R.N., unpublished observations). Thus, TGF-β–induced increase in the expression of key miRs (miR-192 and miR-200 family members) might coordinately downregulate E-box repressors Zeb1 and Zeb2 to increase Col1a2 expression in MCs related to the pathogenesis of DN. The proximal promoter of the Col1a2 gene responds to TGF-β via Smads and SP1 (100). Conversely, the downregulation of ZEB1 and ZEB2 by TGF-β via miR-200 family and miR-192 can affect upstream E-box regions (47). Because E-boxes are also present in the upstream genomic regions of the miR-200 family, miR-200 family members may themselves be regulated by ZEB1 and ZEB2 (136,138). It is possible that the miR-200 family upregulated by TGF-β or in diabetic glomeruli under early stages of the disease can also regulate collagen expression related to diabetic kidney disease by targeting and downregulating E-box repressors. miR-192 might initiate signaling from TGF-β to upregulate miR-200 family members, which subsequently could amplify the signaling by further regulating themselves through downregulation of E-box repressors (Figure 2). Such events could lead to progressive renal dysfunction under pathologic conditions such as diabetes, in which TGF-β levels are enhanced. Conversely, there are several reports that miR-200 family members and miR-192 can be downregulated by TGF-β, and this promotes epithelial-to-mesenchymal transition (EMT) in cancer and other kidney-derived epithelial cell lines via subsequent upregulation of targets ZEB1 and ZEB2 to repress E-cadherin (135–139). Thus, the effects of renal miRs may be cell type specific, and the miR signaling networks that mediate the effects of TGF-β on MCs and epithelial cells and on metastatic and fibrotic EMT may not be identical.

These miR circuits may also operate in cancer, because cyclin-dependent kinase inhibitor p21-mediated cell-cycle arrest but not apoptosis of cancer cells by miR-192 via the p53 pathway was recently reported (140–142). miR-192 activated the promoter of the antiapoptotic Survivin gene (143); therefore, miR-192 likely induces only cell-cycle arrest but not apoptosis. Given that the p53 and Survivin promoters have E-boxes (144,145), these genes may be regulated by ZEB1 and ZEB2 targeted by miR-192 and miR-200 family. Because p21-mediated cell-cycle arrest plays a role in MC hypertrophy (146), miR-192 might also promote glomerular hypertrophy by activating the p21 and p53 pathways.

Wang et al. (48) demonstrated that in cultured human MCs exposed to high glucose or TGF-β, as well as in mouse DN models in vivo, there was a significant upregulation of miR-377 that could downregulate p21-activated kinase and manganese superoxide dismutase and thereby enhance fibronectin production. Thus, miR-377 might also be a key regulator of DN. Recent studies showed that Akt kinase is activated via downregulation of Pten (phosphatase and tensin homolog) targeted by miR-216a and miR-217 that are upregulated by TGF-β in mouse MCs (109). Furthermore, these miRs could promote MC hypertrophy and survival similar to TGF-β (109). It is anticipated that other miRs that are expressed not only in MCs but also in podocytes and tubular and other renal cells and that regulate renal functions under diabetic conditions will be reported in the near future.

Other Kidney Diseases

Studies showed that key miRs are also involved in nondiabetic kidney diseases. miR-15a was reported to modulate the expression of the cell-cycle regulator Cdc25A and affect hepatic cystogenesis in a rat model of polycystic kidney disease (147). A microarray-based study in rats revealed that 30 miRs were differentially expressed in polycystic kidney disease, and two of these, miR-31 and miR-217, were not previously identified in the kidney (148). A comprehensive study of human lupus nephritis identified 66 miRs that were differentially expressed between patients with lupus nephritis and healthy control subjects (149). miR-17-92 cluster targets Pten and Bim and, interestingly, transgenic mice of miR-17-92 cluster had enlarged kidney glomeruli, hypercellularity, mesangial expansion, and proteinuria, features similar to DN (Table 1) (32).

View this table:
Table 1.

miRs with known functions in the kidneya

Recently, a series of reports by investigators who used mice with podocyte-specific deletion of Dicer suggested that miRs play critical roles in podocyte biology and pathology (49–51). It is interesting that all three reports showed major renal abnormalities in these mice. The major phenotypes were proteinuria, podocyte foot process effacement, changes in podocyte genes, and glomerular basement membrane abnormalities. There was a rapid progression of renal disease, glomerulosclerosis and tubulointerstitial fibrosis, and renal failure and death by 6 to 8 wk. The investigators also identified changes in specific miRs (especially miR-30 family) under these conditions and their potential involvement in glomerular disease (Table 1) (49–51). It is interesting that the miR-30 family is reported to target connective tissue growth factor (150), a profibrotic factor that is also downstream of TGF-β. Thus, the targets of these miRs may regulate critical glomerular and podocyte functions. These exciting studies highlight the essential roles of Dicer and miRs in renal physiology and pathology.

Clinical Applications, Therapeutic Strategies, and Perspectives

Overwhelming evidence implicating miRs in the pathology of key human diseases has sparked tremendous interest in development of modalities to block specific miRs and their function in vitro and in vivo. Small non-coding RNAs and miRs, such as miR-192 and miR-377, may be novel targets for DN and other diabetic complications. Vector-based expression of small interfering RNA is a powerful tool to inhibit the expression of targets in vitro and in vivo (151) that has shown promise in the treatment of certain diseases (152,153). Expression of tandem repeats of miR-binding sites (Decoy or Sponge) is an efficient method to inhibit miR action (67,154), as well as chemically modified oligonucleotide (oligo) inhibitors (74,155). Cholesterol-tagged small interfering RNA against 12/15-lipoxygenase, whose expression is enhanced under diabetic conditions (156), ameliorated key features of DN in mice with type 1 diabetes (157). Such cholesterol-tagged antagomirs targeting miR-122 were also very effective in vivo (74). Thus, chemically modified inhibitors (antagomirs, or locked nucleic acid–modified anti-miRs) (44,74,155) targeting key miRs seem to be efficient inhibitors of miR actions in vivo and may be developed as therapies for the prevention and treatment of human DN. Inhibition of miR-122 by 2′-O-methoxyethyl phosphorothioate antisense oligos, cholesterol-tagged 2′-O-Me antisense oligo, or antisense locked nucleic acid (LNA–anti-miR) modified oligos improved hypercholesterolemia in mouse models (73,74,158). LNA–anti-miR-122 also reduced plasma cholesterol levels in a nonhuman primate model without any toxicity, thereby illustrating the potential of such modified oligos to be developed as a new class of therapeutics for disease-associated miRs (155). miR inhibitors, such as LNA–anti-miRs or antagomiRs, injected into animal models of kidney diseases could be evaluated for potential use in similar human kidney disorders (Figure 3).

Figure 3.
Figure 3.

Treating renal disorders in mouse models by targeting specific miRs in vivo with oligonucleotide (oligo) inhibitors such as locked nucleic acid (LNA)-modified anti-miRs or other chemically modified antagomiRs. Cholesterol-tagged anti-miR oligos (antagomiRs) or LNA-modified oligo anti-miRs could be developed as efficient inhibitors of key disease-related miRs. The chemistry of these oligos can be engineered for optimal renal cell targeting, accumulation, and miR inhibition in vivo. Both type 1 and type 2 diabetic mice could be tested by injecting them with specific miR inhibitors or control oligos and examining whether they can prevent or delay key features of DN. Such studies will determine whether the increased rates of DN in the diabetic mice can be attributed at least in part to the aberrant expression of the specific miR being targeted.

It is now clear that key miRs are expressed in specific renal cells; modulate the actions of TGF-β and diabetic conditions; alter MC and podocyte functions; and lead to ECM accumulation, podocyte dysfunction, albuminuria, and EMT and thereby affect renal physiology. This picture is just emerging, and we will undoubtedly see increasing numbers of reports of other miRs and their targets with multiple functions in the kidney. Future studies with overexpression or deletion of individual miRs in a cell-specific manner would provide very useful data on their role in renal biology and pathobiology. Reports of single miR knockouts in a whole-animal or tissue-specific manner have shown dramatic phenotype (24,25,67,68), confirming that miRs can indeed play critical roles in mammalian organ physiology or pathology. Studies in the kidney are still in their infancy but represent an exciting avenue for new therapies for debilitating renal diseases. These translational approaches can be further facilitated by examining the expression of miRs and their targets in renal biopsies obtained from patients with kidney disease. Recently, SNPs that could affect response to miRs were noted in miR target site sequences (159,160). Correlations between miR polymorphisms and SNPs in miR target sites with risk of bladder and colorectal cancers were reported (161–163). Hence, examination of SNPs in miR-binding sites may also provide key insights into various human diseases. Given the unprecedented progress in miR research, we anticipate several discoveries to be reported in the upcoming years.

Glossary of Terms

Apoptosis/apoptotic: Programmed cell death

DGCR8: DiGeorge syndrome critical region gene 8, an essential co-factor for Drosha

Dicer: A key enzyme involved in miR biogenesis in cytoplasm

Drosha: Another key enzyme involved in miR biogenesis in the nucleus

E-box: DNA consensus sequence CANNTG (where N is any nucleotide) that typically lies upstream of a genomic promoter sequence and recruits basic helix-loop-helix transcription factors to regulate the transcription of the downstream gene

ECM: The extracellular tissue/material secreted by cells and can provide mechanical support for the cells in addition to performing various other important functions.

Glomeruli/glomerulus: A capillary tuft surrounded by Bowman's capsule in renal nephrons

Glomerular basement membrane: Filtration structure consisting of the basal laminal portion of the glomerulus

Hypertrophy: An increase in size of an organ/cell or in a select area of the tissue

miR: Short, non-coding RNAs that regulate gene expression

Nephropathy: Damage to or disease of the kidney

Non-coding RNA: RNAs that are not translated to proteins; they serve to regulate gene expression or other cellular processes

Oligonucleotide: A short nucleic acid polymer, usually containing ≤20 bases

Phosphorylation: Addition of a phosphate group to a protein/chemical

Podocyte: Cells in the renal visceral epithelium forming part of the glomerular filtration barrier

Progenitor cell: A cell that can differentiate into a specific type of cell

Promoter: Regulatory genomic DNA sequence that regulates transcription, typically located upstream of the gene

Proteinuria: Presence of excess serum proteins in the urine, a hallmark of DN and typically indicative of renal malfunction

Renal cortex: Portion of the kidney between the renal capsule and the renal medulla

Renal medulla: Innermost part of the kidney

Target gene: Gene regulated by a given miR

TRBP: Transactivating response RNA-binding protein: A co-factor for Dicer

Transcription: The process of copying DNA to RNA by an enzyme called RNA polymerase

Translation: The first stage of protein biosynthesis, via the production of proteins by decoding mRNA and generating an amino acid polymer.

Tubules: Small renal structures that filter blood and produce urine.

Disclosures

None.

Acknowledgments

The authors are supported by grants from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, and the Juvenile Diabetes Research Foundation.

We are grateful to members of the Natarajan laboratory for helpful discussions and suggestions.

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