Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron.

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.

Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs.

MicroRNA biomarkers in clinical renal disease: from diabetic nephropathy renal transplantation and beyond.

Chronic Kidney Disease (CKD) is a common health problem affecting 1 in 12 Americans. It is associated with elevated risks of mortality, cardiovascular disease, and high costs for the treatment of renal failure with dialysis or transplantation. Advances in CKD care are impeded by the lack of biomarkers for early diagnosis, assessment of the extent of tissue injury, estimation of disease progression, and evaluation of response to therapy. Such biomarkers should improve the performance of existing measures of renal functional impairment (estimated glomerular filtration rate, eGFR) or kidney damage (proteinuria). MicroRNAs (miRNAs) a class of small, non-coding RNAs that act as post-transcriptional repressors are gaining momentum as biomarkers in a number of disease areas. In this review, we examine the potential utility of miRNAs as promising biomarkers for renal disease. We explore the performance of miRNAs as biomarkers in two clinically important forms of CKD, diabetes and the nephropathy developing in kidney transplant recipients. Finally, we highlight the pitfalls and opportunities of miRNAs and provide a broad perspective for the future clinical development of miRNAs as biomarkers in CKD beyond the current gold standards of eGFR and albuminuria.

Nephron segment specific microRNA biomarkers of pre-clinical drug-induced renal toxicity: Opportunities and challenges.

Drug-induced nephrotoxicity is a common drug development complication for pharmaceutical companies. Sensitive, specific, translatable and non-invasive biomarkers of renal toxicity are urgently needed to diagnose nephron segment specific injury. The currently available gold standard biomarkers for nephrotoxicity are not kidney-specific, lack sensitivity for early detection, and are not suitable for renal damage localization (glomerular vs tubulointerstitial injury). MicroRNAs (miRNAs) are increasingly gaining momentum as promising biomarkers of various organ toxicities, including drug induced renal injury. This is mostly due to their stability in easily accessible biofluids, ease of developing nucleic acids detection compared to protein detection assays, as well as their interspecies translatability. Increasing concordance of miRNA findings by standardizing methodology most suitable for their detection and quantitation, as well as characterization of their expression pattern in a cell type specific manner, will accelerate progress toward validation of these miRNAs as biomarkers in pre-clinical, and clinical settings. This review aims to highlight the current pre-clinical findings surrounding miRNAs as biomarkers in two important segments of the nephron, the glomerulus and tubules.

Identification of Promising Urinary MicroRNA Biomarkers in Two Rat Models of Glomerular Injury.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate protein levels posttranscriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Here, changes in miRNA expression patterns are described in 2 different rodent models of glomerular injury (acute puromycin aminonucleoside nephropathy and passive Heymann nephritis). By employing 2 different modes of glomerular insult, oxidative stress and immune-mediated toxicity, miRNA changes in both isolated glomeruli as well as urine specimens allow for identification of urinary miRNA biomarkers that are suggestive of drug-induced injury specifically to the glomerulus. Subsets of glomerular urinary miRNAs associated with these different modes of glomerular toxicity seem to be dependent on the mechanism of the induced injury, while 9 miRNAs that changed early in both glomerular and urine specimens were common to both studies. We further show that the miRNAs identified as mechanism-specific early glomerular injury biomarkers target key pathways and transcripts relevant to the type of insult, while the insult-independent changes might serve as ideal glomerular injury biomarkers.

Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Next-generation sequencing (NGS) is increasingly employed in biomedical investigations. Although concordance between this platform and qRT-PCR based assays has been reported in high quality specimens, information is lacking on comparisons in biofluids especially urine. Here we describe the changes in miRNA expression patterns in a rodent model of renal tubular injury (gentamicin). Our aim is to compare RNA sequencing and qPCR based miRNA profiling in urine specimen from control and rats with confirmed tubular injury. RESULTS: Our preliminary examination of the concordance between miRNA-seq and qRT-PCR in urine specimen suggests minimal agreement between platforms probably due to the differences in sensitivity. Our results suggest that although miRNA-seq has superior specificity, it may not detect low abundant miRNAs in urine samples. Specifically, miRNA-seq did not detect some sequences which were identified by qRT-PCR. On the other hand, the qRT-PCR analysis was not able to detect the miRNA isoforms, which made up the majority of miRNA changes detected by NGS. CONCLUSIONS: To our knowledge, this is the first time that miRNA profiling platforms including NGS have been compared in urine specimen. miRNAs identified by both platforms, let-7d, miR-203, and miR-320, may potentially serve as promising novel urinary biomarkers for drug induced renal tubular epithelial injury.

miR-122 regulates tumorigenesis in hepatocellular carcinoma by targeting AKT3.

MicroRNAs (miRNAs) have been implicated in the orchestration of diverse cellular processes including differentiation, proliferation, and apoptosis and are believed to play pivotal roles as oncogenes and tumor suppressors. miR-122, a liver specific miRNA, is significantly down-regulated in most hepatocellular carcinomas (HCCs) but its role in tumorigenesis remains poorly understood. Here we identify AKT3 as a novel and direct target of miR-122. Restoration of miR-122 expression in HCC cell lines decreases AKT3 levels, inhibits cell migration and proliferation, and induces apoptosis. These anti-tumor phenotypes can be rescued by reconstitution of AKT3 expression indicating the essential role of AKT3 in miR-122 mediated HCC transformation. In vivo, restoration of miR-122 completely inhibited xenograft growth of HCC tumor in mice. Our data strongly suggest that miR-122 is a tumor suppressor that targets AKT3 to regulate tumorigenesis in HCCs and a potential therapeutic candidate for liver cancer.

miR-221 promotes tumorigenesis in human triple negative breast cancer cells.

Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis. TNBCs are characterized by their resistance to apoptosis, aggressive cellular proliferation, migration and invasion, and currently lack molecular markers and effective targeted therapy. Recently, miR-221/miR-222 have been shown to regulate ERα expression and ERα-mediated signaling in luminal breast cancer cells, and also to promote EMT in TNBCs. In this study, we characterized the role of miR-221 in a panel of TNBCs as compared to other breast cancer types. miR-221 knockdown not only blocked cell cycle progression, induced cell apoptosis, and inhibited cell proliferation in-vitro but it also inhibited in-vivo tumor growth by targeting p27(kip1). Furthermore, miR-221 knockdown inhibited cell migration and invasion by altering E-cadherin expression, and its regulatory transcription factors Snail and Slug in human TNBC cell lines. Therefore, miR-221 functions as an oncogene and is essential in regulating tumorigenesis in TNBCs both in vitro as well as in vivo.

Morphine- and CaMKII-dependent enhancement of GIRK channel signaling in hippocampal neurons.

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines. In vivo administration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca(2+) and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the µ-selective (DAMGO) and δ-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba(2+)-sensitive basal K(+) currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine.

Nek6 mediates human cancer cell transformation and is a potential cancer therapeutic target.

We investigated the role of Nek6, a member of the NIMA-related serine/threonine kinase family, in tumorigenesis. Transcript, protein, and kinase activity levels of Nek6 were highly elevated in the malignant tumors and human cancer cell lines compared with normal tissue and fibroblast cells. Expression of exogenous wild-type Nek6 increased anchorage-independent growth of a variety of human cancer cell lines, whereas overexpression of the kinase-dead Nek6 and RNAi knockdown of endogenous Nek6 suppressed cancer cell transformation and induced apoptosis. Additionally, in in vivo xenograft nude mouse model, knockdown of Nek6 in HeLa cells resulted in reduction of tumor size relative to control siRNA tumors. Most importantly, knocking down endogenous Nek6 levels or exogenous expression of the kinase-dead form did not inhibit cell proliferation, nor did it induce apoptosis in normal fibroblast cells. Taken together, our data indicate a pivotal role for Nek6 in tumorigenesis and establish Nek6 as a potential target for treatment of a variety of human cancers.

A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction.

G protein-gated potassium (Kir3) channels are important for controlling neuronal excitability in the brain. Using a proteomics approach, we have identified a unique rodent intracellular protein, sorting nexin 27 (SNX27), which regulates the trafficking of Kir3 channels. Like most sorting nexins, SNX27 possesses a functional PX domain that selectively binds the membrane phospholipid phosphatidylinositol-3-phosphate (PI3P) and is important for trafficking to the early endosome. SNX27, however, is the only sorting nexin to contain a PDZ domain. This PDZ domain discriminates between channels with similar class I PDZ-binding motifs, associating with the C-terminal end of Kir3.3 and Kir3.2c (-ESKV), but not with that of Kir2.1 (-ESEI) or Kv1.4 (-ETDV). SNX27 promotes the endosomal movement of Kir3 channels, leading to reduced surface expression, increased degradation and smaller Kir3 potassium currents. The regulation of endosomal trafficking via sorting nexins reveals a previously unknown mechanism for controlling potassium channel surface expression.

Subunit-specific regulation of Kir3 channels by sorting nexin 27.

G protein-gated inwardly rectifying potassium (Kir3) channels are involved in regulating membrane excitability in the brain. Kir3 channels have been shown to play a role in learning, analgesia and drug addiction. Little is known about the cell surface regulation of Kir3 channels. Using a proteomics approach, we recently discovered that sorting nexin 27 (SNX27) associates with a subset of Kir3 channels. Sorting nexins have been implicated in trafficking of proteins through endosomal compartments. The single PDZ domain of SNX27 binds directly to the PDZ binding motif of Kir3 channels leading to their downregulation. Here, we examined the functional effect of SNX27b expression on different subunit combinations of the Kir3 family. Our results show that regulation of Kir3 channels by SNX27 depends critically on the combination of Kir3 subunits. This type of subunit-specific regulation could be important for determining the extent of Kir3 inhibition in normal as well as diseased states, such as drug addiction.

Efficacy of camptothecin analog DX-8951f (Exatecan Mesylate) on human pancreatic cancer in an orthotopic metastatic model.

We determined the antitumor and antimetastatic efficacy of the camptothecin analogue DX-8951f in an orthotopic metastatic mouse model of pancreatic cancer. DX-8951f showed efficacy against two human pancreatic tumor cell lines in this model. These cell lines were transduced with the green fluorescent protein, enabling high-resolution visualization of tumor and metastatic growth in vivo. The DX-8951f studies included both an early and advanced cancer model. In the early model, using the human pancreatic cancer lines MIA-PaCa-2 and BxPC-3, treatment began when the orthotopic primary tumor was approximately 7 mm in diameter. DX-8951f was significantly effective against both MIA-PaCa-2 and BxPC-3. In contrast, 2', 2'-difluorodeoxycytidine (gemcitabine), the standard treatment for pancreatic cancer, did not have significant efficacy against MIA-PaCa-2. Although gemcitabine showed significant activity against BxPC-3 primary tumor growth, it was not effective on metastasis. In the model of advanced disease, using BxPC-3, treatment started when the orthotopic primary tumor was 13 mm in diameter. DX-8951f was significantly effective in a dose-response manner on the BxPC-3 primary tumor. DX-8951f also demonstrated antimetastatic activity in the late-stage model, significantly reducing the incidence of lymph node metastasis while eliminating lung metastasis. In contrast, gemcitabine was only moderately effective against the primary tumor and ineffective against metastasis at both sites in the late-stage model. Therefore, DX-8951f was highly effective against primary and metastatic growth in this very difficult-to-treat disease and showed significantly higher efficacy than gemcitabine, the standard treatment of pancreatic cancer. DX-8951f, therefore, has important clinical promise and has more positive features than the currently used camptothecin analogue CPT-11, which requires metabolic activation and is toxic.

Bax-induction gene therapy of pancreatic cancer.

BACKGROUND: Bax is a strong pro-apoptotic gene that induces programmed cell death when expressed. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit for telomerase, an enzyme found to be active in more than 85% of human cancers. Recently, a binary adenoviral system (Ad/GT-Bax + Ad/hTERT-GV16) was constructed using the hTERT promoter to induce Bax gene expression in tumor cells. METHODS: To test whether human pancreatic tumor cells would respond to this system of Bax-induced apoptosis, we compared the effects of Bax gene induction with that of LacZ gene induction using the same binary system. RESULTS: Lysates of the human pancreatic cell lines PANC-28, MIA PaCa-2, and BxPC-3 showed significantly elevated levels of human telomerase using the PCR-based TRAP assay. As early as 24 h after treatment with Bax-induction gene therapy, growth inhibition was observed. Overexpression of the Bax protein was confirmed by Western blotting. Extensive apoptosis on FACS analysis at 48 h was seen after Bax induction. In addition, cytosolic cytochrome c levels increased compared to mitochondrial levels after Bax induction. Levels of caspase-3, a key downstream enzyme involved in apoptosis, also increased significantly compared to controls after treatment. None of these effects were seen with LacZ. CONCLUSION: Our results suggest that the binary adenoviral vector system, Ad/GT-Bax + Ad/hTERT-GV16, induces high levels of Bax expression that induce apoptosis in human pancreatic cancer cells.

Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.

We report here whole-body optical imaging, in real time, of genetically fluorescent pancreatic tumors growing and metastasizing to multiple sites in live mice. The whole-body optical imaging system is external and noninvasive. Human pancreatic tumor cell lines, BxPC-3 and MiaPaCa-2, were engineered to stably express high-levels of the Aequorea victoria green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell lines were surgically orthotopically implanted as tissue fragments in the body of the pancreas of nude mice. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions that developed in the spleen, bowel, portal lymph nodes, omentum, and liver. Intravital images in the opened animal confirmed the identity of whole-body images. The whole-body images were used for real-time, quantitative measurement of tumor growth in each of these organs. Intravital imaging was used for quantification of growth of micrometastasis on the liver and stomach. Whole-body imaging was carried out with either a trans-illuminated epi-fluorescence microscope or a fluorescence light box, both with a thermoelectrically cooled color CCD camera. The simple, noninvasive, and highly selective imaging made possible by the strong GFP fluorescence allowed detailed simultaneous quantitative imaging of tumor growth and multiple metastasis formation of pancreatic cancer. The GFP imaging affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals without the need for anesthesia, substrate injection, contrast agents, or restraint of animals required by other imaging methods. The GFP imaging technology presented in this report will facilitate studies of modulators of pancreatic cancer growth, including inhibition by potential chemotherapeutic agents.