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MicroRNAs are differentially expressed in the serum and renal tissues of cats with experimentally induced chronic kidney disease: a preliminary study

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  • 1 Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA
  • | 2 Athens Veterinary Diagnostic Laboratory, Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA

Abstract

OBJECTIVE

To identify differentially expressed microRNA in the serum and renal tissues of cats with experimentally induced chronic kidney disease (CKD).

SAMPLE

Banked renal tissues and serum from 4 cats.

PROCEDURES

Cats previously underwent 90-minute unilateral ischemia with delayed contralateral nephrectomy 3 months after ischemia. Tissues were collected from the contralateral kidney at the time of nephrectomy and from the ischemic kidney 6 months after nephrectomy (study end). Serum was collected prior to ischemia (baseline serum) and at study end (end point serum). Total RNA was isolated from tissues and serum, and microRNA sequencing was performed with differential expression analysis between the contralateral and ischemic kidney and baseline and end point serum.

RESULTS

20 microRNAs were differentially expressed between ischemic and contralateral kidneys, and 52 microRNAs were differentially expressed between end point and baseline serum. Five microRNAs were mutually differentially expressed between ischemic and contralateral kidneys and baseline and end point serum, with 4 (mir-21, mir-146, mir-199, and mir-235) having increased expression in both the ischemic kidney and end point serum and 1 (mir-382) having increased expression in the ischemic kidney and decreased expression in end point serum. Predicted target search for these microRNA revealed multiple genes previously shown to be involved in the pathogenesis of feline CKD, including hypoxia-inducible factor-1α, transforming growth factor-β, hepatocyte growth factor, fibronectin, and vascular endothelial growth factor A.

CLINICAL RELEVANCE

MicroRNAs were differentially expressed after CKD induction in this preliminary study. Regulation of renal fibrosis in feline CKD may occur through microRNA regulation of mRNAs of pro- and anti-fibrotic genes.

Abstract

OBJECTIVE

To identify differentially expressed microRNA in the serum and renal tissues of cats with experimentally induced chronic kidney disease (CKD).

SAMPLE

Banked renal tissues and serum from 4 cats.

PROCEDURES

Cats previously underwent 90-minute unilateral ischemia with delayed contralateral nephrectomy 3 months after ischemia. Tissues were collected from the contralateral kidney at the time of nephrectomy and from the ischemic kidney 6 months after nephrectomy (study end). Serum was collected prior to ischemia (baseline serum) and at study end (end point serum). Total RNA was isolated from tissues and serum, and microRNA sequencing was performed with differential expression analysis between the contralateral and ischemic kidney and baseline and end point serum.

RESULTS

20 microRNAs were differentially expressed between ischemic and contralateral kidneys, and 52 microRNAs were differentially expressed between end point and baseline serum. Five microRNAs were mutually differentially expressed between ischemic and contralateral kidneys and baseline and end point serum, with 4 (mir-21, mir-146, mir-199, and mir-235) having increased expression in both the ischemic kidney and end point serum and 1 (mir-382) having increased expression in the ischemic kidney and decreased expression in end point serum. Predicted target search for these microRNA revealed multiple genes previously shown to be involved in the pathogenesis of feline CKD, including hypoxia-inducible factor-1α, transforming growth factor-β, hepatocyte growth factor, fibronectin, and vascular endothelial growth factor A.

CLINICAL RELEVANCE

MicroRNAs were differentially expressed after CKD induction in this preliminary study. Regulation of renal fibrosis in feline CKD may occur through microRNA regulation of mRNAs of pro- and anti-fibrotic genes.

Introduction

Chronic kidney disease (CKD) is highly prevalent in geriatric cats and is characterized by persistent and progressive changes in renal function and architecture.1,2 Several factors have been postulated to contribute to the development of CKD in cats, including aging, increased cellular senescence, and telomere shortening.1,3 Increased activity of proinflammatory and profibrotic cells and factors, including myofibroblasts, transglutaminase 2, and transforming growth factor-β (TGF-β), have been detected in cats with CKD.47 The final common consequence of CKD is tubulointerstitial inflammation and fibrosis, and the severity of these lesions correlates with the degree of renal function impairment.1,8 Fibrosis leads to expansion of the extracellular matrix and loss of normal renal tissue, and increasing levels of fibrosis correlate with increasing serum creatinine concentrations and worsening of CKD.1,8

Early diagnosis of kidney disease in cats is challenging, as currently available diagnostic techniques are insensitive until a substantial percentage of kidney function is lost. Loss of urine concentrating ability occurs with ≥ 66% loss of functional renal mass, and serum creatinine concentration measurements exceeding the upper limit of the reference interval occur with ≥ 75% loss of functional renal mass.9 Measurement of glomerular filtration rate (GFR) may allow for earlier diagnosis, but is not commonly performed in clinical practice due to technical difficulty and relative expense.10 A circulating marker of GFR, symmetric dimethylarginine (SDMA), has been advocated as more sensitive than serum creatinine for diagnosis of CKD.10 However, the specificity and positive predictive value of serum SDMA for a 30% decrease in GFR are lower than for serum creatinine. In addition, the time between increases in serum SDMA and increases in serum creatinine varies among individuals, and these factors may increase in varying order among individuals. The effects of pre- and postrenal factors on serum SDMA concentrations are currently undefined.10,11

An ideal biomarker for CKD would allow for reliable, early identification of renal injury and would provide a method for clinicians to track subtle changes in function, facilitating treatment decisions. A promising group of biomarker candidates for kidney injury are microRNAs (miRNAs), which are small, noncoding RNAs that affect posttranscriptional regulation via interactions with mRNAs.12,13 These miRNAs regulate cellular responses via binding of mRNAs, either preventing their translation or causing their degradation.12,13 A single miRNA may regulate many mRNAs, thereby having effects on the expression of numerous genes.12 There is substantial alteration of miRNA expression in diseased cells compared with their normal counterparts.14 Further, miRNAs show tissue- and disease-state-specific expression patterns and have excellent stability in biofluids, making them excellent biomarker candidates.12,15

Altered miRNA expression has been demonstrated in people with CKD. One group found dysregulation of 24 different miRNAs in fibrotic kidneys compared with normal, healthy kidneys.16 One specific miRNA, mir-21, has been found to promote renal fibrosis by regulating a metabolic switch that initiates fibrogenesis.16 Although several studies1719 have demonstrated altered gene regulation in feline CKD, the role of miRNAs in the pathogenesis and progression of feline CKD has not been critically evaluated. The objective of the present study was to evaluate miRNA expression in the kidneys and serum of cats with experimentally induced CKD. The hypothesis was that miRNAs would be differentially expressed between serum obtained prior to and after experimental induction of CKD and between kidneys previously subjected to transient renal ischemia and the contralateral kidneys from the same cats.

Materials and Methods

Samples

Banked renal tissue and serum samples were used from 4 purpose-bred, adult male neutered cats subjected to transient renal ischemia and delayed contralateral nephrectomy as a model of CKD for an unrelated study (unpublished). All study procedures were approved by the University of Georgia Institutional Care and Use Committee. The study did not require approval due to use of banked samples without live animal involvement.

All cats were castrated male domestic shorthairs with a median age of 1.6 years (range, 1.0 to 1.7 years) and a median weight of 6.7 kg (range, 5.6 to 7.9 kg). Physical examination, CBC, serum biochemistry, urinalysis, and urine protein-to-creatinine ratio were normal at baseline in all cats. General methods used to induce transient renal ischemia via ventral midline celiotomy have been published in detail elsewhere.20,21 Cats were fasted for 12 hours prior to anesthesia. Cats were sedated with acepromazine (0.01 mg/kg, IM), buprenorphine (0.04 mg/kg, IM), and ketamine (7 mg/kg, IM) and were intubated and maintained on isoflurane in oxygen. Cats received IV isotonic crystalloids (0.9% NaCl solution, 10 mL/kg/h) throughout the procedure. A hot water blanket was used to maintain body temperature.

Surgery was performed in a sterile surgery suite via ventral midline celiotomy. Ischemia of the right kidney was induced by cross-clamping of the renal artery and vein for 90 minutes; the left kidney was undisturbed. Postoperative analgesia was provided by placement of a transdermal fentanyl patch (25 µg/h; Noven Pharmaceuticals Inc) on the lateral abdomen. Postoperative analgesia was augmented via transmucosal buprenorphine (0.3 mg/kg) as needed based on a discomfort score22 in the 24 hours following application of the fentanyl patch. Three months following the unilateral renal ischemia surgery, cats were anesthetized with the same protocol described above, nephrectomy of the contralateral, undisturbed (left) kidney was performed, and tissue samples from this kidney were immediately flash frozen in liquid nitrogen and stored at −80 °C for later analysis. Renal tissue samples were also placed in neutral-buffered 10% formalin for histologic evaluation. In all instances, care was taken to include equal proportions of cortex, medulla, and corticomedullary junction. Nine months following unilateral renal ischemia (ie, 6 months following contralateral nephrectomy), cats were euthanized, and tissues from the previously ischemic (right) kidney were collected within 1 hour, preserved in the same manner as for the contralateral kidney, and stored at −80 °C for later analysis.

Fourteen days prior to (ie, at baseline) and 270 ± 1 days after (ie, at study end) surgery for transient renal ischemia, blood was obtained by peripheral venipuncture; these samples were designated as baseline and end point serum, respectively. Samples were transferred to blood collection tubes, allowed to clot, and centrifuged at room temperature within 1 hour of collection. Serum was collected and stored at −80 °C for later analysis. In addition, serum creatinine and BUN were measured at baseline, 180 days, and study end. Serum creatinine concentration was also measured 1 day prior to contralateral nephrectomy (day 89) and 1 day following contralateral nephrectomy (day 91). Urine was collected via free catch in an empty litter box or via cystocentesis at baseline, day 89, day 91, day 180, and study end, and urine specific gravity was measured with a refractometer.

Formalin-fixed, paraffin-embedded tissue samples from all kidneys were histologically evaluated by a single board-certified veterinary pathologist (DRR) for degree of interstitial inflammation, fibrosis, and tubular atrophy using previously described techniques and a previously described scoring system.17,21 Briefly, each histologic abnormality was graded on a scale of 0 to 3, with 0 indicating no evidence of the abnormality, 1 indicating mild changes, 2 indicating moderate changes, and 3 indicating severe changes.

RNA isolation

Total RNA was isolated from tissue (Quick-RNA MiniPrep [Plus]; Zymo Research) and serum (Quick-cfRNA serum and plasma; Zymo Research) samples according to manufacturer directions. Approximately 30 mg of renal tissue and 250 to 350 μL of serum were used for extraction. Renal tissue samples were analyzed for RNA purity via spectrophotometry (Nanodrop 2000; Thermo Fisher Scientific) with 260 of 280 ratios of ≥ 2 considered acceptable for further analysis. Serum samples were not evaluated via spectrophotometry due to known poor purity and low yield.23 Samples underwent further quality control assessment with a bioanalyzer system (2100 Bioanalyzer Instrument; Agilent). An RNA integrity number of > 6.5 was considered acceptable for further analysis.

RNA sequencing

Library preparation and RNA sequencing were performed (Georgia Genomics and Bioinformatics Core). Briefly, sample concentration was normalized in 10.5 µL of nuclease-free water, and libraries were prepared with the following steps: 3′and 5′ adapters were selectively ligated to small RNA molecules, and cDNA was synthesized using a primer specific to the 3′ adapter sequence. Index sequences were added to each library during library PCR. Final libraries were size selected to enrich for small RNA products. Prepared libraries were quality control analyzed using qubit and fragment analyzer to determine the concentration and size of the libraries.

Statistical analysis

Descriptive statistics were generated for clinicopathologic and histologic scoring data using commercially available software (Excel version 2016; Microsoft Corp). Data are presented as median (range).

Bioinformatics analysis

Bioinformatics was performed (Georgia Genomics and Bioinformatics Core). Briefly, raw read data were quality trimmed (Trimmomatic version 0.39; Cutadapt version 2.10), as previously described.24,25 Read filtering to the reference feline genome (Felis_catus_9.0) was performed (UEA small RNA Workbench version 4.5); reads that did not align to the cat genome or that were transfer RNA, ribosomal RNA, or homopolymers were discarded. Next, miRNA reads were identified (UEA small RNA Workbench version 4.5) in the animal miRNA database (miRbase release 22.1). No cat-specific mature hairpin miRNA accessions are available in the database (miRbase release 22.1), so data were mapped to all available animal miRNA accessions. Finally, differential expression analysis was performed with pairwise comparisons (ischemic vs contralateral kidneys and baseline vs end point serum; DESeq2 version 1.28.0). After normalization, a logistic regression model was fit for each gene using the Wald test. P values were corrected for multiple testing using the Benjamini and Hochberg method to calculate false discovery rate values. A false discovery rate cutoff of ≤ 0.100 was used. A log2 (fold change) > 0 between miRNAs was considered to be upregulation, and a log2 (fold change) < 0 between miRNAs was considered to be downregulation.

Predicted target search

Predicted target search was performed to identify potential gene targets for the identified miRNA (TargetScan 7.2). A search of the database was performed for each miRNA, and predicted targets were exported into a spreadsheet (Excel version 2016; Microsoft Corp). The spreadsheet for each miRNA was searched for genes previously identified to be dysregulated in feline CKD.4,5,7,1719

Results

All cats had a numeric increase in serum creatinine and BUN and a decrease in urine specific gravity at study end compared with baseline (Table 1). Histologic examination of the contralateral kidney showed normal tissue architecture, with a histologic score of 0 for fibrosis, inflammation, and tubular atrophy. Histologic examination of the ischemic kidneys confirmed the presence of fibrosis, inflammation, and tubular atrophy, with median (range) scores of 1 (1 to 3) for each.

Table 1

Selected serum and urine biochemical parameters in 4 cats with experimentally induced chronic kidney disease (CKD).

ParameterBaselineDay 89aDay 91bDay 180cEnd point
Serum creatinine (mg/dL; RI, 0.6–1.8)
 Cat 11.31.32.31.51.6
 Cat 21.01.32.41.71.6
 Cat 31.41.32.21.61.6
 Cat 41.01.13.46.04.6
BUN (mg/dL; RI, 21–36)
 Cat 1213933
 Cat 2183028
 Cat 3182423
 Cat 4179487
Urine specific gravity
 Cat 11.0531.0591.0371.0461.047
 Cat 21.0471.0591.0391.0541.032
 Cat 31.058> 1.0611.0311.0511.041
 Cat 41.0501.0501.0291.0091.012

Cats underwent 90 minutes of unilateral renal ischemia with contralateral nephrectomy at 3 months. Baseline and end point samples were obtained 14 days prior to and 270 ± 1 days after transient unilateral renal ischemia, respectively.

— = Not applicable. RI = Reference interval.

aOne day prior to contralateral nephrectomy. bOne day after contralateral nephrectomy. cThree months after contralateral nephrectomy.

Differential expression analysis revealed 20 miRNAs differentially expressed between the ischemic and contralateral kidneys (Table 2) and 52 miRNAs differentially expressed between end point and baseline serum (Table 3). Five miRNA (mir-21, mir-92, mir-146, mir-199, and mir-382) were mutually differentially expressed between the ischemic and contralateral kidney and also end point and baseline serum (Table 4). For mir-21, mir-92, mir-146, and mir-199, increased expression occurred in both ischemic kidneys and end point serum, compared with the contralateral kidneys and baseline serum, respectively. Expression of mir-382 was increased in ischemic kidneys compared with contralateral kidneys, but decreased in end point serum compared with baseline serum. Predicted target search revealed multiple potential gene targets previously identified to be involved in the pathogenesis of feline CKD through proinflammatory and profibrotic pathways (Supplementary Table S1).46,1719

Table 2

Significantly (P < 0.05; false discovery rate < 0.10) differentially expressed microRNAs (miRNAs) identified between the ischemic and contralateral kidneys from the cats of Table 1.

miRNAFold change
mir-101–1.88
mir-106–2
mir-1226.55
mir-124718.9
mir-1426.53
mir-1463.21
mir-15027.69
mir-1992.09
mir-212.91
mir-2144.04
mir-2234.52
mir-2357.46
mir-30–1.7
mir-3829.2
mir-4097.09
mir-43213.66
mir-48517.21
mir-5097.5
mir-5147.5
mir-5743.14
Table 3

Significantly differentially expressed miRNAs identified between end point and baseline serum from the cats of Table 1.

miRNAFold change
mir-1031.72
mir-1071.73
mir-12246.47
mir-12415.5
mir-128–2.42
mir-1296–29.32
mir-133–10.68
mir-1388–2.34
mir-143–2.12
mir-145–2.52
mir-1461.94
mir-1835.79
mir-18391.94
mir-1851.98
mir-186–1.75
mir-187–15.43
mir-190–17.28
mir-1923.91
mir-1944.72
mir-1952.39
mir-1992.19
mir-2002.83
mir-2032.44
mir-2043.83
mir-206–5.18
mir-211.76
mir-2114.54
mir-2183.66
mir-23510.37
mir-241.78
mir-2478–41.4
mir-25–1.97
mir-330–2
mir-3355.8
mir-340–7.58
mir-350–5.99
mir-3611.52
mir-3625.65
mir-365–2.47
mir-378–14.35
mir-381–10.31
mir-382–11.19
mir-4454–18.64
mir-483126.9
mir-487–7.98
mir-491–18.6
mir-4974.36
mir-5001.95
mir-532–1.7
mir-6601.97
mir-889–32.63
mir-99953.68
Table 4

MicroRNAs that were mutually significantly differentially expressed between both ischemic and contralateral kidneys and end point and baseline serum from the cats of Table 1.

miRNAFold change 1Fold change 2
mir-211.541.76
mir-1461.681.94
mir-1991.072.19
mir-2352.9010.37
mir-3829.20–11.19

Fold change 1 represents the difference between the ischemic kidney and the contralateral kidney. Fold change 2 represents the difference between the end point serum sample and baseline serum sample.

Discussion

Five miRNAs were differentially expressed in both the serum and tissues of cats with experimentally induced CKD; thus, the hypothesis was accepted. The samples included in the present study were obtained from cats that underwent unilateral, transient ischemia and reperfusion with delayed contralateral nephrectomy as a model of CKD. Cats experienced serum and urinary biochemical changes consistent with kidney injury, as well as histologic changes to the ischemic kidney mimicking those of naturally occurring CKD (ie, varying degrees of interstitial fibrosis, inflammation, and tubular atrophy), as described previously in similar models.17,21

The present study identified increased expression of mir-21 in the renal tissues and serum. In human renal disease, mir-21 has been studied extensively and is known to be a profibrotic miRNA.26,27 In 1 study28 of human renal transplant recipients, urine levels of mir-21 correlated with grade of interstitial fibrosis and tubular atrophy, serum creatinine concentrations, and GFR. Hypoxia/reoxygenation of mouse renal tubular epithelial cells increased mir-21 expression in another study.29 Furthermore, antagonism of mir-21 with antagomirs or increased expression of mir-21 target genes reduced levels of renal fibrosis in several studies,27,29,30 further demonstrating the importance of mir-21 in renal fibrosis.

In addition, the present study found that mir-92, mir-146, and mir-199 had increased expression in both ischemic kidneys and baseline serum samples compared with the contralateral kidneys and end point serum samples. In rats with diabetic nephropathy, mir-92 is upregulated in the kidneys and has been shown to suppress Smad7,31 which is known to block TGF-β–induced renal fibrosis.32 Additionally, mir-92 was upregulated in rats with Adriamycin-induced nephrotic syndrome,33 which is associated with tubulointerstitial fibrosis and inflammation.34 The mir-146 family of miRNA, comprised of mir-146a and 146b, has been shown to be involved with renal injury, with mir-146a appearing to be protective and mir-146b appearing to promote renal injury. In 1 study,35 delivery of mir-146a via nanoparticles attenuated renal fibrosis in mice through inhibition of profibrotic and inflammatory pathways, such as those mediated by TGF-β and nuclear factor-κB. Another group found that mir-146b was highly expressed in mice during renal fibrosis in the late stages (weeks) after injury.36 In mice with renal ischemia-reperfusion injury, mir-146 was upregulated and was found to play a protective role by regulating apoptosis37; the increase in expression of mir-146 in the present study is therefore unsurprising given the similar mechanism of CKD induction. In the present study, mir-199 was also upregulated in ischemic kidneys and in end point serum. In diabetic rats and TGF-β1–treated human proximal tubule cells, mir-199 is upregulated, and blockade of mir-199 reduces epithelial-to-mesenchymal transition and renal fibrosis through attenuation of TGF-β1.38

In the cats of this study, expression of mir-382 was increased in ischemic kidneys compared with contralateral kidneys but was decreased in end point serum compared with baseline serum. In mice with diabetic nephropathy, mir-382 is upregulated in renal tissues, and downregulation of this miRNA reduces extracellular matrix accumulation.39 In human patients with renal interstitial fibrosis, mir-382 is also upregulated.40 Following unilateral renal obstruction in mice, mir-382 expression increases,40,41 and blockade of mir-382 reduces medullary fibrosis.41 The role of circulating mir-382 is unknown; however, levels of expression of miRNA in tissues do not always correlate with the level of expression of the same miRNA in serum and may reflect factors such as the intra- or extracellular location42 or whether miRNAs are located within extracellular vesicles or in Ago protein complexes.43,44 Additional studies are needed to determine whether circulating mir-382 influences renal injury in feline CKD and the mechanism by which this may occur.

Predicted gene targets for the 5 differentially expressed miRNAs of the present study are in agreement with previously reported works evaluating both naturally occurring and experimentally induced CKD in cats.17,18 Specifically, matrix metalloproteinase-16 (MMP16) was a predicted target for all 5 miRNAs that were differentially expressed in renal tissues and serum in the present study. In a similar unilateral ischemia model of CKD, MMP16 expression was greater in the ischemic kidneys compared with the contralateral, undisturbed kidneys.19 Matrix metalloproteinases are heavily involved in extracellular matrix regulation,45 and regulation of MMPs by miRNAs has been shown previously. In 1 study,46 mir-146a was shown to target MMP16 in human breast cancer. Another group found that miRNAs were involved with TGFB1-mediated upregulation of MMP16 in human bladder cancer cell lines, although only a single miRNA (mir-200b) was evaluated and the specific miRNAs identified in the present study were not evaluated.47 The specific role of MMP16 in the pathogenesis of CKD is unknown, but is likely related to its role in extracellular matrix regulation via its effects on collagen and fibronectin.45

Another predicted target in the present study, hypoxia inducible factor 1 subunit α (HIF1A), was identified for mir-92, mir-146, and mir-199. This gene has also been previously found to have a direct relationship with mir-21 in mice undergoing renal ischemic preconditioning.48 In normoxic conditions, HIF1A has a short half-life, but stabilizes in hypoxic conditions.49 Its activation is controlled through posttranslational methods, including miRNA interactions, which have been shown to regulate its expression in acute kidney injury.50 In the initial phase of acute kidney injury, HIF1A is protective, acting initially to reduce apoptosis and necrosis of renal tubular epithelial cells, decrease inflammation, and reduce levels of reactive oxygen species.51,52 During the repair phase of acute kidney injury, HIF1A assists in kidney repair.50 In an experimental model of feline CKD similar to the one reported here, HIF1A expression was not significantly different between ischemic kidneys, contralateral kidneys, or kidneys from healthy cats not subjected to unilateral ischemia, although there was a positive correlation between HIF1A expression and interstitial fibrosis.17 Conversely, in the renal tissues of cats with naturally occurring CKD that had, on average, more severe histologic changes than cats with experimentally induced CKD, transcript levels of HIF1A were significantly greater than in renal tissues from healthy control cats.18 Failure to identify significant differences in HIF1A expression in previously reported transient unilateral renal ischemia models may be explained, in part, by the fact that the contralateral, undisturbed kidney experiences altered expression of other profibrotic genes when compared with kidneys from healthy controls. Thus, the contralateral kidney is an imperfect control due to crosstalk with the ischemic kidney due to systemic alterations aimed at maintaining total GFR.

Another common predicted target of the differentially expressed miRNA identified in this study, specifically mir-21, mir-92, and mir-199, was TGFB, which acts upon Smad in the TGF-β signaling pathway to drive renal fibrosis.53 The role of mir-21 in the regulation of TGFB is well studied, and increasing levels of this miRNA correlate with severity of fibrosis.54,55 In cats, TGFB transcript levels are increased in renal tissues of cats with naturally occurring CKD18 and ischemic kidneys of cats with experimentally induced CKD via unilateral renal ischemia,17 both compared with kidneys from healthy control cats. Although TGFB was not a predicted target for mir-382 in the present study, a previous study56 confirmed that in TGFB-treated human renal epithelial cells, mir-382 is upregulated and suppresses superoxide dismutase 2 to sustain the oxidative stress response to TGFB.

The predicted target hepatocyte growth factor (HGF) has a known protective effect against renal fibrosis through blockade of TGF-β57,58 and was previously identified as a molecular function target in a similar experimentally induced model of CKD in cats.19 In the present study, HGF was a predicted target of mir-21, mir-92, mir-146, and mir-199. Both HGF and TGF-β increase after renal injury; in acute injuries, HGF predominates, whereas in chronic injuries, HGF activity wanes as TGF-β activity increases.59 More work needs to be done to determine how the balance of HGF and TGF-β drives fibrosis in this model of feline CKD.

Fibronectin (FN1) is also involved in a TGF-β–mediated pathway.60 When TGF-β acts upon renal myofibroblasts, fibronectin, extracellular mesenchymal matrix proteins, and collagen I and III are highly expressed, promoting fibrosis.60 FN1 was identified as an upregulated gene in a similar model of experimentally induced CKD in cats.19 In the present study, FN1 was identified as a potential target of mir-21, mir-146, and mir-199.

An additional predicted target in the present study, vascular endothelial growth factor A (VEGFA), is heavily involved in angiogenesis and is known to assist in repair in the early stages of renal disease.61 However, levels of VEGFA decrease with chronicity of disease, particularly as inflammation and fibrosis worsen.62 This has recently been demonstrated in a similar model of experimentally induced feline CKD in which decreased expression of VEGFA was found in both the ischemic and contralateral kidneys compared with healthy control kidneys, but not compared with one another.19 In the present study, VEGFA was a predicted target of mir-146 and mir-199, both of which were more highly expressed in the end point serum as compared with baseline. These circulating miRNAs may have resulted in decreased expression of VEGFA in the contralateral kidney while both circulating and renal tissue miRNA likely affected the ischemic kidney. Further work should be performed to determine how miRNA released into circulation from the ischemic kidney work to effect change within the contralateral, undisturbed kidney.

Limitations of this study include the small number of biological replicates, although this is a common sample size for pilot studies for molecular investigations.6367 Renal tissue is not routinely sampled in the diagnostic evaluation of cats with nonproteinuric CKD due to the invasive nature of obtaining samples, the need for sedation or anesthesia, and the fact that obtaining renal tissue further reduces the functional nephron capacity of the patient. For this preliminary study, the ability to compare serum results with tissue provided a method for selecting the miRNAs that were altered in both sample types. Future studies may not require tissue samples and should include urine in addition to serum, as these are more easily obtained samples for diagnosis and monitoring of cats with CKD. In some studies, urine concentrations correlate with disease severity,28 making this an attractive sample type for future analysis. Tissue samples from kidneys taken prior to unilateral renal ischemia were not available for evaluation of tissue miRNA expression. Such tissues would have been a true control and a more appropriate comparator than the contralateral kidney. Previous work in a similar experimental model of CKD has identified changes to gene transcript levels in the contralateral kidney despite no direct insult to this kidney,17,19 suggesting crosstalk between the ischemic and contralateral kidney. This may also explain why fewer miRNAs were differentially expressed between ischemic and contralateral kidneys than between end point serum and baseline serum.

This study evaluated limited time points due to the use of previously banked samples but provides preliminary information with which future studies can be conducted. Such future studies should evaluate additional time points to determine whether these altered miRNA expression patterns occur early on in the disease process. In the present study, target analysis for differentially expressed miRNAs was based on predicted target searching, mapping to genes previously associated with feline CKD. Additional work should be done to determine whether these miRNAs are definitively acting on these predicted targets in this CKD model. Differential expression of miRNAs has been identified in many diseases, and overlap of dysregulated miRNA occurs between cancerous and noncancerous processes. Because the present study used each cat as its own internal comparator, these results likely reflected changes occurring due to the induction of CKD, rather than a separate disease process. However, studies confirming the predicted targets could also help confirm that dysregulation occurs specific to renal disease. Studies should also be performed in cats with naturally occurring CKD to determine whether differential expression of miRNA is similar to this model of CKD.

Results of this preliminary study identified 5 miRNAs (mir-21, mir-92, mir-146, mir-199, and mir-382) that were mutually differentially expressed between ischemic and contralateral kidneys and baseline and end point serum. All 5 of these miRNAs have been previously associated with renal disease in humans and mice. Furthermore, all 5 miRNAs had predicted target genes known to be dysregulated in experimentally induced and naturally occurring feline CKD. Additional work is needed to determine how expression patterns change with disease progression and whether these miRNAs may be potential diagnostic or therapeutic targets for feline CKD.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org

Acknowledgments

Funded by a grant from the Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

The authors declare that there were no conflicts of interest.

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Supplementary Materials

Contributor Notes

Corresponding author: Dr. Grimes (jgrimes@uga.edu)