Evaluation of profibrotic gene transcription in renal tissues from cats with naturally occurring chronic kidney disease

Abstract Background Increased gene transcription of hypoxia‐induced mediators of fibrosis in renal tissue has been identified in experimentally induced, ischemic chronic kidney disease (CKD). Objective To characterize hypoxia‐induced profibrotic pathways in naturally occurring CKD in cats. Animals Twelve client‐owned cats with CKD and 8 healthy control cats. Methods In this prospective, cross‐sectional study, bilateral renal tissue samples were assessed histologically for inflammation, tubular atrophy, and fibrosis, and by reverse transcription‐quantitative PCR for characterization of transcript levels of hypoxia‐inducible factor‐1α (HIF1A), matrix metalloproteinases‐2 (MMP2), ‐7 (MMP7), and ‐9 (MMP9), tissue inhibitor of metalloproteinase‐1 (TIMP1), transforming growth factor‐β1 (TGFB1), and vascular endothelial growth factor‐A (VEGFA). Linear mixed models were used to compare gene transcription between diseased and healthy kidneys, and to examine the association between transcript levels and serum creatinine concentration for all cats, and between transcript levels and histologic scores of diseased kidneys. Results Kidneys from cats with CKD had significantly higher transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 (all P < .001), and lower levels of VEGFA (P = .006) than those from control cats. Transcript levels of MMP7 (P = .05) and TIMP1 (P = .005) were positively associated with serum creatinine in cats with CKD, but not in control cats. In diseased kidneys, transcript levels of MMP2 (P = .002), MMP7 (P = .02), and TIMP1 (P = .02) were positively, whereas those of VEGFA (P = .003) were negatively, associated with histologic score severity. Conclusion and Clinical Significance Evaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats.

Conclusion and Clinical Significance: Evaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats.

K E Y W O R D S
feline, hypoxia-inducible factor, matrix metalloproteinases, tissue inhibitor of metalloproteinase, transforming growth factor, tubulointerstitial fibrosis, vascular endothelial growth factor 1 | INTRODUCTION Tubulointerstitial fibrosis, a key histologic feature of chronic kidney disease (CKD) in cats, 1 is correlated with the degree of functional renal impairment. 2,3 Across mammalian species, this pattern of histologic change is commonly observed in the advanced and end-stages of CKD. 4,5 However, in cats, renal fibrosis is present from the early stages of disease. 1,3 Growing evidence has emphasized the critical role of tubulointerstitial hypoxia in the development and progression of renal fibrosis and CKD. [6][7][8] Although the processes leading to fibrosis can initially promote normal tissue repair in response to renal injury, 9 the development of fibrosis has been proposed as a maladaptive response. Fibrosis can alter renal microcirculation and increase the distance for oxygen diffusion between renal tubules and capillaries, thereby promoting chronic renal hypoxia. 7,10 On a molecular level, the cellular and tissular responses to hypoxia are orchestrated by the complex interaction of cytokines and transcription factors.
Accumulation of hypoxia-inducible factor (HIF)-1α in hypoxic regions initiates hypoxia-adaptive responses, 8 and is a key driving force for the progression of CKD in rodents. 11 Vascular endothelial growth factor (VEGF)-A, a potent proangiogenic factor, is stimulated by renal hypoxia via HIF-1α. 12,13 Both underexpression and overexpression of VEGF-A have been associated with worsening of kidney disease, demonstrating this cytokine's complex pathophysiologic role. 14 Other fibrogenic factors that have been suggested as central to the development of renal fibrosis under hypoxic conditions include transforming growth factor (TGF)-β1, 7,15 the matrix metalloproteinases (MMPs), particularly MMP-2, -7, and -9, and their tissue inhibitors (TIMPs). 16,17 Although alterations in urinary TGF-β1 and VEGF-A occur in cats with CKD, [18][19][20][21] the roles of HIF-1α and MMPs remain to be characterized in spontaneous renal disease in cats. There is increased transcription of the genes for MMP-2 (MMP2), MMP-7 (MMP7), MMP-9 (MMP9), TIMP-1 (TIMP1), and TGF-β1 (TGFB1), and decreased transcription of the gene for VEGF-A (VEGFA), in the kidneys of cats subjected to unilateral, transient renal ischemia, relative to those of healthy controls. 22 In that study, although there was no difference in transcript abundance of HIF1A between groups, renal transcript levels of HIF1A, MMP2, MMP7, and TIMP1 were positively and strongly correlated with worsening degrees of fibrosis in kidneys exposed to transient ischemia.
The objective of the present study was to characterize the renal transcription of hypoxia-induced profibrotic pathways in naturally occurring CKD in cats. It was hypothesized that as in ischemiainduced experimental CKD, and compared to tissues from healthy control cats, gene transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 would be increased, and those of VEGFA would be decreased, in renal tissues from cats with naturally occurring CKD. A secondary objective was to examine the association between profibrotic gene transcription and histologic renal lesions. It was hypothesized that transcript levels of the profibrotic mediators would be positively, whereas those of the proangiogenic factor VEGFA would be negatively, associated with severity of histologic changes in affected kidneys.

| Study design
This was a prospective, cross-sectional study performed on renal tissue samples obtained from client-owned cats diagnosed with naturally occurring CKD (CKD group) and from healthy control cats. The University of Georgia Institutional Animal Care and Use Committee approved all activities related to this study (Animal Use Protocol A2017 05-008-Y3-A1).

| Animals
Renal tissue samples from cats of the CKD group were obtained immediately postmortem from cats that presented for euthanasia, or within consistent with CKD. Cats may have been euthanized or died of natural causes related to renal or extrarenal disease. Cats were excluded if they received a renin-angiotensin-aldosterone system antagonist (ie, an angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, or mineralocorticoid receptor antagonist) or a short-acting corticosteroid in the 14 days preceding euthanasia or natural death, or if they received a depot corticosteroid injection in the 6 months before euthanasia or natural death. Cats were also excluded if they were affected by uncontrolled hyperthyroidism (total T4 > upper limit of laboratory reference range at most recent sampling) or congestive heart failure. All owners were required to read and sign a form consenting to their pet's participation in the study.
Samples from the control group were collected from adult cats that were euthanized as part of population control measures at a local animal control facility, and from adult purpose-bred research cats participating in unrelated terminal studies having no impact on renal structure and/or function. These cats were considered to be healthy based on normal findings of physical exam and necropsy, and were deemed to have normal renal function and structure on the basis of renal histology, serum biochemistry, and urine analyses (ie, sCr <1.6 mg/dL, SDMA ≤14 μg/dL, USG >1.035, and UPC <0.4).
For healthy intact cats, an UPC < 0.6 was considered acceptable for inclusion, provided all other renal function biomarkers (ie, sCr, serum urea nitrogen [SUN], SDMA, and USG) and renal histology were normal. 23 Cats recruited from the animal control facility population were additionally screened for retroviral infections using a combined feline immunodeficiency virus antibody and feline leukemia virus antigen test (SNAP FIV/FeLV Combo Test, IDEXX Laboratories, Westbrook, Maine), and were excluded if positive for either.

| Blood and urine sample collection and processing
For control cats identified at the animal control facility, blood and urine samples were collected via cardiocentesis and cystocentesis, respectively, immediately after euthanasia with pentobarbital. For purposebred control cats, blood and urine samples were collected via jugular venipuncture and cystocentesis, respectively, after sedation with intramuscular buprenorphine (0.03 mg/kg), acepromazine (0.1 mg/kg), and midazolam (0.3 mg/kg), immediately before euthanasia with pentobarbital. Serum, ethylenediaminetetraacetic acid-anticoagulated blood, and urine samples were submitted for analysis within 1 hour of collection.
Client-owned cats enrolled in the CKD group did not undergo blood and urine sampling. Information pertaining to each cat's renal function was obtained from review of the individual's medical record.

| Clinical laboratory analyses
Complete blood count and serum biochemistry profile including SDMA, urinalysis, and UPC measurement were performed for each control cat.   After overnight incubation at 4 C, tissues were removed from RNA stabilization solution, homogenized with a mortar and pestle, divided into 30 mg aliquots, and stored at −80 C until further analysis.

| Gene transcription analysis
Reverse transcription and quantitative PCR were performed as previously described. 22 For each sample, total RNA was extracted from Gene specific primers were selected from previously reported studies (Table 1). 22,[27][28][29][30] Primers were previously validated for reverse transcription-quantitative PCR analysis in feline renal tissue homogentes. 22  hematoxylin. For each kidney, 10 consecutive ×20 fields of cortex and corticomedullary junction were scored for the degree of fibrosis, inflammation, and tubular atrophy. A consensus score was arrived at by conference of 2 board-certified veterinary pathologists (C. A. B. and D. R. R.), as previously described. 22,31 Briefly, the degree of fibrosis was evaluated in Masson's trichrome-stained sections and scored as 0 (absent; no increase compared to normal), 1 (mild, rare foci/segments of fibrosis involving <20% of the examined field), 2 (moderate, fibrotic segments involving 20%-30% of the examined field), or 3 (severe, fibrotic segments involving >30% of the examined field). Inflammation (ie, presence of lymphocytes, plasma cells, macrophages, or some combination of these) was evaluated in hematoxylin and eosin-stained slides and scored as 0 (absent, no inflammatory cells), 1 (mild, <10% of the examined field affected), 2 (moderate, 10%-50% of the examined field affected), or 3 (severe, >50% of the examined field affected). Lastly, tubular atrophy was scored in periodic acid-Schiff-and hematoxylin-stained sections as 0 (no atrophy), 1 (mild, fewer than 10 scattered atrophic tubules per field), 2 (moderate, linear streaks of tubular atrophy often with fibrosis and inflammation), or 3 (severe, 2 or more streaks of tubular atrophy per field). Numeric scores of the 20 (10 cortical and 10 corticomedullary) examined fields were used to calculate median scores for inflammation, tubular atrophy, and fibrosis for each kidney.

| Gross and histologic renal evaluation
Median scores that fell between 2 classification categories were rounded to the nearest integer.    and concurrent leukocytosis and bacteriuria (n = 1). One CKD cat was excluded because of having received a depot corticosteroid injection 10 days before euthanasia. Twelve cats with CKD and 8 healthy control cats met the enrollment criteria and were included in the present study; all included cats underwent euthanasia.

| Statistical analysis
A summary of demographic and clinical data of included cats is presented in Table 2. Although the ages of 4 feral control cats were estimated, cats of the CKD group were significantly older than healthy controls. There was a significant difference in sex and neuter status between groups, with the control group having a greater proportion of sexually intact cats. There were no significant differences in body weight or breed distribution between groups. Consistent with the inclusion criteria and definitions used to designate cats as affected by CKD, significant differences in SUN, sCr, USG, and UPC were observed between groups. All neutered cats included in the control group had a UPC ≤0.19.
All cats in the CKD group were euthanized for clinical signs presumed to be related to kidney disease, including persistent lethargy, inappetence, chronic weight loss, or some combination of these.
Acute-on-CKD was considered likely for 2 of these cats. As biochemical data from client-owned cats were not acquired during the same hospital visit as euthanasia in all instances, but rather obtained via review of medical record, the median (range) time between the date of last creatinine measurement and euthanasia was longer for CKD Medications prescribed in the 6 weeks preceding euthanasia included mirtazapine (n = 2), amoxicillin/clavulanic acid (n = 2), atenolol (n = 1), chitosan and calcium carbonate (n = 1), aluminum hydroxide (n = 1), ranitidine (n = 1), sucralfate (n = 1), and lactulose (n = 1). Subcutaneous fluids were being administered regularly by the owner in 2 cases.
Six cats in the CKD group were fed a commercially available pre-    Table 3.

| Gross and histologic renal evaluation
Marked asymmetry in renal size and morphology was present in 3 CKD cats (ie, "big kidney-little kidney" syndrome). In each, the right kidney was abnormally large, whereas the left kidney was abnormally small.
Histologically, cats with CKD were diagnosed with typical ischemic CKD (ie, chronic tubular atrophy and tubulorrhexis with interstitial inflammation, lipid, and fibrosis; n = 7), ischemic CKD with oxalosis (n = 2), amyloidosis (n = 1), membranoproliferalive glomerulonephritis (n = 1), and possible renal maldevelopment with secondary ischemic T A B L E 2 Clinical and clinicopathologic data from cats with chronic kidney disease (n = 12) and from healthy control cats (n = 8). Numerical data are presented as mean ± SD or median (range), where appropriate. Number of cats for which data are available is provided if different from the number of individuals in each group changes (n = 1). Urothelial proliferative changes were noted in the papilla and pelvis of 7 cats with CKD.

| Association between gene transcript levels and histologic scores of diseased kidneys
In the kidneys from the CKD group cats, there was a significant, positive association between transcript levels of MMP7 and median tubular atrophy scores, and between transcript levels of each of MMP2, MMP7, and TIMP1 and median fibrosis scores (Table 4; Figure 3). In tissues from cats with CKD, transcript abundance of MMP2 and MMP7 was correlated with worsening degrees of renal fibrosis.
Tubulointerstitial fibrosis is highly correlated with functional impairment. 2,3 Thus, it was not surprising that transcript levels of MMP7 were also positively associated with sCr in the CKD group in the present study. In human beings, MMP-7 is recognized as both an important mediator of fibrosis in CKD, as well as a urinary biomarker of renal fibrosis. 17,33 Our data suggest that MMP-7 may similarly represent a useful biomarker for this histologic pattern of change in cats; however, whether increased transcription of the MMP7 gene is accompanied by increased urinary levels of this protein remains to be investigated.

T A B L E 3
Association between serum creatinine concentration (mg/dL) and natural log-transformed bilateral renal tissue homogenate gene transcript levels in healthy control cats (n = 8) and cats with chronic kidney disease (n = 11) using a linear mixed model Notes. Linear mixed model with fixed effect = group, creatinine, group*creatinine; random effect = cat. Slopes represent the change in ln(transcript levels) corresponding to a 1 mg/dL increase in serum creatinine. One cat in the chronic kidney disease group did not have a serum creatinine measurement within 91 days of euthanasia and was not included in the analysis. Abbreviations: HIF1A, gene for hypoxia-inducible factor-1α; MMP2, gene for matrix metalloproteinase-2; MMP7, gene for matrix metalloproteinase-7; MMP9, gene for matrix metalloproteinase-9; RPS7, gene for ribosomal protein S7; TGFB1, gene for transforming growth factor-β1; TIMP1, gene for tissue inhibitor of metalloproteinase-1; VEGFA, gene for vascular endothelial growth factor-A. The gelatinase MMP-2 also appears to play a pivotal role in the progression of interstitial fibrosis. 34 Serum concentrations of both MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 were increased in children with CKD when compared to age-matched controls, and these concentrations were elevated in proportion to disease stage. 35 Conversely, in adult human beings with CKD, serum activity of MMP-2 was increased, whereas activity of MMP-9 was decreased relatively to control subjects. 36 In that report, sCr was directly correlated with MMP-2 activity and inversely correlated with MMP-9 activity. Although no significant association between renal transcript levels of MMP2 and sCr was observed in the present study (P = .06), the number of subjects evaluated was small. Also, this study did not In a previous study, renal transcription of HIF1A was not different between cats with experimentally induced, ischemic CKD and control cats; however, it was positively correlated with worse fibrosis scores in diseased kidneys. 22 Conversely, in the present study, which evaluated kidneys from cats with more severe histologic and functional derangements than those of the earlier study, a significant difference in transcription abundance of HIF1A was observed in renal tissues from cats with CKD as compared to those from normal controls; nonetheless, an association with histologic scores was not found.
These data suggest that chronic renal hypoxia might be an ongoing feature of CKD in cats but that HIF1A transcription might not be consistently proportional to disease severity. Work performed in cultured renal cells and knockout mice models suggests that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease by promoting fibrogenesis. 40,41 Therefore, HIF-1α signaling might warrant evaluation as a therapeutic target in cats.
In the present study, transcription of TGFB1 was upregulated in CKD. Two prior veterinary reports document increased urinary TGF-β1 levels (expressed as urinary TGF-β1-to-creatinine ratio) in cats with naturally occurring CKD, as compared to healthy control cats. 18,21 Recently, a positive correlation between this ratio and sCr was identified. 18 We failed to find a similar association in the present study and although TGF-β1 is a prototypical fibrogenic cytokine, there was no significant association between its transcript levels and the median histologic lesion scores. As regulation of gene expression occurs at the level of both transcription and translation, 42  In addition to documenting increased urinary TGF-β1-to-creatinine ratio in cats with CKD, Habenicht and colleagues noted that cats with CKD had significantly lower urinary VEGF-to-creatinine ratio than did T A B L E 4 Association between renal histologic lesion scores for inflammation, tubular atrophy, and fibrosis, and natural log-transformed gene transcript levels in bilateral renal tissue samples from cats with chronic kidney disease (n = 12) using a linear mixed model Note. Linear mixed model with fixed effect = histologic score; random effect = cat. Abbreviations: HIF1A, gene for hypoxia-inducible factor-1α; MMP2, gene for matrix metalloproteinase-2; MMP7, gene for matrix metalloproteinase-7; MMP9, gene for matrix metalloproteinase-9; RPS7, gene for ribosomal protein S7; TGFB1, gene for transforming growth factor-β1; TIMP1, gene for tissue inhibitor of metalloproteinase-1; VEGFA, gene for vascular endothelial growth factor-A. a The coefficient for the VEGFA fibrosis model represents the change in ln(VEGFA transcript levels) for kidneys with a fibrosis score of 2 (moderate) or 3 (severe) compared to kidneys with a fibrosis score of 1 (mild). The coefficients for all other models represent the change in ln(transcript levels) corresponding to a one-unit increase in the linear histologic score. normal cats. 18 In a separate study, urinary VEGF-to-creatinine ratio was inversely associated with the development of azotemia after treatment of hyperthyroid cats. 19 These findings are in accordance with the downregulation of renal transcription of VEGFA described in the CKD cats of present study. It has been hypothesized that decreased production of VEGF may promote loss of peritubular capillary density and contribute to progressive renal disease in CKD. 43 Nevertheless, the role of this growth factor in the pathophysiology of renal disease is complex.
Conflicting data from different models and distinct forms of renal disease describe it as deleterious in some disease settings and protective in others. 44 There are limitations to this study. First, cats with CKD were significantly older and more likely to be sexually altered (via orchiectomy or ovariohysterectomy) than control cats. Although age was not significantly associated with the transcript levels of any gene in our study, the impact of neuter status was not explored. Furthermore, as posttranscriptional regulation of gene expression was not examined, any impact of age or sex in the regulation of gene expression would have been missed. Additionally, cats with CKD were obtained from a population of client-owned cats, whereas control cats were obtained from feral or purpose-bred cat populations. Therefore, results may be influenced by differences in dietary or environmental factors, or both.
Of relevance, renal tissues were procured after death or euthanasia and a bias toward animals that may have been experiencing relatively rapid disease progression is likely in the CKD group. To this point, an acute exacerbation of renal disease was not able to be distinguished from stable CKD in all cases. It is possible that an overrepresentation of acute-on-chronic disease biased our results toward patterns observed in acute kidney injury. As blood and urine sampling was not performed in client-owned cats, clinicopathologic data from cats with CKD were collected at various time points before euthanasia. Thus, these data may not accurately reflect disease state at the time of collection, either by representing an earlier time point, or by being acquired during a period of decompensation, when the patient may have been more likely to experience prerenal azotemia. To address this, the authors limited the analyses measuring association between gene transcription and sCr to cats for which biochemical data was obtained within 3 months of euthanasia. Nonetheless, the potential impact of these factors must be considered when interpreting the reported correlations between gene transcript levels and sCr. Importantly, the number of individuals examined in the present study was small. Therefore, the study may have been underpowered to detect additional significant associations between gene transcription and biochemical or histologic F I G U R E 3 Scatter plot of natural log of normalized gene transcript levels of MMP7 and tubular atrophy scores (A), MMP2 and fibrosis scores (B), MMP7 and fibrosis scores (C), TIMP1 and fibrosis scores (D), and VEGFA and fibrosis scores (E) in tissue samples from each kidney of cats with chronic kidney disease (n = 12) parameters in the CKD population. Furthermore, the associations noted here may not remain significant if examined in a larger population of cats. Finally, the present study's assessment of the evaluated pathways was limited to the quantification of gene transcript levels using RT-qPCR. As the expression of the proteins coded by these genes was not quantified, the definitive role of these markers in the pathophysiology of tubulointerstitial fibrosis in cats remains to be clarified.
In conclusion, kidneys from cats with CKD included in the present study showed upregulation of transcription of profibrotic cytokines and growth factors known to be induced by ischemic injury. Additionally, transcript levels of MMP2, MMP7, and TIMP1 were positively associated with the severity of histologic scores. Future evaluation of the proteins corresponding to the genes evaluated here in a larger population of cats is warranted to explore their potential as biomarkers of renal fibrosis and as targets for therapeutic intervention in feline CKD.