Left ventricular hypertrophy in experimental chronic kidney disease is associated with reduced expression of cardiac Kruppel-like factor 15

Female Sprague–Dawley rats (body weight 200–225 g) were purchased from Monash University (Melbourne, Australia) and were housed at 23–25 °C in a 12 h:12 h light–dark cycle, with ad libitum food containing 0.4–0.6% NaCl (Norco, NSW, Australia) and water. Rats were weighed and allocated to one of three groups (Control, STNx vehicle or ramipril) according to body weight in order to ensure the mean body weight per group was similar pre-operatively. There were no significant differences in pre-operative body weight between the 3 groups (mean ± SEM, Control (201 ± 3.7 g); STNx-vehicle (207 ± 1.3 g); STNx- ramipril (211 ± 2.3 g), P = 0.163). One animal in the Control group died unexplained prior to sham surgery. There were no further adverse events. In brief, rats were anesthetized by an intraperitoneal injection of pentobarbitone sodium (60 mg/kg/body weight, Boehringer Ingelheim, NSW, Australia), and STNx (n = 20) or sham surgery (Control, n = 9) was performed as described previously [15‐17] by removal of the right kidney, and infarction of two-thirds of the left kidney. Following surgery, STNx rats received daily vehicle (n = 10) or the ACE inhibitor, ramipril (1 mg/kg/day, n = 10) by gavage for 28 days. Sham-operated Control rats received vehicle (n = 9). Investigators were not blinded to the disease groups or treatment. However, the cardiac assessment was performed by an investigator who was blinded to both model and treatment, and all biochemical, gene expression and histological analysis were performed in a blinded manner.

On day 27, rats were housed in metabolic cages and a urine sample collected. On day 28, rats were anaesthetised with intraperitoneal sodium pentobarbitone (60 mg/kg/body weight), and cardiac haemodynamics were assessed as previously described [15]. Heart rate, systolic and diastolic blood pressure, mean arterial pressure (MAP), LV end-diastolic pressure (LVEDP) and the maximal and minimal rate of ventricular contraction (dP/dt_max, dP/dt_min) and the time constant of isovolumic relaxation (Tau) were measured. Rats were killed by a lethal dose of sodium pentobarbitone, decapitated, trunk blood collected, centrifuged, and plasma snap-frozen and stored at -80 °C. The heart and left kidney was removed and weighed, and the left ventricle (LV) was transversely dissected with one piece fixed in 4% paraformaldehyde and embedded in paraffin for histopathology, and the remainder snap frozen in isopentane and stored at -80 °C for mRNA extraction.

Plasma and urine creatinine and plasma urea were measured using an autoanalyser (Beckman Instruments, Palo Alto, CA, USA). Urinary protein was determined using the bicinchoninic acid (BCA) method with a commercially available Pierce™ BCA protein assay kit (ThermoFisher Scientific, Victoria, Australia). Plasma ACE activity was measured as previously described [18].

Collagen was assessed as previously described [15‐17] using LV (4 μm) sections which were deparaffinized, rehydrated, and stained with 0.1% Sirius Red (Polysciences Inc) in saturated picric acid (picrosirius red) for 1 h, differentiated in 0.01% HCl for 30 s, and rapidly dehydrated. Interstitial collagen volume fraction was determined by measuring the area of stained tissue within a given field, excluding vessels, artefacts, minor scars or incomplete tissue fields; 15–20 fields were analysed per animal (n = 6/gp). The area stained was calculated as a percentage of the total area within a given field.

Immunohistochemical staining for cardiac KLF15 (polyclonal antibody, Aviva, Sapphire Bioscience, Australia, diluted 1:100) was performed in LV (4 μm) sections. Sections were deparaffinized, rehydrated, and then endogenous peroxidase activity quenched for 30 min by incubation in 3% H₂O₂ in methanol. The primary antibody was applied and left to incubate overnight at 4 °C. The Elite Vectastain ABC kit (Vector Laboratories, CA, USA) followed by 3,3-Diaminobenzidine (Sigma, St Louis, MO, USA) were used to visualize antibody binding. Slides were counterstained with haematoxylin and cover-slipped. Negative control slides were incubated with normal goat serum in the absence of the primary antibody. Staining was quantified using computerized image analysis (MCID, Imaging Research, Ontario, Canada). Images were imported into the AIS Imaging program (AIS Imaging, Ontario, Canada) using a colour video camera and a standard light microscope (magnification × 20). The detection level threshold for positively stained areas (brown for 3,3-Diaminobenzidine staining) was set so that the processed image accurately reflected the positively stained areas as visualized by light microscopy on the unprocessed digital image. KLF15 staining was quantitated by measuring the area of stained tissue within a given field, excluding vessels, artifacts, minor scars or incomplete tissue fields. Fifteen fields were analysed per animal (n = 6/gp). The density x area of staining was determined by calculating the number of selected pixels (positively stained areas) in the total area within a given field.

Total RNA was isolated from LV using the RNeasy kit method (Qiagen). cDNA was synthesized with a reverse transcriptase reaction using standard techniques (Superscript III kit; Life Technologies) as described previously [15]. Rat cardiac KLF15, ANP, BNP, CTGF, GATA4 and MEF2c gene expression was determined using TaqMan assays (ThermoFischer Scientific, Victoria, Melbourne). TaqMan gene expression assay identifiers used were as follows: KLF15 - Rn00585508_m1; BNP - Rn04219558_g1; ANP - Rn00664637_g1; CTGF - Rn01537279_g1; GATA4 - Rn01530459_m1 and MEF2A - Rn01478096_m1). Cardiac ACE gene expression was determined using primers and probes as previously described [19]. Quantitative RT-PCR was performed using the TaqMan system based on real-time detection of accumulated fluorescence (7500 Real Time PCR System, Applied Biosystems, CA, USA). Gene expression was normalized to 18S VIC and reported as the fold change compared with expression in vehicle treated Control rats which were given an arbitrary value of one.

At 4 weeks after STNx, rats had reduced body weight (P < 0.05) and kidney impairment with elevated plasma creatinine (P < 0.001) and plasma urea, proteinuria (P < 0.01) and reduced creatinine clearance (P < 0.001) compared to Control rats (Table 1). In STNx rats, treatment with ramipril reduced plasma ACE activity (< 0.0001) and proteinuria (P < 0.05) but did not change plasma creatinine and urea or creatinine clearance.

STNx rats had significant and marked increases in systolic and diastolic blood pressure (Table 2, P < 0.01) and MAP (P < 0.001). CKD led to adverse effects on cardiac function with hypercontractility (P < 0.05), and diastolic dysfunction with impaired active relaxation (Tau, P < 0.05), and increased LVEDP (P < 0.01). Ramipril had significant cardiovascular benefits to decrease blood pressure, Tau and LVEDP (all P < 0.01) (Table 2).

STNx rats had LV hypertrophy (P < 0.001, Fig. 1a) and interstitial fibrosis (P < 0.05, Table 2) compared to Control rats. In STNx rats, ACE inhibition reduced LV mass (P < 0.001, Fig. 1a) and interstitial fibrosis (P < 0.05, Table 2) compared to vehicle treatment.

Changes in LV gene expression with STNx and the effect of ramipril are shown in Table 3. Cardiac ACE gene expression was unchanged in STNx rats compared to Control, and reduced by ramipril (P < 0.01). STNx rats had a significant increase in gene expression of markers of cardiac hypertrophy (ANP, BNP, both P < 0.01). Expression of ANP and BNP is regulated by pro-hypertrophic transcriptional factors including MEF2A and GATA4, and STNx rats had increased expression of GATA4 mRNA (P < 0.05), but no change in MEF2A. STNx rats also had increased cardiac expression of the pro-fibrotic marker, CTGF (P < 0.05). Ramipril reduced ACE gene expression, ANP (P < 0.01), BNP (P < 0.05) and CTGF (P < 0.01) but had no effect on GATA4 or MEF2A mRNA expression. Ramipril had no effect on either GATA4 or MEF2A mRNA expression in STNx rats.

There was a 50% reduction in cardiac KLF15 gene expression in STNx rats with LVH compared to Control rats, there (P < 0.05; Fig. 1b) with a corresponding fall in KLF15 protein (P < 0.05, Fig. 1c). Treatment with ramipril led to increases in both cardiac KLF15 mRNA (P < 0.05, Fig. 1b) and protein expression (P < 0.01, Fig. 1c).

There was a significant negative correlation between LV mass and LV KLF15 gene (rho = − 0.53, P = 0.029; Fig. 2a) and protein expression (rho = − 0.78, P = 0.003; Fig. 2b) when assessed in vehicle treated Control and STNx rats. KLF15 gene and protein expression was lowest in rats with the highest LV mass. Figure 3 shows KLF15 immunohistochemical staining in the cytoplasm and nuclei of myocytes, with reduced staining in STNx rats.