Dysregulation of microRNAs and renin-angiotensin system in high salt diet-induced cardiac dysfunction in uninephrectomized rats

Abstract

Uninephrectomy is not associated with major adverse events in cardiovascular and renal functions of live kidney donors. The effect of high salt diet on the quality of life of live kidney donors is largely unknown. Hence in this study, we aimed to determine the effect of high salt diet on the alterations of renin-angiotensin system and microRNAs leading to CV and renal dysfunction in uninephrectomized rats. In order to mimic clinical scenario, uninephrectomized male Sprague Dawley rats were fed initially with normal pellet diet for 12 weeks and then for 20 weeks with high salt (10% w/w NaCl) diet. At the end of the study, biochemical, functional, histological and molecular parameters were measured. High salt diet feeding resulted in renal dysfunction & fibrosis, decreased baroreflex sensitivity, increased in vivo cardiovascular reactivity to angiotensin II owing to upregulation of angiotensin II type 1 receptors and L-type calcium channels leading to cardiovascular dysfunction in uninephrectomized rats (UNX+HSD) worse than that of normal (binephric) rats fed with high salt diet (HSD). Protein expression of functional and hypertrophic protein markers revealed decreased SERCA, p-AMPK and increased p-AKT. Interestingly, levels of miR-25, miR-451 and miR-155 increased and miR-99 decreased in heart of uninephrectomized rats fed with high salt. However, circulating miR-25 and miR-451 levels decreased and miR-99b increased in these animals. Our study points out that since tissue and circulating levels of miRNAs are not similar, caution must be exercised during the usage of miRs as diagnostic or prognostic biomarkers. To our knowledge, we are the first to show that epigenetic alterations result in cardiac dysfunction in uninephrectomized rats fed with high salt diet.

MicroRNA-941 regulates the proliferation of breast cancer cells by altering histone H3 Ser 10 phosphorylation

“…For quality control and individual LNA RT-PCR assays, reverse transcription was carried out using the Universal cDNA Synthesis kit II (Exiqon) as described 25 , 26 . Real time PCR was performed at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s/60 °C for 1 min using a Light Cycler 2.0 (Roche Diagnostics, USA).…”

“…Cytoplasmic and histone proteins were isolated and estimated according to the methods as described 27 . After isolation, equal amounts of protein were run on SDS PAGE and were electrophoretically transferred onto nitrocellulose membrane using semi-dry transfer apparatus (Bio-Rad) as described 26 . Immunoblot analysis was performed using anti-E-cadherin (rabbit, 1:500, Santa Cruz Biotechnology, CA, USA), anti-MMP-13 (rabbit, 1:1000, Santa Cruz Biotechnology, CA, USA), anti-CyclinD1 (rabbit, 1:500, Santa Cruz Biotechnology, CA, USA) anti-p21 (rabbit, 1:1000, Santa Cruz Biotechnology, CA, USA), anti-p-MSK-1 (Thr 581) (rabbit, 1:500,Cell signalling technology, USA), anti-MKP-1 (rabbit, 1:500, Santa Cruz Biotechnology, CA, USA), anti-PP2B-B1/2 (mouse, 1:1000, Santa Cruz Biotechnology, CA, USA), anti-histone H3 ser 10 phosphorylation (rabbit, 1:500, Cell signalling technology, USA), anti-H3 (goat, 1:1000 Sigma, St. Louis, MO, USA), anti-thymidylate synthetase (mouse, 1:1000, Santa Cruz Biotechnology, CA, USA)and anti α-tubulin (mouse, 1:1000, Santa Cruz Biotechnology, CA, USA) primary antibodies.…”

Dietary Supplementation of Methyl Donor l‐Methionine Alters Epigenetic Modification in Type 2 Diabetes

“…Histological slides were prepared as described by Amara et al . Briefly, the liver was fixed in 10% neutral buffer formalin, dehydrated gradually in ethanol and xylene, and then embedded in paraffin.…”

Liver mitochondrial membrane permeability modulation in insulin-resistant, uninephrectomised male rats by Clerodendrum volubile P. Beauv and Manihot esculenta Crantz

“…The rats underwent uni-nephrectomy of the left kidney in an aseptic condition, under ketamine (70 mg/kg, i. p.)/ xylazine (7 mg/kg, i. p.) anesthesia, before injection with saline (20 ml/kg, s.c.) according to the method described by Holte et al [52] with slight modifications [53]. Briefly, a 2 cm flank incision penetrating the abdominal cavity was made after the area had been cleaned and shaved.…”