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Flanagan, E and Johns, EJ;
Physiological Society General meeting
siRNA modulation of TGF-β: impact in a rat model of chronic renal failure
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Chronic renal failure is an insidious disease which presents as a gradual fibrosis of the tubular and vascular elements of the kidney. The initial insult to the kidney is unclear, but is associated with hypertension and dyslipidaemias of obesity and type II diabetes. There is liberation of growth factors and cytokines, a key one being TGF-β, which induce extracellular matrix production, fibrosis and eventually chronic renal failure if the insult persists. The aim of this study was to examine whether supression of TGF-β production, using siRNA, could attenuate the development of chronic renal failure. Male Wistar rats, 250-300g, received weekly injections of puromycin, 20mg/kg ip, for 6 weeks. At weekly intervals, groups of animals (n=6) were placed in metabolic cages and 24 h urine and a blood sample were collected. Vehicle and 1 mg/kg siRNA for TGF-β (Locus ID: 59086; Refseq number: NM-021578) dissolved in buffer and DOTAP was given iv 48h after each puromycin injection beginning at week 3. The animals were then killed and kidneys taken for analysis. Electrolyte excretion and creatinine clearance, urinary, cortical and medullary TGF-β levels were estimated. Data, means▒S.E.M, were analysed using Student┐s `t┐ test and significance taken at P<0.05. After 6 weeks of puromycin treatment creatinine clearance was lower, 14▒3 versus 50▒5 ml/kg/h and fractional sodium excretion was higher, at 5.6▒0.7 versus 1.2▒0.3% (both P<0.05) compared to the control group. In the puromycin group, urinary excretion of TGF-β was elevated, 7.7▒1.8 versus 1.9▒0.6 ng/24h and both cortical and medullary TGF-β was higher, 5.2▒0.8 versus 2.1▒0.1 pg/mg and 7.0▒0.5 versus 2.3▒0.1 pg/mg, respectively, compared to the control group (all P<0.01). In the groups of rats receiving both puromycin plus the siRNA, there was a blunting of the fall in creatinine clearance, the rise in fractional sodium excretion and the increases in urinary, cortical and medullary TGF-β of some 13 to 30% (all P<0.05). These data demonstrate that in this puromycin model of chronic renal failure the reduction in renal haemodynamic and excretory function was associated with a marked increase in renal tissue and urinary excretion of TGF-β. Administration of siRNA directed against TGF-β mRNA, partially ameliorated TGF-β production with a concomitant improvement in kidney function. It may well be that modification and adjustment of the siRNA dosing regime may result in a more effective suppression of TGF-β and hence improvement in kidney function.

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