Ab7900 machine

Total RNA was extracted from kidney tissue using Trizol. cDNA was synthesised using Superscript III (Life Technologies) first strand synthesis. The cDNA was subjected to standard curve measurement to ensure efficiency prior to real time PCR, which was done using SYBR green (Life Technologies, Australia) for MCP-1, fibronectin, TGFβ, collagen IV, GLUT1 and GLUT2 using actin as the endogenous control. PCR for SGLT2 and SGLT1 were done using Taqman PCR Universal Mastermix (Applied Biosystems). The RTPCR was performed on the AB7900 machine (Applied Biosystems, Australia). Gene expression was quantified relative to actin. The primers are listed in Table 2.

Total RNA was extracted from whole kidney tissue using the RNeasy Plus Mini extraction kit (Qiagen Valencia, CA, USA). cDNA was synthesised using the Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). Predesigned primers (Sigma-Aldrich, NSW Australia) for fibronectin, collagen I and IV, αSMA, E-Cadherin and actin are listed in Table 2. Quantitative real-time PCR was performed with SensiMix SYBR hiRox (Bioline, NSW Australia) on the AB7900 machine (Applied Biosystems, Australia). Gene expression is presented as fold-change compared with control after normalisation to the housekeeping gene actin.Table 2

PCR primer sequences.

Target	Forward (5′-3′)	Reverse (5′-3′)
LOXL2	ATTAACCCCAACTATGAAGTG	CTGTCTCCTCACTGAAGGCTC
Fibronectin	ACAGAAATGACCATTGAAGG	TGTCTGGAGAAAGGTTGATT
COL1A1	CATGTTCAGCTTTGTGGACCT	GCAGCTGACTTCAGGGATGT
Col IV	TTAAAGGACTCCAGGGACCAC	CCCACTGAGCCCTGTCACAC
aSMA	ATAGGTGGTTTCGTGGATGC	ACTCTCTTCCAGCCATCTTTCA
E-Cadherin	CAAAGTGACGCTGAAGTCCA	TACACGCTGGGAAACATGAG
β-Actin	CTAAGGCCAACCGTGAAAAG	ACCAGAGGCATACAGGGACA

Total RNA was extracted from kidney tissue using Qiagen RNEasy Mini kit on an automated RNA extraction protocol using Qiacube. cDNA was synthesised using Roche Transcriptor First Strand cDNA synthesis kit (Roche, USA). The real time PCR was done using SYBR green (Bioline, Australia) for fibronectin (forward-CACGGAGGCCACCATTACT and reverse-CTTCAGGGCAATGACGTAGAT) using actin (forward-CAGCTGAGAGGGAAATCGTG and reverse-CGTTGCCAATAGTGATGACC) as the endogenous control. Primers were sourced from Sigma. The RT-PCR was performed on the AB7900 machine (Applied Biosystems, Australia).