Hydrogen sulphide-synthesising enzymes in pig kidney cortex and medulla

Richard Roberts, Matthew Bradbury, Agalyah Krishnan, Hemaben Mistry, Jennifer Phelps, Hnin Ei Phyu, Gulveer Sehmbey, Ching Yee Yeap, Michael Garle, Liaque Latif, Steve Alexander. University of Nottingham, Nottingham, UK

Hydrogen sulphide (H2S) is generated by three enzymatic routes (1). Two enzymes are able to use pyridoxal phosphate-dependent metabolism of L-cysteine, CBS (cystathionine β-synthase) and CSE (cystathionine γ-lyase). The third pathway involves cysteine aminotransferase to generate 3-mercaptopyruvate, which is utilised by MPST (3-mercaptopyruvate sulphurtransferase), a pyridoxal phosphate-independent enzyme. Infusion of an H2S donor into the rat renal artery has been reported to lead to increased renal blood flow, glomerular filtration rate and urinary cation excretion (2). This effect was mimicked by L-cysteine infusion and inhibited by amino-oxyacetic acid (AOAA) and L-propargylglycine (PPG) inhibitors of CBS and CSE. We have, therefore, examined the expression of hydrogen sulphide-synthesising enzymes in pig kidney cortex and medulla.

Cortex and medulla were dissected from porcine kidneys (obtained from a local abattoir) and differential centrifugation was used to generate cytosol, microsomes and mitochondria. A modification of the methylene blue method was used to estimate H2S production, while immunoblotting was employed to identify protein levels (3). Statistical analysis was conducted using ANOVA with Bonferroni’s multiple comparison on preparations from four animals (data presented are mean ± SEM).

10 mM L-cysteine-mediated H2S generation in the medulla cytosol was significantly inhibited in the presence of 100 µM AOAA, but not 100 µM PPG (control 7.5 ± 1.1; +AOAA 4.2 ± 0.8 (P<0.01); +PPG 6.4 ± 1.0 nmol/mg protein/90 min). In the medulla microsomal fraction, neither inhibitor was able to alter L-cysteine-mediated H2S generation (control 4.7 ± 1.0; +AOAA 3.4 ± 0.3; +PPG 4.4 ± 0.6 nmol/mg protein/90 min). Medulla mitochondrial L-cysteine-mediated H2S generation was variable and unaltered in the presence of either AOAA or PPG (control 9.8 ± 5.1; +AOAA 9.9 ± 5.5; +PPG 10.5 ± 4.9 nmol/mg protein/90 min). Using 0.3 mM 3-mercaptopyruvate as substrate in medullary fractions, H2S production was undetectable in microsomes or mitochondria, while cytosolic production was variable (4.0 ± 2.9 nmoles/mg protein/30 min).

In the kidney cortex, cytosolic H2S production from L-cysteine was also inhibited by AOAA, but not PPG (control 7.8 ± 1.4; +AOAA 1.1 ± 0.5 (P<0.001); +PPG 6.2 ± 0.9 nmol/mg protein/90 min). Cortical microsomal L-cysteine-mediated H2S generation was also significantly inhibited by AOAA, but not PPG (control 5.3 ± 0.9; +AOAA 2.6 ± 0.9 (P<0.05); +PPG 3.7 ± 1.0 nmol/mg protein/90 min). Cortical mitochondrial H2S generation from L-cysteine was also inhibited by AOAA, but not PPG (control 4.6 ± 2.0; +AOAA 2.4 ± 1.3 (P<0.05); +PPG 4.5 ± 2.2 nmol/mg protein/90 min). As in the medulla, cortical H2S production using 3-mercaptopyruvate as substrate was undetectable in microsomes or mitochondria, while cytosolic production was measurable (9.0 ± 0.9 nmoles/mg protein/30 min).

Immunoblotting suggested predominant expression of CBS at 63 kDa in cytosolic fractions from medulla and cortex, while CSE at 45 kDa was located in cortex cytosol, but not microsomes or mitochondria. MPST at 22-25 kDa was predominantly located in mitochondria from cortex and medulla.

In conclusion, there is a concordance between the pattern of immunoreactivity and enzyme activity for the L-cysteine-metabolising enzymes CBS and CSE in the pig kidney, while 3-mercaptopyruvate metabolism and MPST immunoreactivity are at odds. Whether the distribution pattern of these enzymes has an impact on renal function remains to be determined.

1. Papapetropoulos et al. (2014). Br J Pharmacol in press. PM:24909294

2. Xia et al. (2009). J Pharmacol Exp Ther 329: 1056-1062. PM:19246614

3. Rashid et al. (2013). Br J Pharmacol 168: 1902-1910. PM:23215842