Introduction
Hydrogen sulfide (H
2S) has traditionally been considered a toxic gas with the smell of rotten eggs, but is now also known as a third gasotransmitter, along with nitric oxide (NO) and carbon monoxide (CO) [
1,
2]. The physiological and pathological functions of H
2S include neurotransmission [
3], vascular relaxation [
3,
4], insulin secretion [
5], cell proliferation and apoptosis [
6].
The key enzymatic production of H
2S from
l-cysteine in mammalian tissues is catalyzed by cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) [
1,
3]. CSE is distributed in smooth muscle cells, liver and pancreas [
5,
7,
8], whereas CBS is found in the brain, liver, kidney and pancreas [
5,
8]. However, the roles of these two enzymes in the kidney remain unclear.
We previously reported that hyperglycemia reduces the level of endothelial NO synthase (eNOS) expression in the diabetic kidney, thereby reducing NO production and subsequently inducing endothelial dysfunction [
9]. We postulated that hyperglycemia would also decrease CSE expression in the kidney, which may cause renal microcirculation injury and renal ischemia. To investigate the roles of CSE and CBS in the kidney, the present study examined the localization of both enzymes in the normal kidney and the effect of the H
2S donor sodium hydrosulfide (NaHS) in the renal peritubular capillary (PTC) under conditions of diabetic nephropathy, using pancreatic β-cell-specific calmodulin-overexpressing transgenic (CaMTg, also called as OVE26) mice as a model of diabetes [
9].
Discussion
We detected CSE and CBS in the proximal tubules, but not in the glomeruli or distal tubules, and NaHS administration was found to increase PTC blood flow and PTC diameter using a reliable CCD system. Importantly, CSE expression was markedly decreased in the diabetic kidney with advanced lesions, whereas CBS expression was unaffected. Progressive diabetic nephropathy caused vasoconstriction and a loss of blood flow, which was ameliorated by NaHS treatment. These findings suggest that CSE might regulate PTC microcirculation in the tubulointerstitium and play a key role in the development of advanced diabetic nephropathy.
H
2S protects several tissues from various types of cell damage through anti-atherosclerotic effects and preservation of mitochondrial function [
16,
17]. However, the precise roles of H
2S in the kidney remain unknown. CSE expression is also reported to be predominant in the vascular smooth muscle cells (VSMCs) [
18], although earlier studies did not detect CSE expression in endothelial cells [
4,
19]. In our study, however, CSE expression was not found on VSMCs in PTCs and renal arterioles. In the renal system, PTCs are composed only of endothelial cells and play important roles in reabsorption and secretion between blood and the inner lumen of the nephron through active transport, secondary active transport or transcytosis. Instead of PTCs, tubular epithelial cells may express CSE protein alongside PTCs in the kidney.
Previous studies have reported that H
2S could induce vasorelaxation by directly opening K
ATP channels in VSMCs [
4,
20]. Unfortunately, we cannot directly verify in vivo the effect of H
2S through its measurement and treatment. We therefore demonstrated that administration of the H
2S donor NaHS increased blood flow by PTC dilation. As VSMCs are not present in PTCs, H
2S may act on the PTC endothelial cells. In addition, H
2S may be generated in the proximal tubules, and H
2S thus produced may reach PTC endothelial cells via membrane permeation. Given our results and the findings from several reports that endogenous H
2S has protective effects on renal ischemia/reperfusion injury [
21], H
2S produced by CSE and/or CBS in the tubular epithelial cells might have beneficial effects on the tubulointerstitium through anti-apoptotic effects and the regulation of hemodynamics. Anti-apoptotic effects of H
2S have also been reported in other types of cells [
12,
22].
Next, we evaluated whether biosynthesis of H
2S was altered in spontaneously diabetic CaMTg mice, which exhibit hallmarks of human diabetic nephropathy such as hyalinosis of the afferent and efferent arteries with neovascularization. We have previously reported that hyperglycemia reduces eNOS expression, thereby reducing the level of NO production and subsequently inducing endothelial cell proliferation injury [
9]. Upregulation of VEGF and downregulation of NO (‘uncoupling’ of the VEGF–NO axis) result in the progression of extra vessels and of extravasation from immature vessels, leading to the development of diabetic nephropathy. Other investigators have demonstrated that both CSE and CBS activities in the pancreas and liver, as well as plasma H
2S and
l-cysteine levels, are increased in streptozotocin-treated diabetic rats [
10,
23]. Intriguingly, CSE expression, like eNOS expression, in the proximal tubules was reduced in that diabetic model. H
2S also promotes angiogenesis through VEGF signaling pathways such as the PI3 K-Akt pathway [
24]. As the biological features of H
2S resemble those of NO, modulation of H
2S production might be involved in diabetic tubulointerstitial ischemia. High glucose further induces the CSE expression in the β cells in pancreas, in contrast to the renal proximal tubules [
10]. Interestingly,
l-cysteine or NaHS suppressed apoptosis in pancreatic islet under diabetic status. Indeed, pretreatment with
l-cysteine improved the secretory responsiveness following stimulation with glucose. These suggest that H
2S may protect β cells from glucotoxicity, eventually leading to the promotion of insulin secretion [
5].
We emphasize that the diabetic model used in this study showed decreased PTC blood flow velocity and blood flow, in spite of PTC neovascularization. Endothelial dysfunction attributable to eNOS reduction and insulin deficiency might induce decreased PTC blood flow, resulting in tubulointerstitial ischemia and injury [
25,
26]. Eventually, this state would create a vicious cycle such as activation of the renin–angiotensin system. We further demonstrated that NaHS administration increased blood flow by PTC dilation. Indeed, CSE reduction results in decreased H2S formation [
18]. These findings suggest that CSE in the proximal tubules may regulate the interstitial microcirculation via H
2S production. As the sensitivity of PTCs to NaHS was maintained in this diabetic model, H
2S may offer a useful target for the treatment of diabetic nephropathy.
Acknowledgments
This paper was supported by the Aichi Diabetes Rheumatoid Gout Foundation, KAKENHI (21591146 and 22790255) from Japan Society Promotion of Science and Research Fund at the Discretion of President, Oita University, a Grant-in-Aid for Diabetic Nephropathy Research, from the Ministry of Health, Labor and Welfare of Japan, and grants from Oita Broadcasting System Cultural Foundation and from Oita University Venture Business Laboratory.