Hydrogen sulfide attenuates sFlt1-induced hypertension and renal damage by upregulating vascular endothelial growth factor

Abstract

Soluble fms-like tyrosine kinase 1 (sFlt1), a circulating antiangiogenic protein, is elevated in kidney diseases and contributes to the development of preeclampsia. Hydrogen sulfide is a vasorelaxant and proangiogenic gas with therapeutic potential in several diseases. Therefore, we evaluated the potential therapeutic effect and mechanisms of action of hydrogen sulfide in an animal model of sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis created by adenovirus-mediated overexpression of sFlt1 in Sprague-Dawley rats. We injected sFlt1-overexpressing animals intraperitoneally with the hydrogen sulfide-donor sodium hydrosulfide (NaHS) (50 µmol/kg, twice daily) or vehicle (n=7 per group). Treatment with NaHS for 8 days significantly reduced sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis. Measurement of plasma protein concentrations with ELISA revealed a reduction of free plasma sFlt1 and an increase of free plasma vascular endothelial growth factor (VEGF) after treatment with NaHS. Renal VEGF-A mRNA expression increased significantly with NaHS treatment. In vitro, NaHS was proangiogenic in an endothelial tube assay and attenuated the antiangiogenic effects of sFlt1. Stimulation of podocytes with NaHS resulted in both short-term VEGF release (120 minutes) and upregulation of VEGF-A mRNA levels (24 hours). Furthermore, pretreatment of mesenteric vessels with a VEGF receptor 2-neutralizing antibody significantly attenuated NaHS-induced vasodilation. These results suggest that hydrogen sulfide ameliorates sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis in rats by increasing VEGF expression. Further studies are warranted to evaluate the role of hydrogen sulfide as a novel therapeutic agent for vascular disorders such as preeclampsia.

Figure 1.

Effect of NaHS on hypertension and proteinuria in rats overexpressing sFlt1. (A) Mean arterial pressure is measured via carotid catheterization under anesthesia after 8 days of treatment with either vehicle or 50 µM/kg NaHS. BP is significantly lower in the NaHS-injected group compared with vehicle. (B) After 7 days of treatment with vehicle or NaHS, rats are placed in metabolic cages to collect 24-hour urine and total albumin is determined. NaHS-treated rats show significant lower albuminuria than vehicle-treated rats. Data are presented as the median (IQR). n=7 per group. *P<0.05.

Figure 2.

NaHS reverses glomerular endotheliosis in rats overexpressing sFlt1. (A) Histopathologic analysis of renal tissue from one representative vehicle-treated rat overexpressing sFlt1 shows marked glomerular endotheliosis with occlusion of capillary lumens. (B) Histopathologic analysis of renal tissue from one representative NaHS-treated rat overexpressing sFlt1 (50 µM/kg) shows open capillaries with absence of proteinaceous deposits and mesangium that is now distinct (stained with hematoxylin and eosin). Electron microscopy is performed for the same rats as shown in A and B. (C) Representative electron micrographs of glomeruli from a vehicle-treated rat overexpressing sFlt1 confirm glomerular endotheliosis. (D) Representative electron micrographs of glomeruli from a NaHS-treated rat overexpressing sFlt1 show ameliorated glomerular endotheliosis. Original magnification, ×40 in B; ×10,000 in D.

Figure 3.

NaHS is proangiogenic in vitro. (A) An endothelial tube assay is performed using recombinant sFlt1 or NaHS (600 µM) to treat the cells. A representative experiment is shown for the following conditions: control, recombinant sFlt1, and recombinant sFlt1 in combination with NaHS. (B) Quantification of the endothelial tube assay. The tubes are quantified by counting branching points and are normalized to the control condition (100%). Statistical analysis is performed using the Kruskal–Wallis test. After administration of recombinant sFlt1 to the endothelial cells, significantly decreased angiogenesis is shown. NaHS is able to restore this decrease, but angiogenesis in the sFlt1 plus NaHS condition is still significantly lower compared with control. Experiments were repeated five times. Data are presented as the median (IQR). *P<0.05.

Figure 4.

Effect of NaHS on sFlt1 and VEGF expression in rats. (A) A standard curve for recombinant VEGF protein is generated in the absence (standard) or in the presence of 1 or 100 ng VEGF using a murine ELISA for measurement of sFlt1 levels as described in Concise Methods. (B) Free plasma sFlt1 (ng/ml) before and after 8 days of treatment with either vehicle or NaHS. NaHS-treated rats (50 µM/kg) show a significant reduction in free plasma sFlt1 after treatment, whereas vehicle-treated rats do not. The free plasma sFlt1 levels in the NaHS-treated rats are significantly lower compared with the vehicle group. (C) Free plasma VEGF (pg/ml) after 8 days of treatment with NaHS (left). After treatment with NaHS, VEGF levels are significantly higher compared with vehicle treatment. Renal VEGF mRNA levels are shown with respect to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT; right). Renal VEGF mRNA expression is significantly higher in NaHS-treated rats compared with the vehicle group. n=7 per group. Data are presented as the median (IQR). *P<0.05.

Figure 5.

Stimulation of human podocytes with NaHS causes VEGF release and upregulation of VEGF mRNA. (A) Human podocytes treated with PMA (positive control, 50 ng/ml), PBS (control), 100 µM NaHS, or 1000 µM NaHS. VEGF (pg/ml) release is increased after 120 minutes of stimulation in the 100 and 1000 µM NaHS-treated groups compared with the PBS-treated group. (B) After 24 hours of stimulation with 100 µM NaHS, an increase in VEGF-A mRNA is present compared with PBS (control). Data are presented as the median (IQR). *P<0.05. n=5 per group. Pos, positive control; PMA, phorbol 12-myristate 13-acetate.

Figure 6.

The role of VEGF signaling in NaHS-mediated vascular relaxation. (A) Treatment with the VEGFR2 neutralizing antibody DC101 (50 µg/ml) attenuates VEGF-mediated relaxation compared with vehicle-treated vessels (IgG antibody; 50 µg/ml). (B) Treatment with DC101 (50 µg/ml) attenuated NaHS-mediated relaxation compared with vehicle-treated vessels (IgG antibody; 50 µg/ml). Responses are expressed as the percentage of relaxation from preconstriction and each value represents the mean±SEM. n=3–4 mice per group. *P<0.05 vehicle-treated versus DC101-treated mice; **P<0.01 vehicle-treated versus DC101-treated mice.