Elevated tissue factor expression contributes to exacerbated diabetic nephropathy in mice lacking eNOS fed a high fat diet

Abstract

Summary Background Human eNOS (NOS3) polymorphisms that lower its expression are associated with advanced diabetic nephropathy (DN), and the lack of eNOS accelerates DN in diabetic mice. Diabetes is associated with fibrin deposition. Lack of nitric oxide and fatty acids stimulate the NF-kB pathway, which increases tissue factor (TF). Objectives To test the hypothesis that TF contributes to the severity of DN in the diabetic eNOS-/- mice fed a high fat (HF) diet. Methods We made eNOS-/- and wild type mice diabetic with streptozotocin. Half of them were placed on a high fat (HF) diet. Results Blood glucose levels were not affected by either the diet or eNOS genotype. Lack of eNOS in the diabetic mice increased urinary albumin excretion, glomerulosclerosis, interstitial fibrosis, and glomerular basement membrane thickness. HF by itself did not affect DN in the wild type mice, but significantly enhanced DN in eNOS-/- mice. More than half of diabetic eNOS-/- mice on HF died prematurely with signs of thrombotic complications. Diabetic kidneys contained fibrin and TF, and their levels were increased by the lack of eNOS and by HF in an additive fashion. The HF diet increased the kidney expression of inflammatory genes. The increase in TF preceded DN, and administration of an anti-mouse TF antibody to diabetic mice reduced the expression of inflammatory genes. Conclusion Together, these data indicate a causal link between TF and the exacerbation of DN in eNOS-/- mice. The condition is significantly worsened by enhanced inflammatory responses to a HF diet via TF.

A modest decrease in endothelial NOS in mice comparable to that associated with human NOS3 variants exacerbates diabetic nephropathy

“…In this context, it is notable that B6 mice made diabetic by streptozotocin develop variable degrees of tubulointerstitial fibrosis (11,22). In agreement with this result, we find that our eNOS +/+ Akita diabetic mice developed mild tubulointerstitial fibrosis at age 7 mo (Fig.…”

“…The 894T NOS3 gene produces about 30% of the amount of NO produced by the more common 894G allele when expressed in CHO cells in vitro (4). Although complete absence of eNOS accelerates DN in mice (8)(9)(10)(11), this result does not tell us whether production of eNOS at reduced levels comparable to those associated with the NOS3 polymorphisms is sufficient to worsen DN. To investigate this possibility, we first crossed C57BL/6J (B6) female eNOS +/− heterozygous mice with B6 males heterozygous for both the eNOS disruption (eNOS +/− ) and the diabetogenic Akita mutation (Ins2 C96Y/+ ).…”

“…Several previous studies using homozygous eNOS −/− diabetic mice, including ours, have demonstrated that absence of eNOS worsens DN (8)(9)(10)(11). However, there have been no reports of humans homozygous for absence of eNOS.…”

“…5 and 6). Furthermore, in a previous study of B6 mice made diabetic by lowdose streptozotocin, we showed that anti-TF neutralizing antibody decreases renal expression of inflammatory and fibrogenic genes (11). This increase in TF in the eNOS +/− Akita mice may be one of the ways whereby decreased expression of eNOS worsens DN.…”

“…NO inhibits coagulation (12), and diabetes leads to fibrin deposition in glomeruli (13). eNOS −/− mice made diabetic by low doses of streptozotocin have increased fibrin deposition and tissue factor (TF, F3) expression in their glomeruli (11). Accordingly, we examined fibrin deposition and TF expression in the kidneys of Akita diabetic mice.…”