Angiotensin II contributes to diabetic renal dysfunction in rodents and humans via Notch1/Snail pathway

Abstract

In nondiabetic rat models of renal disease, angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. Herein, we wanted to explore the role of Ang II in diabetic nephropathy by a translational approach spanning from in vitro to in vivo rat and human studies, and to dissect the intracellular pathways involved. In isolated perfused rat kidneys and in cultured human podocytes, Ang II down-regulated nephrin expression via Notch1 activation and nuclear translocation of Snail. Hairy enhancer of split-1 was a Notch1-downstream gene effector that activated Snail in cultured podocytes. In vitro changes of the Snail/nephrin axis were similar to those in renal biopsy specimens of Zucker diabetic fatty rats and patients with advanced diabetic nephropathy, and were normalized by pharmacological inhibition of the renin-angiotensin system. Collectively, the present studies provide evidence that Ang II plays a relevant role in perpetuating glomerular injury in experimental and human diabetic nephropathy via persistent activation of Notch1 and Snail signaling in podocytes, eventually resulting in down-regulation of nephrin expression, the integrity of which is crucial for the glomerular filtration barrier.

Figure 1

Infusion of Ang II in rat isolated perfused kidneys affects glomerular barrier function and integrity. A: Proteinuria levels at baseline (white bars) and after a 10-minute infusion with 16 ng/minute Ang II or vehicle (grey bars). ^∗P < 0.01 versus baseline. B: Representative images of nephrin immunofluorescence (red) in glomeruli of rats after Ang II (left panel) or vehicle (right panel) infusion. C: Transmission electron micrographs of the capillary wall ultrastructure of rats infused with Ang II (left panel) or vehicle (right panel). Arrowhead, podocyte effacement; arrows, podocyte slits with or without the linear diaphragm between neighboring podocytes.

Figure 2

Ang II reduces nephrin expression through the involvement of Notch1 in cultured human podocytes. A: Representative images of nephrin staining in human podocytes exposed for 3 hours to 100 nmol/L Ang II. B: Nephrin mRNA expression in human podocytes exposed to Ang II alone or in combination with 1 μmol/L γ-secretase inhibitor, GSI X. ^∗P < 0.01 versus control podocytes; ^†P < 0.01 versus Ang II alone. C, top panel: Representative images of ICN1 staining (red) and cell nuclei (DAPI, blue). Bottom panel: Quantification of the percentage of nuclei positive for ICN1 at different time intervals. Transforming growth factor (TGF)-β (5 ng/mL for 120 minutes) was used as a positive control. ^∗∗P < 0.001 versus control podocytes.

Figure 3

Role of the Notch1 pathway on Snail activation in Ang II–treated podocytes. A: Snail protein expression in podocytes exposed to Ang II in the presence or absence of 1 μmol/L γ-secretase inhibitor, GSI X, at different time intervals. Representative images of Snail staining (red) and nuclei (DAPI, blue) and quantification of Snail-positive nuclei (%). ^∗P < 0.001 versus control; ^†P < 0.001 versus Ang II. B: Snail mRNA expression in podocytes exposed to Ang II for 1 hour in the presence or absence of GSI X and in podocytes knocked down for HES1 with siHES1 or control nontarget siRNA (siNULL). ^∗P < 0.001 versus control; ^†P < 0.001 versus Ang II or Ang II + siNULL. C: Representative images of Snail staining (red with DAPI in blue) in siNULL- or siHES1-transfected podocytes exposed to Ang II at different time intervals and relative quantification of Snail-positive nuclei (%). ^∗P < 0.001 versus control; ^†P < 0.001 versus Ang II + siNULL.

Figure 4

Ramipril reduced proteinuria, improved renal histological features, and restored nephrin and Snail expression in ZDF rats. A: Urinary protein excretion rate in ZDF rats receiving vehicle or ACE inhibitor and controls measured at 4 (pretreatment) and 8 months. ^∗P < 0.01 versus controls; ^†P < 0.01 versus untreated. B: Representative images of PAS reagent–stained kidney sections of control and diabetic animals that received ramipril or did not receive ramipril and were sacrificed at 8 months. C: Photomicrographs of nephrin (red) and Snail (green) immunofluorescence on frozen kidney sections. Nuclei were stained with DAPI.

Figure 5

Ramipril did not reduce the area of sclerosis in glomeruli of patients with type 2 diabetes. Representative images of PAS reagent–stained kidney biopsy specimens from control and diabetic patients untreated or treated with ramipril. Scale bars are indicated.

Figure 6

Ramipril restores nephrin and Snail expression in patients with type 2 diabetes to control levels. A: Representative images of immunofluorescence of nephrin (red) in glomeruli of diabetic patients, untreated or treated with ACE inhibitor. Control subjects were included for comparisons. B: Snail expression evaluated by immunofluorescence (green). C: Double immunofluorescence of nephrin and Snail. Insets, colocalization of nephrin with Snail (yellow, merged signal; twofold enlargement of boxed area).

Figure 7

Ramipril restores Notch1 expression in diabetic patients. Representative images of Notch1 glomerular expression assessed by ICN1 immunoperoxidase. Insets, twofold enlargement of boxed area. Arrows, mild and cytoplasmic signal in controls that increases and becomes nuclear in diabetic glomeruli. The staining is negative in the absence of primary antibody (minus). Double immunofluorescence of ICN1 (green) and WT1 (red) reveals Notch1 activation in podocytes of untreated diabetic patients. Arrows, colocalization of the two signals (yellow). The histogram shows the percentage of ICN1-positive nuclei. ^∗P < 0.05 versus control group; ^†P < 0.01 versus diabetic patients.

Figure 8

Ang II activates the Notch1/Snail signaling, which is responsible for the pathogenesis of proteinuria in diabetic nephropathy. Schematic drawing summarizes the molecular pathway by which Ang II, after binding with angiotensin type 1 receptor (AT₁R), induces increased activation of Notch1. The active Notch1 (ICN1) via the downstream target gene, HES1, increases the expression of Snail and its translocation into the nucleus, leading to nephrin down-regulation. This series of events is responsible for the molecular podocyte lesions and loss of the glomerular permeability in diabetes.