Angiotensin II Stimulation of DPP4 Activity Regulates Megalin in the Proximal Tubules

Abstract

Proteinuria is a marker of incipient kidney injury in many disorders, including obesity. Previously, we demonstrated that megalin, a receptor endocytotic protein in the proximal tubule, is downregulated in obese mice, which was prevented by inhibition of dipeptidyl protease 4 (DPP4). Obesity is thought to be associated with upregulation of intra-renal angiotensin II (Ang II) signaling via the Ang II Type 1 receptor (AT₁R) and Ang II suppresses megalin expression in proximal tubule cells in vitro. Therefore, we tested the hypothesis that Ang II will suppress megalin protein via activation of DPP4. We used Ang II (200 ng/kg/min) infusion in mice and Ang II (10(-8) M) treatment of T35OK-AT₁R proximal tubule cells to test our hypothesis. Ang II-infused mouse kidneys displayed increases in DPP4 activity and decreases in megalin. In proximal tubule cells, Ang II stimulated DPP4 activity concurrent with suppression of megalin. MK0626, a DPP4 inhibitor, partially restored megalin expression similar to U0126, a mitogen activated protein kinase (MAPK)/extracellular regulated kinase (ERK) kinase kinase (MEK) 1/2 inhibitor and AG1478, an epidermal growth factor receptor (EGFR) inhibitor. Similarly, Ang II-induced ERK phosphorylation was suppressed with MK0626 and Ang II-induced DPP4 activity was suppressed by U0126. Therefore, our study reveals a cross talk between AT₁R signaling and DPP4 activation in the regulation of megalin and underscores the significance of targeting DPP4 in the prevention of obesity related kidney injury progression.

Keywords: AT1R; DPP4; EGFR; ERK; megalin; obesity related kidney disease; proteinuria.

Figure 1

Ang II infusion activates the renin-angiotensin system (RAS) and dipeptidyl peptidase 4 (DPP4) and suppresses megalin protein levels in mice: (A) Quantification of differential mRNA expression of RAS in the kidney (Ai–Aiv) and depiction of actual bands that were used for quantification (Av); (B) DPP4 activity in the kidney expressed as relative light units (RLUs); and (C) megalin protein expression by immunoblot of kidney lysates. n = 3–4; * p < 0.05; AGT: Angiotensinogen; AT_1AR: Angiotensin type 1A receptor; AT_1BR: Angiotensin type 1B receptor; AT2R: Angiotensin type 2 receptor; 18s: 18s ribosomal RNA; Con: Saline-infused mice; Ang II: Ang II-infused mice (200 ng/kg/min).

Figure 2

DPP4 enzymatic activity and protein levels in T35OK-AT₁R proximal tubule cells after acute stimulation with Ang II: (A) DPP4 activity after stimulation (30 min) with Ang II (10⁻⁸ M) and blockade (60 min prior to Ang II) with olmesartan (10⁻⁶ M) or MK0626 (5 × 10⁻⁶ M); and (B) DPP4 protein levels after stimulation (30 min) with Ang II and blockade with olmesartan and MK0626. n = 3–5; * p < 0.05; Olme: Olmesartan; MK0626: Rodent DPP4 inhibitor (Merck & Co., Inc.).

Figure 3

Ang II regulates megalin protein expression via DPP4 activation. (A) Megalin protein expression by immunoblot in T35OK-AT₁R proximal tubule cells. Proximal tubule cells were stimulated with Ang II (10⁻⁸ M) for 24 h and pre-treated 1 h with olmesartan (10⁻⁶ M), AG1478 (10⁻⁵ M), U0126 (10⁻⁵ M) and MK0626 (5 × 10⁻⁵ M); (B) ERK1/2 activation was assessed by pThr²⁰²Tyr²⁰⁴-ERK1/2 increase. Proximal tubule cells were stimulated with Ang II (10⁻⁸ M) for 30 min and the ratio of pThr²⁰²Tyr²⁰⁴-ERK1/2 to total ERK1/2 ratio was calculated. Proximal tubule cells were pre-treated for 1 h with olmesartan (10⁻⁶ M), AG1478 (10⁻⁵ M), U0126 (10⁻⁵ M) and MK0626 (5 × 10⁻⁵ M); (C) DPP4 activity in conditions of chronic Ang II stimulation. T35OK-AT₁R proximal tubule cells were stimulated for 24 h with Ang II (10⁻⁸ M) and olmesartan (10⁻⁶ M), AG1478 (10⁻⁵ M), U0126 (10⁻⁵ M) and MK0626 (5 × 10⁻⁵ M) were tested for blockade of Ang II-mediated increase in DPP4 activity. n = 3–6; * p < 0.05 when compared to control; † p < 0.05 when compared to Ang II.