Adrenomedullin-RAMP2 System Suppresses ER Stress-Induced Tubule Cell Death and Is Involved in Kidney Protection

<div><p>Various bioactive peptides have been implicated in the homeostasis of organs and tissues. Adrenomedullin (AM) is a peptide with various bioactivities. AM-receptor, calcitonin-receptor-like receptor (CLR) associates with one of the subtypes of the accessory proteins, RAMPs. Among the RAMP subisoforms, only RAMP2 knockout mice (−/−) reproduce the phenotype of embryonic lethality of AM−/−, illustrating the importance of the AM-RAMP2-signaling system. Although AM and RAMP2 are abundantly expressed in kidney, their function there remains largely unknown. We used genetically modified mice to assess the pathophysiological functions of the AM-RAMP2 system. RAMP2+/− mice and their wild-type littermates were used in a streptozotocin (STZ)-induced renal injury model. The effect of STZ on glomeruli did not differ between the 2 types of mice. On the other hand, damage to the proximal urinary tubules was greater in RAMP2+/−. Tubular injury in RAMP2+/− was resistant to correction of blood glucose by insulin administration. We examined the effect of STZ on human renal proximal tubule epithelial cells (RPTECs), which express glucose transporter 2 (GLUT2), the glucose transporter that specifically takes up STZ. STZ activated the endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK). AM suppressed PERK activation, its downstream signaling, and CCAAT/enhancer-binding homologous protein (CHOP)-induced cell death. We confirmed that the tubular damage was caused by ER stress-induced cell death using tunicamycin (TUN), which directly evokes ER stress. In RAMP2+/− kidneys, TUN caused severe injury with enhanced ER stress. In wild-type mice, TUN-induced tubular damage was reversed by AM administration. On the other hand, in RAMP2+/−, the rescue effect of exogenous AM was lost. These results indicate that the AM-RAMP2 system suppresses ER stress-induced tubule cell death, thereby exerting a protective effect on kidney. The AM-RAMP2 system thus has the potential to serve as a therapeutic target in kidney disease.</p></div

Cisplatin induces enhanced renal injury in RAMP2+/−.

<p>(A) Serum BUN and creatinine (Cr) in wild-type and RAMP2+/−3 days after administration of vehicle (Control) or cisplatin (CDDP) (20 mg/kg). (B) H&E staining of the renal cortex in CDDP-treated wild-type and RAMP2+/− kidneys. Scale bars = 100 µm. Sections from RAMP2+/− kidneys showed severe tubular injury. (C) Quantitative real-time PCR analysis of the indicated gene expression. Data from the wild-type control group were assigned a value of 1. n = 5 in each group. Bars are means ± SEM. *p<0.05.</p

Western blot analysis of ER stress-related factors in STZ-treated RPTECs.

<p>(A) Western blot analysis of ER stress sensor molecules in STZ-treated RPTECs. (B) Optical densities of Western blots were quantified, and the relative expression of BiP and ATF6 and the p-PERK/PERK and p-IRE1/IRE1 ratios (PERK and IRE1 activation level) are shown. (C) Western blot analysis of the mediators downstream of PERK. (D) Quantification of the results in (C). The experiments were repeated 3 times. Data from the vehicle group were assigned a value of 1. Bars are means ± SEM. *p<0.05, ***p<0.001.</p

Renal tubular changes induced by the STZ treatment.

<p>(A) Cross-sections of kidneys collected from wild-type and RAMP2+/− mice 4 weeks after STZ administration (H & E staining). (B) Higher magnification of the renal cortex. STZ-administered RAMP2+/− and AM+/− showed substantial degeneration of tubule cells, which was not detected in RAMP2+/− without STZ administration. (C) Electron micrograph of a section of proximal tubule shows destruction of the brush border (protrusion of a tubule cellular membrane into the lumen) in a RAMP2+/− kidney. Scale bars = 100 µm (B), 10 µm (C).</p

STZ enhances tubule cell autophagy in RAMP2+/− kidneys.

<p>(A) Fluorescent immunostaining of LC3 (green) and DAPI (blue) in proximal renal tubules 4 weeks after STZ administration to wild-type and RAMP2+/− mice. (B) Comparison of autophagosome numbers between wild-type and RAMP2+/− kidneys. Numbers of LC3-positve particles per microscope field were counted in LC3 immunostained sections. A total of 10 fields were analyzed. (C) Electron micrograph showing autophagosomes in a renal tubule cell. Bars are means ± SEM. ***p<0.001. Scale bars = 10 µm (A), 1 µm (C).</p

AM suppresses STZ-induced apoptosis among human renal tubule cells (RPTECs).

<p>(A) RT-PCR analysis showing the expression of GLUT2 in RPTECs. The arrow indicates the GLUT2 PCR product. (B) TUNEL assays of cultured RPTECs treated with vehicle, AM, STZ, or STZ+AM. Arrows show the TUNEL-positive cells. Scale bars = 100 µm. (C) Numbers of TUNEL-positive cells per 1,000 cultured cells. The experiments were repeated 3 times. Bars are means ± SEM. *p<0.05, **p<0.01.</p

Primers and probes used for quantitative real-time RT-PCR.

<p>Primers and probes used for quantitative real-time RT-PCR.</p

Blood glucose and glomerular changes in the STZ-induced diabetic nephropathy model.

<p>(A) Bar graphs showing relative gene expression determined by quantitative real-time PCR. Gene expression of RAMP2 was downregulated in RAMP2+/− kidney. (B) Effect of STZ treatment on blood glucose levels. In both the control and STZ groups, blood glucose levels did not differ between wild-type and RAMP2+/− mice 2 weeks after STZ administration. Data were obtained at 10∶00 a.m. without fasting. (C-E) Comparison of glomerular lesions in wild-type and RAMP2+/− kidneys following STZ treatment. (C) PAS staining of glomeruli 12 weeks after STZ administration. Scale bars = 10 µm. (D) Glomerular area. (E) Mesangial area/glomerular area ratios. n = 5 in each group. Bars are means ± SEM. ***p<0.001. n.s. = not significant.</p

Tunicamycin (TUN) induces tubular injury and upregulation of ER stress-related factors in RAMP2+/− kidneys.

<p>(A) Serum BUN and creatinine (Cr) in wild-type and RAMP2+/−4 days after administration of vehicle (Control) or TUN (0.5 mg/kg). (B) H&E staining of the renal cortex in TUN-treated wild-type and RAMP2+/− kidneys. Scale bars in upper panels = 100 µm, lower panels = 50 µm. Sections from RAMP2+/− kidneys showed severe tubular injury with cell vacuolation. (C) Quantitative real-time PCR analysis of BiP and CHOP gene expression. TUN-treated RAMP2+/− kidneys showed upregulation of ER stress-related factors. Data from the wild-type control group were assigned a value of 1. n = 5 in each group. Bars are means ± SEM. *p<0.05.</p

STZ-induced tubule cell death was enhanced in RAMP2+/− kidneys.

<p>(A) TUNEL assays of the renal cortex 4 weeks after STZ administration showed tubule cell death to be increased in RAMP2+/− kidneys. (B) Quantitative real-time PCR analysis of samples from the renal cortex. Shown are the relative gene expression levels of Bax and Bcl-2 (upper panels). Data from the wild-type control group were assigned a value of 1. Note the downregulation of Bcl-2 in RAMP2+/− kidneys indicated by the higher Bax/Bcl-2 ratio (lower panel). n = 5 in each group. Bars are means ± SEM. *p<0.05. (C) H&E staining of the renal cortex from RAMP2+/− kidneys 2 weeks after STZ administration, with or without insulin (Ins) treatment. Although insulin-treated RAMP2+/− mice had normal blood glucose levels, they showed the same level of tubular injury as mice not receiving insulin. Arrows indicate vacuolated proximal tubules. Scale bars = 50 µm (A), (C).</p