GRP78 contributes to the beneficial effects of SGLT2 inhibitor on proximal tubular cells in DKD

Beneficial effects of SGLT2 inhibitors on kidney function are well-known; however, their molecular mechanisms are not fully understood. We focused on 78 kDa glucose-regulated protein (GRP78) and its interaction with SGLT2 and Integrin β1 beyond the chaperone property of GRP78. In STZ-induced diabetic mouse kidneys, GRP78, SGLT2, and Integrin β1 increased in the plasma membrane fraction, while they were suppressed by canagliflozin. The altered subcellular localization of GRP78/Integrin β1 in STZ mice promoted epithelial mesenchymal transition (EMT) and fibrosis, which were mitigated by canagliflozin. High glucose conditions reduced intracellular GRP78, increased its secretion, and caused EMT-like changes in cultured HK2 cells, which were again inhibited by canagliflozin. Urinary GRP78 increased in STZ mice, and in vitro experiments with recombinant GRP78 suggested that inflammation spread to surrounding tubular cells and canagliflozin reversed this effect. Under normal glucose culture, canagliflozin maintained SERCA activity, promoted endoplasmic reticulum (ER) robustness, reduced ER stress response impairment, and protected proximal tubular cells. In conclusion, canagliflozin restored subcellular localization of GRP78, SGLT2 and Integrin β1 and inhibited EMT and fibrosis in DKD. In non-diabetic CKD, canagliflozin promoted ER robustness by maintaining SERCA activity and preventing ER stress response failure, and contribute to tubular protection.</p

Orphan receptors expressed in renal cell lines.

<p>(A) Refer to the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085594#pone-0085594-g003" target="_blank">Figure 3A</a> for details. (B) Representative photomicrographs of immunofluorescent staining. Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) was expressed in both MES13 and mProx24 cell lines.</p

Comparative expression levels of NHRs in renal cell lines under high-glucose conditions.

<p>Mesangial (MES13), podocyte (MPC), proximal tubular epithelial (mProx24), and collecting duct (mIMCD3) cell lines. Each panel displays NHR expression under high-glucose conditions compared with low-glucose conditions. Values depict the means ± SEM of three independent samples. HG, high-glucose. LG, low-glucose.</p

Representative photomicrographs of double immunofluorescent staining in diabetic C57BL/6 mice.

<p>NOR1 expression was localized in mesangial, proximal tubular epithelial, and collecting duct cells, but not in podocytes in the kidneys of diabetic C57BL/6 mice.</p

Endocrine receptors expressed in renal cell lines.

<p>(A) The relative mRNA levels are depicted for mesangial (MES13), podocyte (MPC), proximal tubular epithelial (mProx24), and collecting duct (mIMCD3) cell lines. All values are expressed relative to 18S and arithmetically adjusted to depict the lowest-expressing sample as a unit of 1. Values represent the means ± SEM of three independent samples of each cell lines, and the results are representative of two independent studies. (B) Representative photomicrographs of immunofluorescent staining. Vitamin D receptor (VDR) was predominantly expressed in mProx24 cells, and to a lesser extent in MES13 cells.</p

Adopted orphan receptors expressed in renal cell lines.

<p>(A) Refer to the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085594#pone-0085594-g003" target="_blank">Figure 3A</a> for details. (B) Representative photomicrographs of immunofluorescent staining. Peroxisome proliferator-activated receptor-δ (PPARδ) was expressed in both MES13 and mProx24 cell lines.</p

Comparative expression levels of 49 nuclear hormone receptors (NHRs) in mouse kidney tissue and renal cell lines.

<p>The relative mRNA levels are depicted for mouse kidney (C57BL/6 (A) and db/m (B)) and mesangial (MES13) (C), podocyte (MPC) (D), proximal tubular epithelial (mProx24) (E) and collecting duct (mIMCD3) (F) cell lines. All values are expressed relative to 18S and arithmetically adjusted to depict the highest-expressed NHR for each tissue/cell line as a unit of 100. Values represent the means ± SEM of three independent samples of each tissue or cell line. Setting arbitrary cutoffs at Ct<31 (present) or Ct>31 (absent), as shown by broken lines in the C57BL/6 mouse kidney panel, reveals that 25 NHRs were expressed and six NHRs were not detected in C57BL/6 mouse kidney.</p

Localization of endogenous VASH2 in glomeruli.

<p>(A) The mRNA level of VASH2 in the kidney cortex from wild-type mice was assessed by real-time PCR. VASH2 expression was increased in diabetic condition compared with non-diabetic mice kidney. n = 6 for each group. *<i>P</i><0.05 versus wild-type mice. (B) VASH2 expression is detected with immunofluorescence for β-galactosidase. No immunoreactivity in glomeruli is seen in wild-type mice (left panel), whereas glomeruli from non-diabetic and diabetic VAHS2 knockout (VASH2^{<i>LacZ/LacZ</i>}) mice (middle and right panel, respectively) show a β-galactosidase-positive area (original magnification, ×400). (C) Double immunofluorescence for β-galactosidase and markers for glomerular component cells in VASH2 knockout mice show that the localization of β-galactosidase-positive area are consistent with platelet-derived growth factor receptor-β (PDGFRβ)-positive mesangial cells, but not CD31-positive endothelial cells and zonula occludens-1 (ZO-1)-positive podocytes.</p

Alterations of glomerular endothelial area and VEGF-A expression in diabetic VASH2 knockout mice.

<p>(A) The distribution of CD31, a marker for endothelial cells, was determined by immunofluorescence in non-diabetic wild-type, non-diabetic VASH2 knockout, diabetic wild-type, and diabetic VASH2 knockout mice (original magnification, ×400). (B) In quantitative analysis, CD31-positive glomerular endothelial area was expanded in diabetic wild-type mice, but it was significantly prevented in diabetic VASH2 knockout mice. No difference was found in endothelial area between non-diabetic wild-type and non-diabetic VASH2 knockout mice. (C, D) Immunoblot for vascular endothelial growth factor-A (VEGF-A; C) and VEGF receptor-2 (VEGFR2; D). Each lane was loaded with 40 μg of protein obtained from the renal cortex. Each band was scanned and subjected to a densitometric analysis. Increased VEGF-A level induced by diabetes showed no difference between wild-type and VASH2 knockout mice, whereas increased VEGFR2 expression seen in diabetic wild-type mice was significantly suppressed in diabetic VASH2 knockout mice. n = 6 for each group. *<i>P</i><0.05 versus non-diabetic WT or VASH2 knockout mice, ^#<i>P</i><0.05 versus diabetic WT mice. Each column shows the mean ± SE.</p

Flowchart of the study design.

<p>Flowchart of the study design.</p