Expression of chemokines/cytokines in renal cortex and urine of D₂−/− mice. A

<p>. Expression of Ltα, MCP-2, and NFkB1 mRNA was quantified by qRT-PCR; results were corrected for expression of GAPDH mRNA and expressed as fold change in comparison to their expression in D₂+/+ mice. *P<0.03 vs. D₂+/+ mice. <b>B</b>. Protein expression of MCP-1 (17 kDa) and TNFα protein (25 kDa) was semi-quantified by immunoblotting. Inset shows one set of immunoblots. Results were corrected for expression of actin and expressed as percentage of the expression in D₂+/+ mice, *P<0.02 vs. D₂+/+ mice, n = 5/group. <b>C.</b> Protein expression of IL-6 (25 kDa) and IL-10 (20 kDa) protein semi-quantified by immunoblotting. Results were corrected for expression of actin and expressed as percentage of the expression in D₂+/+, <b>*</b> P<0.05 vs. D₂+/+ mice, n = 5/group Urinary excretion of IL-6 and IL-10 was quantified by ELISA. *P<0.02 vs. D₂+/+ mice, n = 5/group.</p

Renal inflammation and injury in D₂−/− mice.

<p>Masson stained sections of D₂+/+ mouse kidney (<b>A</b> and <b>B</b>) and D₂−/− mouse kidney (<b>C</b> and <b>D</b>). H-E stained sections of D₂+/+ mouse kidney (<b>E</b>) and D₂−/− mouse kidney (<b>F</b>). G: glomerulus. PCT: proximal convoluted tubule. Proteinaceous casts are marked with arrows (<b>F</b>). Sections from 3 mouse kidneys per group were studied. <b>G</b> and <b>H</b>: Inflammatory cell infiltration. Kidney sections from D₂+/+ (<b>G</b>) and D₂−/− (<b>H</b>) mice were immunostained for the presence of macrophages and monocytes (arrows). The number of positive cells in 10 randomly selected fields was greater in D2−/− (68±3) than in D2+/+ (15±1, P<0.01) mice. Sections from 3 mouse kidneys per group were studied. <b>I</b>. Renal cortical expression of Col 1α1 mRNA determined by qRT-PCR. Results were corrected for expression of GAPDH mRNA and expressed as fold change in comparison to their expression in D₂+/+ mice. *P<0.05 vs D₂+/+; n = 5/group. <b>J</b>. Urinary microalbuminuria. Urine samples were collected for 24 h from mice in metabolic cages. Albumin was measured by ELISA. *P<0.04 vs. D₂+/+; n = 5/group. Magnification: A and C: 100X; B, D, G and H: 400X; E-F: 200X.</p

Effect of selective renal silencing of D₂R in the remaining kidney of uni-nephrectomized mice on blood pressure and expression on inflammatory factors in the kidney and liver.

<p>Renal cortical <i>Drd2</i> was silenced by the renal subcapsular infusion for seven days of <i>Drd2</i> siRNA, via an osmotic minipump in uni-nephrectomized adult male C57BL/6J mice (see Methods). A. Expression of D₂R protein (55 kDa band) in renal cortex and liver was semi-quantified by immunoblotting. Results were corrected for GAPDH and expressed as % of non-silencing siRNA treated kidneys. * P<0.05 vs non-silencing (NS) siRNA; n = 5/group. B. Systolic blood pressure measured under anesthesia in mice before and seven days after <i>Drd2</i> siRNA infusion. * P<0.05 vs, NS siRNA; n = 5/group. C. Renal cortical expression of TNFα, Ltα, NFkB1, MCP-1, MCP-2, IL-10, IL-11 osteopontin, and Col 1α1 mRNA was quantified by qRT-PCR, results corrected for expression of GAPDH mRNA, and expressed as fold change in comparison to their expression in mice treated with NS siRNA. *P<0.05 vs. NS; n = 5/group.</p

D₂R function in moue renal proximal tubule cells A.

<p>Effect of silencing of D₂R on the expression of pro-inflammatory cytokines/chemokines in mouse RPTCs. Cells were cultured to 60–70% confluence and transfected with non-silencing (NS siRNA) or <i>Drd2</i> siRNA. After 48 h the cells were washed and lysed. Protein expression of D₂R (55 kDa), TNFα (25 kDa), and MCP-1(17 kDa) was semi-quantified by immunoblotting. Inset shows one set of immunoblots. NFkB activation was analyzed via the transient expression of a NFkB-luciferase reporter system by reverse transfection Results are expressed as percentage of NS siRNA or fold activation compared to NS siRNA. *P<0.05 vs. NS (non-silencing) siRNA, n = 4/group. <b>B</b>. Effects of Ang II and D₂R stimulation on TNFα and MCP-1 in mouse RPTCs. Cells were serum starved for 2 h before treatment for 24 h in serum-free medium with vehicle (PBS) or 100 nM Ang II, in the presence or absence of 1 μM quinpirole (D₂R/D₃R agonist) or 1 μM quinpirole plus 1 μM L-741,262 (D₂R antagonist). Expression of TNFα (25 kDa) and MCP-1 (17 kDa) protein was semi-quantified by immunoblotting. Inset shows one set of immunoblots. Results were corrected for actin and expressed as % of vehicle. * P<0.05 vs. vehicle; n = 6/group.</p

Effect of selective renal silencing of D₂R in one kidney of mice without uni-nephrectomy on blood pressure and expression of inflammatory factors in the kidney and liver.

<p>Renal cortical D₂R was silenced by the renal subcapsular infusion in the left kidney for seven days of <i>Drd2</i> siRNA, via an osmotic minipump in adult male C57BL/6J mice (see Methods). A. Expression of D₂R protein (55 kDa band) in renal cortex and liver was semi-quantified by immunoblotting. Results were corrected for GAPDH and expressed as % of NS siRNA treated kidneys. * P<0.05 vs non-silencing NS siRNA; n = 4/group. B. Systolic blood pressure measured under anesthesia in mice before and seven days after <i>Drd2</i> siRNA infusion; n = 5/group. C. Renal cortical expression of TNFα, Ltα, NFkB1, MCP-1, MCP-2, IL-10, IL-11, osteopontin and collagen 1α1 mRNA was quantified by qRT-PCR, results corrected for expression of GAPDH mRNA, and expressed as fold change in comparison to their expression in mice treated with NS siRNA. *P<0.05 vs. NS; n = 5/group.</p

Effect of apocynin on renal cortical expression of TNFα, MCP-1, and IL-6, and urinary excretion of IL-6.

<p> Expression of TNFα (25 kDa) and MCP-1 (17 kDa) protein in renal cortex was semi-quantified by immunoblotting. Inset shows one set of immunoblots. Results were corrected for expression of GAPDH and expressed as percentage of D₂+/+ mice treated with vehicle, *P<0.05 vs. vehicle or apocynin treated D₂+/+; n = 5/group. Renal expression, semi-quantified by immunoblotting (25 kDa), and urinary excretion of IL-6 quantified by ELISA in 24 h urine samples. Results are expressed as percentage of D₂+/+ mice treated with vehicle. *P<0.05 vs. vehicle or apocynin treated D₂+/+; n = 5/group.</p

Gene expression profiling of cytokines, chemokines and receptors in the kidney of D₂+/+ and D₂−/− mice.

<p>Fold-change was calculated by the Δ Ct method. n = 3/group.</p

Expression of cytokines and chemokines in the heart left ventricle of D₂+/+ and D₂−/− mice determined by qRT-PCR.

<p>Fold-change was calculated by the ΔΔCt method. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038745#pone-0038745-t001" target="_blank">Table 1</a>. NS = not significant; n = 5/group.</p