Deficient dopamine D2 receptor function causes renal inflammation independently of high blood pressure

Abstract

Renal dopamine receptors participate in the regulation of blood pressure. Genetic factors, including polymorphisms of the dopamine D(2) receptor gene (DRD2) are associated with essential hypertension, but the mechanisms of their contribution are incompletely understood. Mice lacking Drd2 (D(2)-/-) have elevated blood pressure, increased renal expression of inflammatory factors, and renal injury. We tested the hypothesis that decreased dopamine D(2) receptor (D(2)R) function increases vulnerability to renal inflammation independently of blood pressure, is an immediate cause of renal injury, and contributes to the subsequent development of hypertension. In D(2)-/- mice, treatment with apocynin normalized blood pressure and decreased oxidative stress, but did not affect the expression of inflammatory factors. In mouse RPTCs Drd2 silencing increased the expression of TNFα and MCP-1, while treatment with a D(2)R agonist abolished the angiotensin II-induced increase in TNF-α and MCP-1. In uni-nephrectomized wild-type mice, selective Drd2 silencing by subcapsular infusion of Drd2 siRNA into the remaining kidney produced the same increase in renal cytokines/chemokines that occurs after Drd2 deletion, increased the expression of markers of renal injury, and increased blood pressure. Moreover, in mice with two intact kidneys, short-term Drd2 silencing in one kidney, leaving the other kidney undisturbed, induced inflammatory factors and markers of renal injury in the treated kidney without increasing blood pressure. Our results demonstrate that the impact of decreased D(2)R function on renal inflammation is a primary effect, not necessarily associated with enhanced oxidant activity, or blood pressure; renal damage is the cause, not the result, of hypertension. Deficient renal D(2)R function may be of clinical relevance since common polymorphisms of the human DRD2 gene result in decreased D(2)R expression and function.

Figure 1. Renal inflammation and injury in D₂−/− mice.

Masson stained sections of D₂+/+ mouse kidney (A and B) and D₂−/− mouse kidney (C and D). H-E stained sections of D₂+/+ mouse kidney (E) and D₂−/− mouse kidney (F). G: glomerulus. PCT: proximal convoluted tubule. Proteinaceous casts are marked with arrows (F). Sections from 3 mouse kidneys per group were studied. G and H: Inflammatory cell infiltration. Kidney sections from D₂+/+ (G) and D₂−/− (H) 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. I. 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. J. 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.

Figure 2. Expression of chemokines/cytokines in renal cortex and urine of D₂−/− mice. A

. 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. 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. C. 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+/+, * 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.

Figure 3. Effect of apocynin on renal cortical expression of TNFα, MCP-1, and IL-6, and urinary excretion of IL-6.

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.

Figure 4. D₂R function in moue renal proximal tubule cells A.

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 Drd2 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. 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.

Figure 5. 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.

Renal cortical Drd2 was silenced by the renal subcapsular infusion for seven days of Drd2 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 Drd2 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.

Figure 6. 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.

Renal cortical D₂R was silenced by the renal subcapsular infusion in the left kidney for seven days of Drd2 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 Drd2 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.