David L. Mattson

Influence of Dietary Sodium Intake on Renal Medullary Nitric Oxide Synthase

We previously reported that chronic systemic treatment of rats with a nitric oxide synthase inhibitor leads to a selective decrease in renal medullary blood flow, retention of sodium, and the development of hypertension. In the present studies, we used protein blotting techniques to determine the whole tissue distribution and relative quantitation of the different nitric oxide synthase isoforms in the renal cortex and medulla of Sprague-Dawley rats maintained on a low (0.4% NaCl) or high (4.0% NaCl) dietary salt intake. Neural, endothelial, and inducible nitric oxide synthase were readily detectable in homogenized renal inner and outer medullas. Only endothelial nitric oxide synthase was detectable in the renal cortex. Densitometric comparison of Western blots from equal amounts of total inner medullary tissue protein indicated that endothelial, inducible, and neural nitric oxide synthase were increased by 145%, 49%, and 119%, respectively, in rats maintained on a high NaCl diet compared with rats on a low NaCl diet. No significant differences in nitric oxide synthase levels were detected in the outer medulla, renal cortex, or aorta of rats maintained on low and high NaCl diets. In separate studies, continuous intravenous infusion of N(G)-nitro-L-arginine methyl ester (8.6 mg/kg per day) for 11 days in chronically instrumented rats increased mean arterial pressure 32 +/- 3 mm Hg in rats on a high NaCl diet (n=5) but only increased pressure 17 +/- 3 mm Hg in rats on a low NaCl diet (n=6). These data indicate that increased levels of renal medullary nitric oxide synthase may be important in the chronic adaptation to increased sodium intake.

The Renal Medulla and Hypertension

We review evidence supporting the conclusion that renal dysfunction underlies the development of all forms of hypertension in humans and experimental animals. Indexes of global renal function are generally normal in the early stages of most genetic forms of hypertension, but renal function is clearly impaired in long-established hypertension. Studies in our laboratory over the past decade summarized below have established that the renal medulla plays an important role in sodium and water homeostasis and in the long-term control of arterial pressure. Development of implanted optical fibers for measurement of cortical and medullary blood flows with laser-Doppler flowmetry and techniques for delivery of vasoactive compounds into the medullary interstitial space enabled us to examine determinants of medullary flow (nitric oxide, atrial natriuretic peptides, kinins, eicosanoids, vasopressin, renal sympathetic nerves, etc). We have shown in spontaneously hypertensive rats that the initial changes of renal function begin as a reduction of medullary blood flow in the absence of changes of cortical flow. Long-term medullary interstitial infusion of captopril, which preferentially increased medullary blood flow, resulted in a lowering of arterial pressure. In normal Sprague-Dawley rats, selective reduction of medullary flow with medullary interstitial or intravenous infusion of small amounts of NG-nitro-L-arginine methyl ester resulted in hypertension. These and other studies we review show that although blood flow to the inner renal medulla comprises less than 1% of the total renal blood flow, changes in flow to this region can have a major effect on sodium and water homeostasis and on the long-term control of arterial blood pressure.

Role of nitric oxide in renal papillary blood flow and sodium excretion.

Renal medullary interstitial infusion of NG-nitro-L-arginine (120 μg/hr, n = 7) decreased papillary blood flow to 71±5% of control without altering outer cortical flow. Before NG-nitro-L-arginine infusion, interstitial acetylcholine administration (200 μg/hr) increased cortical and papillary blood flow to 134±6% and 113±2% of control, respectively. After NG-nitro-L-arginine administration, the vasodilator response to acetylcholine was abolished. In clearance experiments, renal medullary infusion of NG-nitro- L-arginine (120 μg/hr, n = 7) significantly decreased total renal blood flow by 10%, renal interstitial fluid pressure by 23%, sodium excretion by 34%, and urine flow by 39% without altering glomerular filtration rate, fractional sodium and water excretion, blood pressure, or urine osmolality. These data indicate that selective inhibition of nitric oxide in the renal medullary vasculature reduces papillary blood flow, which is associated with decreased sodium and water excretion. We conclude that nitric oxide exerts a tonic influence on the renal medullary circulation.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney.

This study was designed to quantify nitric oxide synthase (NOS) activity in microdissected glomeruli (Glm), pars convoluta, pars recta, cortical collecting duct, cortical thick ascending limb, outer medullary collecting duct, medullary thick ascending limb and thin limb, inner medullary collecting duct (IMCD) and thin limb, and vasa recta (VR). Total protein from microdissected segments was incubated withl-[3H]arginine and appropriate cofactors, and thel-arginine and convertedl-citrulline were separated by reverse-phase HPLC and radiochemically quantitated. NOS activity was found to be greatest in IMCD (11.5 ± 1.0 fmol citrulline ⋅ mm-1 ⋅ h-1) and moderate in Glm (1.9 ± 0.3 fmol ⋅ glomerulus-1 ⋅ h-1) and VR (3.2 ± 0.8 fmol ⋅ mm-1 ⋅ h-1). All other renal structures studied exhibited significantly less NOS activity. The mRNA for NOS isoforms in the NOS activity-positive segments was then identified by RT-PCR. The IMCD contained mRNA for neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS), but Glm and VR only expressed the mRNA for nNOS and eNOS. These experiments demonstrate that the greatest enzymatic activity for NO production in the kidney is in the IMCD, three- to sixfold less activity is present in the Glm and VR, and minimal NOS activity is found in other segments studied.

T lymphocytes mediate hypertension and kidney damage in Dahl salt-sensitive rats

This study examined mechanisms by which immune cells participate in the development of hypertension and renal disease in Dahl salt-sensitive (SS) rats. Increasing dietary salt from 0.4% to 4.0% NaCl significantly increased renal infiltration of T lymphocytes from 8.8 +/- 1.2 x 10(5) to 14.4 +/- 2.0 x 10(5) cells/2 kidneys, increased arterial blood pressure from 131 +/- 2 to 165 +/- 6 mmHg, increased albumin excretion rate from 17 +/- 3 to 129 +/- 20 mg/day, and resulted in renal glomerular and tubular damage. Furthermore, renal tissue ANG II was not suppressed in the kidneys of SS rats fed 4.0% NaCl. Administration of the immunosuppressive agent mycophenolate mofetil (MMF; 20 mg.kg(-1).day(-1)) prevented the infiltration of T lymphocytes and attenuated Dahl SS hypertension and renal disease. In contrast to vehicle-treated rats, Dahl SS rats administered MMF demonstrated a suppression of renal tissue ANG II from 163 +/- 26 to 88 +/- 9 pg/g of tissue when fed high salt. Finally, it was demonstrated that the T lymphocytes isolated from the kidney possess renin and angiotensin-converting enzyme activity. These data indicate that infiltrating T cells are capable of participating in the production of ANG II and are associated with increased intrarenal ANG II, hypertension, and renal disease. The suppression of T-cell infiltration decreased intrarenal ANG II and prevented Dahl SS hypertension and kidney damage. As such, infiltrating cells are capable of participating in the established phase of Dahl SS hypertension.

Immune Suppression Attenuates Hypertension and Renal Disease in the Dahl Salt-Sensitive Rat

Experiments were performed to determine the importance of activation or infiltration of immune cells in the kidney during the development of hypertension and renal disease in Dahl salt-sensitive rats (SS/Mcw) fed a 4.0% NaCl diet. Compared with vehicle-treated rats, chronic administration of mycophenolate mofetil ([MMF] 30 mg/kg per day, IP), an immunosuppressive agent that has cytostatic effects on T and B cells, decreased cell-specific markers of T and B cells by 50% to 60% in the kidneys of SS/Mcw rats (n=5 per group). Further studies were performed on Dahl SS/Mcw rats, which were instrumented with chronic indwelling catheters and studied after 3 weeks on the 4.0% NaCl diet. Rats were administered MMF or 5% dextrose vehicle daily during the 3-week period of high NaCl intake. Mean arterial blood pressure in the rats administered MMF (122±2 mm Hg; n=11) was significantly decreased compared with vehicle-treated rats (139±4 mm Hg; n=9). Furthermore, the rate of protein (112±13 mg per day) and albumin excretion (15±3 mg per day) in the MMF-treated rats was significantly lower than the protein and albumin excretion rate in vehicle-treated rats (167±25 and 31±7 mg per day, respectively). Creatinine clearance and body weight were not different between the groups, averaging 0.52±0.08 mL/min per gram kidney weight and 322±10 g, respectively, in the MMF-treated group. These experiments indicate that the activation of the immune system or renal infiltration of immune cells plays an important role in the development of hypertension and renal disease in Dahl SS/Mcw rats consuming an elevated NaCl diet.

Neural Nitric Oxide Synthase in the Renal Medulla and Blood Pressure Regulation

We studied the effect of selective inhibition of the neural isoform of nitric oxide synthase in the rat renal medulla in conscious Sprague-Dawley rats. Continuous renal medullar interstitial infusion of an antisense oligonucleotide complementary to the initiation region of the mRNA for neural nitric oxide synthase increased blood pressure 14 +/- 1 mm Hg in rats maintained on a high sodium intake. Medullary interstitial infusion of saline vehicle or a scrambled oligonucleotide probe failed to alter blood pressure in separate groups of high salt control rats. Renal medullary interstitial infusion of the antisense oligonucleotide significantly decreased the level of neural nitric oxide synthase in the renal medulla by 53 +/- 8% and decreased total renal medullary nitric oxide synthase activity by 28 +/- 8%. No alterations were detected in the levels of inducible nitric oxide synthase or beta-actin in the antisense oligonucleotide-infused rats. To confirm the antisense oligonucleotide data, we administered a mechanistically different inhibitor of neural nitric oxide synthase, 7-nitroindazole, to an additional group of rats maintained on a high salt diet. Direct renal medullary interstitial infusion of this selective enzyme inhibitor significantly increased mean arterial pressure (15 +/- 6 mm Hg) and decreased total renal medullary nitric oxide synthase activity by 37 +/- 12% in rats on a high sodium diet. The present experiments demonstrate a role for the neural isoform of nitric oxide synthase in the long-term control of blood pressure in the presence of a high salt diet.

Long-term measurement of arterial blood pressure in conscious mice

This study describes a technique for the direct daily measurement of arterial blood pressure, sampling of arterial blood, and continuous intravenous infusion in free-moving, conscious, Swiss-Webster mice. Catheters were chronically implanted in the femoral artery and vein, tunneled subcutaneously, exteriorized at the back of the neck in a lightweight tethering spring, and attached to a swivel device at the top of the cage. Time-control experiments (n = 8) demonstrated stable values of mean arterial pressure (MAP, 116 +/- 1 mmHg) and heart rate (HR, 627 +/- 21 beats/min) for up to 35 days after catheter implantation. It was further observed that restraining mice (n = 7) increased MAP by 10 +/- 3 mmHg and HR by 78 +/- 8 beats/min from the values observed under free-moving conditions. To demonstrate the chronic use of the venous catheter, intravenous infusion of NG-nitro-L-arginine methyl ester (L-NAME, 8.6 mg.kg-1.day-1, n = 6) for 5 days significantly increased MAP from 117 +/- 4 to 131 +/- 4 mmHg without altering HR. In a final group of mice (n = 5), oral L-arginine (2% in drinking water) increased plasma arginine concentration from 90 +/- 7 to 131 +/- 17 microM and prevented L-NAME hypertension. These experiments illustrate the feasibility of long-term intravenous infusion, direct arterial blood pressure measurements, and arterial blood sampling in conscious mice.This study describes a technique for the direct daily measurement of arterial blood pressure, sampling of arterial blood, and continuous intravenous infusion in free-moving, conscious, Swiss-Webster mice. Catheters were chronically implanted in the femoral artery and vein, tunneled subcutaneously, exteriorized at the back of the neck in a lightweight tethering spring, and attached to a swivel device at the top of the cage. Time-control experiments ( n = 8) demonstrated stable values of mean arterial pressure (MAP, 116 ± 1 mmHg) and heart rate (HR, 627 ± 21 beats/min) for up to 35 days after catheter implantation. It was further observed that restraining mice ( n = 7) increased MAP by 10 ± 3 mmHg and HR by 78 ± 8 beats/min from the values observed under free-moving conditions. To demonstrate the chronic use of the venous catheter, intravenous infusion of N G-nitro-l-arginine methyl ester (l-NAME, 8.6 mg ⋅ kg-1 ⋅ day-1, n = 6) for 5 days significantly increased MAP from 117 ± 4 to 131 ± 4 mmHg without altering HR. In a final group of mice ( n = 5), orall-arginine (2% in drinking water) increased plasma arginine concentration from 90 ± 7 to 131 ± 17 μM and preventedl-NAME hypertension. These experiments illustrate the feasibility of long-term intravenous infusion, direct arterial blood pressure measurements, and arterial blood sampling in conscious mice.

Infiltrating T lymphocytes in the kidney increase oxidative stress and participate in the development of hypertension and renal disease

The present studies examined the role and mechanism of action of infiltrating T lymphocytes in the kidney during salt-sensitive hypertension. Infiltrating T lymphocytes in the Dahl salt-sensitive (SS) kidney significantly increased from 7.2 ± 1.8 × 10(5) cells/2 kidneys to 18.2 ± 3.9 × 10(5) cells/2 kidneys (n = 6/group) when dietary NaCl was increased from 0.4 to 4.0%. Furthermore, the expression of immunoreactive p67(phox), gp91(phox), and p47(phox) subunits of NADPH oxidase was increased in T cells isolated from the kidneys of rats fed 4.0% NaCl. The urinary excretion of thiobarbituric acid-reactive substances (TBARS; an index of oxidative stress) also increased from 367 ± 49 to 688 ± 92 nmol/day (n = 8/group) when NaCl intake was increased in Dahl SS rats. Studies were then performed on rats treated with a daily injection of vehicle (5% dextrose) or tacrolimus (0.25 mg·kg(-1)·day(-1) ip), a calcineurin inhibitor that suppresses immune function, during the period of high-NaCl intake (n = 5/group). In contrast to the immune cell infiltration, increased NADPH oxidase expression, and elevated urine TBARS excretion in vehicle-treated Dahl SS fed high salt, these parameters were unaltered as NaCl intake was increased in Dahl SS rats administered tacrolimus. Moreover, tacrolimus treatment blunted high-salt mean arterial blood pressure and albumin excretion rate (152 ± 3 mmHg and 20 ± 9 mg/day, respectively) compared with values in dextrose-treated Dahl SS rats (171 ± 8 mmHg and 74 ± 28 mg/day). These experiments indicate that blockade of infiltrating immune cells is associated with decreased oxidative stress, an attenuation of hypertension, and a reduction of renal damage in Dahl SS rats fed high salt.