Craig F. Plato

eNOS Mediates l-Arginine–Induced Inhibition of Thick Ascending Limb Chloride Flux

We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0. 5 mmol/L L-Arg to the bath decreased J(Cl) from 105.8+/-17.5 to 79. 2+/-15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J(Cl.) In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J(Cl) of THALs from eNOS knockout mice. When 10 micromol/L SPM was added to the bath of eNOS knockout THALs, J(Cl) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J(Cl) in THALs from iNOS and nNOS knockout mice by 37.7+/-6.4% (P<0.05) and 31.8+/-8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J(Cl).

Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis.

Background & Aims Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC’s anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis. Methods Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC’s antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses. Results CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight. Conclusions CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC’s antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).

Nitric Oxide–Induced Inhibition of Transport by Thick Ascending Limbs From Dahl Salt-Sensitive Rats

The factor responsible for salt sensitivity of blood pressure in Dahl rats is unclear but presumably resides in the kidney. We tested the hypotheses that (1) thick ascending limbs of Dahl salt-sensitive rats (DS) absorb more NaCl than those of Dahl salt-resistant rats (DR) and (2) NO inhibits transport to a lesser extent in thick ascending limbs from DS. We found that basal chloride absorption (J(Cl)) by thick ascending limbs from DR was 105.8+/-10.0 pmol. mm(-1). min(-1) (n=6). Ten and 100 micromol/L spermine NONOate, an NO donor, decreased J(Cl) in DR to 65.8+/-8.5 and 46.8+/-7.0 pmol. mm(-1). min(-1), respectively. Basal J(Cl) in DS was 131.6+/-13.4 pmol. mm(-1). min(-1) (n=7). In DS, 10 and 100 micromol/L spermine NONOate decreased J(Cl) to 111.5+/-12.8 and 46.8+/-6.2 pmol. mm(-1). min(-1), respectively. No difference was observed in basal or NO-inhibited Na absorption by cortical collecting ducts or in basal or NO-inhibited oxygen consumption by inner medullary collecting ducts. Because NO acts via generation of cGMP, we measured cGMP production by thick ascending limbs from DS and DR to see whether a difference in cGMP production could account for the difference in basal or NO-inhibited transport. Basal rates of cGMP production were similar between the 2 strains. Although NO increased cGMP production by thick ascending limbs from both strains, no difference existed between DS and DR. We concluded that the reduced ability of NO to block transport in thick ascending limbs in DS may account for at least part of the salt sensitivity of blood pressure in this strain.

Intestinal Inhibition of the Na+/H+ Exchanger 3 Prevents Cardiorenal Damage in Rats and Inhibits Na+ Uptake in Humans

An inhibitor of intestinal NHE3 reduces absorption of dietary sodium in rats and humans and prevents salt-induced cardiorenal injury in nephrectomized rats. Pass the Salt Whether life arose on earth or elsewhere, our ancestors certainly spent some time in the Earth’s oceans: We still carry the evidence for that in the saltiness of our bodily fluids. When the salty balance of our body goes awry (through diet, for example), disease can result. Now, Spencer and colleagues have found a way to prevent the absorption of dietary sodium through the intestine without requiring the drug be present throughout the body, a safer approach. Normally, most of the sodium chloride that is consumed is taken up by a dedicated transporter in the membranes of intestinal cells. This sodium-proton exchanger, NHE3, pumps one sodium ion in and one proton out, bringing dietary sodium into the body. The authors used an NHE3 inhibitor called tenapanor that does not cross the intestinal barrier. They tested its effects on sodium uptake in normal rats and normal healthy humans, as well as in a rat model of salt-driven hypertension that resembles the patients that might benefit from this treatment. In healthy humans and rats, tenapanor decreased sodium uptake from the intestine and increased its concentrations in the stool, as expected for an NHE3 inhibitor acting exclusively in the intestine. It had no other damaging effects to the animals or subjects. The rats with salt-driven hypertension exhibited numerous problems—heart hypertrophy, excess fluid, arterial stiffening, and high blood pressure. Treatment with the drug before the disease onset and—important for a therapeutic agent—after disease onset improved all of these measures. A combination of tenapanor and the popular antihypertensive drug enalapril (which acts to inhibit angiotensin-converting enzyme) was more effective against certain outcomes than either drug alone. The combination improved cardiac function (left ventricular hypertrophy and arterial stiffness) and kidney function markedly, which enalapril alone did not. Modern humans often eat too much salt or have other reasons to limit the amount of sodium they consume. The inhibition of sodium uptake transporters in the intestine may prove a better way to combat these problems than our current approaches. The management of sodium intake is clinically important in many disease states including heart failure, kidney disease, and hypertension. Tenapanor is an inhibitor of the sodium-proton (Na+/H+) exchanger NHE3, which plays a prominent role in sodium handling in the gastrointestinal tract and kidney. When administered orally to rats, tenapanor acted exclusively in the gastrointestinal tract to inhibit sodium uptake. We showed that the systemic availability of tenapanor was negligible through plasma pharmacokinetic studies, as well as autoradiography and mass balance studies performed with 14C-tenapanor. In humans, tenapanor reduced urinary sodium excretion by 20 to 50 mmol/day and led to an increase of similar magnitude in stool sodium. In salt-fed nephrectomized rats exhibiting hypervolemia, cardiac hypertrophy, and arterial stiffening, tenapanor reduced extracellular fluid volume, left ventricular hypertrophy, albuminuria, and blood pressure in a dose-dependent fashion. We observed these effects whether tenapanor was administered prophylactically or after disease was established. In addition, the combination of tenapanor and the blood pressure medication enalapril improved cardiac diastolic dysfunction and arterial pulse wave velocity relative to enalapril monotherapy in this animal model. Tenapanor prevented increases in glomerular area and urinary KIM-1, a marker of renal injury. The results suggest that therapeutic alteration of sodium transport in the gastrointestinal tract instead of the kidney—the target of current drugs—could lead to improved sodium management in renal disease.

NITRIC OXIDE, ENDOTHELIN AND NEPHRON TRANSPORT: POTENTIAL INTERACTIONS

1. Nitric oxide (NO) is produced and/or regulates transport in many segments of the nephron, including the proximal convoluted tubule, proximal straight tubule, thick ascending limb, cortical collecting duct and inner medullary collecting duct.

Age exacerbates chronic catecholamine-induced impairments in contractile reserve in the rat

Contractile reserve decreases with advancing age and chronic isoproterenol (ISO) administration is a well-characterized model of cardiac hypertrophy known to impair cardiovascular function. This study evaluated whether nonsenescent, mature adult rats are more susceptible to detrimental effects of chronic ISO administration than younger adult rats. Rats received daily injections of ISO (0.1 mg/kg sc) or vehicle for 3 wk. ISO induced a greater impairment in contractile reserve [maximum of left ventricular pressure development (Δ+dP/dt(max))] in mature adult ISO-treated (MA-ISO) than in young adult ISO-treated rats (YA-ISO) in response to infusions of mechanistically distinct inotropes (digoxin, milrinone; 20-200 μl·kg(-1)·min(-1)), while basal and agonist-induced changes in heart rate and systolic arterial pressure (SAP) were not different across groups. ISO decreased expression of the calcium handling protein, sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a, in MA-ISO compared with YA, YA-ISO, and MA rats. Chronic ISO also induced greater increases in cardiac hypertrophy [left ventricular (LV) index: 33 ± 3 vs. 22 ± 5%] and caspase-3 activity (34 vs. 5%) in MA-ISO relative to YA-ISO rats. Moreover, β-myosin heavy chain (β-MHC) and atrial natriuretic factor (ANF) mRNA expression was significantly elevated in MA-ISO. These results demonstrate that adult rats develop greater impairments in systolic performance than younger rats when exposed to chronic catecholamine excess. Reduced contractile reserve may result from calcium dysregulation, increased caspase-3 activity, or increased β-MHC and ANF expression. Although several studies report age-related declines in systolic performance in older and senescent animals, the present study demonstrates that catecholamine excess induces reductions in systolic performance significantly earlier in life.