Luminal flow induces NO production in the thick ascending limb (TAL) by activating the enzyme that produces NO, NOS3. Shear stress, cellular stretch and transmural pressure all change when flow increases. Shear stress stimulates NO production in endothelial cells and inner medullary collecting ducts. However, it is unclear whether flow‐induced NO production in TALs is mediated by increases in shear stress, stretch or pressure. We hypothesized that shear stress acts as a mechanical stimulus for NO production in the TAL. We subjected primary cultures of rat TAL cells to shear stresses of 0.02 (low shear stress; LSS) and 0.55 dyne/cm 2 (high shear stress; HSS) and measured intracellular NO using the fluorescent dye DAF‐2 DA. The rate of increase in dye fluorescence is indicative of NO production. TAL cells under LSS produced NO at a rate of 55 ± 10 AU/s. NO production increased to 315 ± 93 AU/s at HSS (p < 0.03, n = 7). Pre‐treatment with the NOS inhibitor L‐NAME (5 mM) inhibited the shear stress‐induced increase in NO production (44 ± 24 vs. 48 ± 12 AU/s for LSS and HSS, respectively; n = 6). The PI3‐kinase inhibitor wortmannin (150 nM) also blocked NO production stimulated by increased shear stress (26 ± 9 AU/s for LSS vs .27 ± 8 AU/s for HSS; n = 5). We conclude that increases in shear stress mediate flow‐induced NO production by NOS via the PI3‐kinase pathway. Changes in flow may play an important role in regulation of TAL function by NO. Research support: NIH Grants HL 28982 and HL 70985
Carbon monoxide (CO) is a physiological messenger with diverse functions in the kidney, including controlling afferent arteriole tone both directly and via tubuloglomerular feedback (TGF). We have reported that CO attenuates TGF, but the mechanisms underlying this effect remain unknown. We hypothesized that CO, acting via cGMP, cGMP-dependent protein kinase, and cGMP-stimulated phosphodiesterase 2, reduces cAMP in the macula densa, leading to TGF attenuation. In vitro, microdissected rabbit afferent arterioles and their attached macula densa were simultaneously perfused. TGF was measured as the decrease in afferent arteriole diameter elicited by switching macula densa NaCl from 10 to 80 mmol/L. Adding a CO-releasing molecule (CORM-3, 5 × 10(-5) mol/L) to the macula densa blunted TGF from 3.3 ± 0.3 to 2.0 ± 0.3 μm (P<0.001). The guanylate cyclase inhibitor LY-83583 (10(-6) mol/L) enhanced TGF (5.8 ± 0.6 μm; P<0.001 versus control) and prevented the effect of CORM-3 on TGF (LY-83583+CORM-3, 5.5 ± 0.3 μm). Similarly, the cGMP-dependent protein kinase inhibitor KT-5823 (2 × 10(-6) mol/L) enhanced TGF and prevented the effect of CORM-3 on TGF (KT-5823, 6.0 ± 0.7 μm; KT-5823+CORM-3, 5.9 ± 0.8 μm). However, the phosphodiesterase 2 inhibitor BAY-60-7550 (10(-6) mol/L) did not prevent the effect of CORM-3 on TGF (BAY-60-7550, 4.07 ± 0.31 μm; BAY-60-7550+CORM-3, 1.84 ± 0.31 μm; P<0.001). Finally, the degradation-resistant cAMP analog dibutyryl-cAMP (10(-3) mol/L) prevented the attenuation of TGF by CORM-3 (dibutyryl-cAMP, 4.6 ± 0.5 μm; dibutyryl-cAMP+CORM-3, 5.0 ± 0.6 μm). We conclude that CO attenuates TGF by reducing cAMP via a cGMP-dependent pathway mediated by cGMP-dependent protein kinase rather than phosphodiesterase 2. Our results will lead to a better understanding of the mechanisms that control the renal microcirculation.
Increased luminal flow enhances nitric oxide (NO) production in thick ascending limbs (TALs). NO produced by NO synthase 3 (NOS3) inhibits Na transport. However, its effect on transport is reduced in Dahl salt-sensitive (SS) vs salt-resistant rats (SR). In Sprague-Dawley rat TALs, angiotensin II can acutely cause NOS3 to uncouple to produce superoxide (O 2 - ) thereby reducing NO production. We hypothesized that flow-induced NO production is decreased in SS TALs and that this is due to NOS3 uncoupling. We measured flow-induced NO in isolated perfused TALs using the fluorescent dye DAF-FM and performed Western blots of renal medullary lysates. Flow-induced NO production was reduced 69% in TALs from SS (11±2 arbitrary units (AU)/min, n=6) vs SR (35±6 AU/min, n=8, p < 0.008). This difference between strains was not due to altered NOS3 expression (NOS3/GAPDH ratio of 0.91 ± 0.08 for SS vs 1.09 ± 0.08 for SR; n = 5 for each). The difference in flow-induced NO between strains was slightly reduced in the presence of the superoxide (O 2 - ) scavenger tempol (19±2 vs 30±5 AU/min for SS and SR, respectively; n=9 for each strain, p < 0.04), suggesting that scavenging of NO by O 2 - plays only a minor role in the difference in flow-induced NO production between SS and SR thick ascending limbs. We next investigated whether NOS3 uncoupling could account for the difference between strains by using the fluorescent dye dihydroethidium to measure flow-induced O 2 - before and after treatment with the NOS inhibitor L-NAME. Blocking NOS3 reduced O 2 - production in SS TALs by 21±7%, from 38±5 to 30±5 AU/min (n=6, p < 0.05) whereas it had no effect in SR TALs (26±6 vs 28±3, n=5). We conclude that the diminished flow-induced NO in SS TALs is not due to differences in NOS3 expression nor acute flow-induced O 2 - , but rather in large part due to uncoupling of NOS3. Impaired flow-induced NO production in TALs could contribute to the Na retention associated with salt-sensitive hypertension.
NO produced by NO synthase type 3 (NOS3) in medullary thick ascending limbs (mTHALs) inhibits Cl(-) reabsorption. Acutely, angiotensin II stimulates thick ascending limb NO production. In endothelial cells, NO inhibits NOS3 expression. Therefore, we hypothesized that angiotensin II decreases NOS3 expression via NO in mTHALs. After 24 hours, 10 and 100 nmol/L of angiotensin II decreased NOS3 expression by 23+/-9% (n=6; P<0.05) and 50+/-5% (n=7; P<0.001), respectively, in primary cultures of rat mTHALs. NO synthase inhibition by 4 mmol/L of N(G)-nitro-L-arginine methyl ester hydrochloride prevented angiotensin II from decreasing NOS3 expression (Delta=-5+/-8%; n=5). In the presence of N(G)-nitro-L-arginine methyl ester hydrochloride, the addition of exogenous NO (1 micromol/L spermine NONOate) restored the angiotensin II-induced decreases in NOS3 expression (-22+/-6%; n=7; P<0.013). In addition, NO scavenging with 10 micromol/L of carboxy-PTIO abolished the effect of angiotensin II in NOS3 expression (Delta=-1+/-8% versus carboxy-PTIO alone; n=6). Angiotensin II increases superoxide, and superoxide scavenges NO. Thus, we tested whether scavenging superoxide enhances the angiotensin II-induced reduction in NOS3 expression. Surprisingly, treatment with 100 micromol/L of Tempol, a superoxide dismutase mimetic, blocked the angiotensin II-induced decrease in NOS3 expression (Delta=-3+/-7%; n=6). This effect was not because of increased hydrogen peroxide. We concluded that angiotensin II-induced decreases in NOS3 expression in mTHALs require both NO and superoxide. Decreased NOS3 expression by angiotensin II in mTHALs could contribute to increased salt retention observed in angiotensin II-induced hypertension.
Luminal flow augments Na+ reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na+, playing a key role in its homeostasis. Whether flow elevations enhance Na+-K+-2Cl- cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.
Dietary Fructose is implicated in the development of hypertension, diabetes and metabolic syndrome. Rats supplemented with 20% fructose develop hypertension, hypertriglyceridemia, and hyperglycemia after 8 weeks (Mamikutty N,et al. Biomed Res Int. '14). We hypothesized rats given a fructose‐enriched diet mimicking the upper range consumption in the US would cause salt‐sensitive hypertension prior to the onset of metabolic abnormalities. Groups of 8‐11 rats were given a normal rat chow diet, supplemented with either 1% NaCl, 20% fructose, or 1% NaCl plus 20% fructose in their drinking water over 4 weeks. Systolic blood pressure and body weights were monitored and 24 hour urine and blood collected at 4 weeks. After 4 weeks, systolic blood pressure was significantly increased by 10 ±4 mmHg (p<0.05) only in the fructose plus salt group. Growth rates were similar in all four groups (5.4 ± 0.4 g/day). Plasma electrolytes and glycemic indices were also similar. Plasma renin activity (PRA) was similar in control vs fructose rats (2.51±0.72 vs 2.71 ±0.90 ng AngI/ml/hr, respectively) but was suppressed by 80% (0.43 ±0.07 ngAng1/ml/hr) in rats fed 1% NaCl. However, fructose feeding prevented high salt induced‐suppression of PRA (1.89 ±0.43 ngAng1/ml/hr). Sodium excretion was elevated in both groups receiving NaCl. We conclude addition of salt to a fructose‐enriched diet induced salt‐sensitive hypertension prior to other pathologies. The blunting of salt‐induced suppression of PRA by fructose suggests it alters the sensitivity of renin secretion to salt intake and this may contribute to the increase in blood pressure.
Increasing Na delivery to the connecting tubule (CNT) stimulates epithelial Na channels (ENaC) and dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone (aldo) enhances CTGF via a nongenomic mechanism that stimulates CNT ENaC via GPR30 and/or mineralocorticoid receptors (MR). Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. Addition of aldo 10 -8 M to the CNT potentiated CTGF, seen as a left-shift in the concentration of NaCl that elicited a half-maximal response (EC 50 ), see Figure. The MR blocker eplerenone (10 -5 M) prevented the enhancement of CTGF by aldo (control EC 50 = 32.4 ± 2.3 mM; aldo + eplerenone EC 50 = 35.4 ± 1.7 mM; n = 7). Neither the transcription inhibitor actinomycin D (5x10 -6 M) nor the translation inhibitor cycloheximide (10 -5 M) prevented the effect of aldo (control EC 50 = 33.0 ± 2.0 mM; aldo + actinomycin D EC 50 = 15.4 ± 1.5 mM; n = 6; P < 0.001, control EC 50 = 33.2 ± 2.4 mM; aldo + cycloheximide EC 50 = 11.2 ± 1.3 mM; n = 6; P < 0.001). We conclude that aldo in the lumen of the CNT enhances CTGF via a nongenomic effect possibly involving MR and/or GPR30 receptors. Enhanced CTGF induced by aldosterone may contribute to renal damage by causing increases in Af-Art dilation and glomerular capillary pressure (glomerular barotrauma). Figure. Control CTGF (○) seen as dilation of norepinephrine-preconstricted Af-Arts induced by increasing NaCl in the CNT. Aldo 10 -8 M (•) enhanced CTGF (n = 6; * P < 0.05; ** P < 0.01; *** P < 0.001; vs . control). Vertical dashed lines indicate EC 50 .
Superoxide (O2−) exerts its physiological actions in part by causing changes in gene transcription. In thick ascending limbs flow-induced O2− production is mediated by NADPH oxidase 4 (Nox4) and is dependent on protein kinase C (PKC). Polymerase delta interacting protein 2 (Poldip2) increases Nox4 activity, but it is not known whether Nox4 translocates to the nucleus and whether Poldip2 participates in this process. We hypothesized that luminal flow causes Nox4 translocation to the nuclei of thick ascending limbs in a PKC-dependent process facilitated by Poldip2. To test our hypothesis, we studied the subcellular localization of Nox4 and Poldip2 using confocal microscopy and O2− production in the absence and presence of luminal flow. Luminal flow increased the ratio of nuclear to cytoplasmic intensity of Nox4 (N/C) from 0.3 ± 0.1 to 0.7 ± 0.1 (P < 0.01) and O2− production from 89 ± 15 to 231 ± 16 AU/s (P < 0.001). In the presence of flow PKC inhibition reduced N/C from 0.5 ± 0.1 to 0.2 ± 0.1 (P < 0.01). Flow-induced O2− production was also blocked (flow: 142 ± 20 AU/s; flow plus PKC inhibition 26 ± 12 AU/s; P < 0.01). The cytoskeleton disruptor cytochalasin D (1 μmol/L) decreased flow-induced Nox4 translocation by 0.3 ± 0.01 (P < 0.01); however, it did not reduce flow-induced O2−. Flow did not alter Poldip2 localization. We conclude that: (1) luminal flow elicits Nox4 translocation to the nucleus in a PKC- and cytoskeleton-dependent process; (2) Nox4 activation occurs before translocation; and (3) Poldip2 is not involved in Nox4 nuclear translocation. Flow-induced Nox4 translocation to the nucleus may play a role in O2−-dependent changes in thick ascending limbs.
We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min (P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min (P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min (P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O(2)(-) scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm(2) produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively (P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.
