Search

The renal afferent arterioles (Af-Arts) account for most of the renal vascular resistance, which is controlled similar to other arterioles and by tubuloglomerular feedback (TGF). The latter signal is generated by sensing sodium chloride concentrations in the macula densa; this in turn results in a signal which acts through the extraglomerular mesangium leading to constriction of the Af-Art. In the outer renal cortex, the connecting tubule (CNT) returns to the glomerular hilus and contacts the Af-Art suggesting that crosstalk may exist here as well. To investigate this, we simultaneously perfused a microdissected Af-Art and adherent CNT. Increasing the sodium chloride concentration perfusing the CNT significantly dilated preconstricted Af-Arts. We called this crosstalk 'connecting tubule glomerular feedback' (CTGF) to differentiate it from TGF. We tested whether entry of Na(+) and/or CI(-) into the CNT is required to induce CTGF by replacing Na(+) with choline(+). Increasing choline chloride concentration did not dilate the Af-Art. To test whether epithelial Na channels (ENaCs) mediate CTGF, we blocked ENaC with amiloride and found that the dilatation induced by CTGF was completely blocked. Inhibiting sodium chloride cotransporters with hydrochlorothiazide failed to prevent Af-Art dilatation. Finally, we tested whether nitric oxide released by the CNT mediates CTGF by the addition of a non-selective nitric oxide synthase inhibitor to the CNT. This potentiated CTGF rather than blocking it. We suggest that crosstalk exists between CNTs and attached Af-Arts, which is initiated by sodium reabsorption through amiloride-sensitive channels and this can contribute to the regulation of renal blood flow and glomerular filtration.

Adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl delivery. However, it is not known whether ATP from the macula densa is broken down to adenosine, or whether this adenosine mediates efferent arteriole (Ef-Art) tubuloglomerular feedback (TGF). We hypothesized that increased macula densa Ca(2+), release of ATP and degradation of ATP to adenosine are necessary for Ef-Art TGF. Rabbit Ef-Arts and adherent tubular segments (with the macula densa) were simultaneously microperfused in vitro while changing the NaCl concentration at the macula densa. The Ef-Art was perfused orthograde through the end of the afferent arteriole (Af-Art). In Ef-Arts preconstricted with norepinephrine (NE), increasing NaCl concentration from 10 to 80 mM at the macula densa dilated Ef-Arts from 7.5+/-0.7 to 11.1+/-0.3 microm. Buffering increases in macula densa Ca(2+) with the cell-permeant Ca(2+) chelator BAPTA-AM diminished Ef-Art TGF from 3.1+/-0.3 to 0.1+/-0.2 microm. Blocking adenosine formation by adding alpha-beta-methyleneadenosine 5'-diphosphate (MADP) blocked Ef-Art TGF from 2.9+/-0.5 to 0.1+/-0.2 microm. Increasing luminal NaCl at the macula densa from 10 to 45 mM caused a moderate Ef-Art TGF response, 1.3+/-0.1 microm. It was potentiated to 4.0+/-0.3 microm by adding hexokinase, which enhances conversion of ATP into adenosine. Our data show that in vitro changes in macula densa Ca(2+) and ATP release are necessary for Ef-Art TGF. ATP is broken down to form adenosine, which mediates signal transmission of Ef-Art TGF.

Background: There is evidence that macula densa nitric oxide (NO) inhibits tubuloglomerular feedback (TGF). However, TGF response is not altered in mice deficient in neuronal nitric oxide synthase (nNOS) (-/-). Furthermore, nNOS expression in the macula densa is inversely related to salt intake, yet micropuncture studies have shown that NOS inhibition potentiates TGF in rats on high sodium intake but not in rats on a low-salt diet. These inconsistencies may be due to confounding systemic factors, such as changes in circulating renin. To further clarify the role of macula densa nNOS in TGF response, independent of systemic factors, we tested the hypothesis that (1) TGF response is inversely related to sodium intake, and (2) during low sodium intake, NO produced by macula densa nNOS tonically controls the basal diameter of the afferent arteriole (Af-Art)., Methods: Af-Arts and attached macula densas were simultaneously microperfused in vitro. TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the macula densa perfusate. TGF response was studied in wild-type (+/+) and nNOS knockout mice (-/-), as well as in juxtaglomerular apparatuses (JGAs) from rabbits fed a low-, normal-, or high-NaCl diet., Results: TGF responses were similar in nNOS +/+ and -/- mice. However, in nNOS +/+ mice, 7-nitroindazole (7-NI) perfused into the macula densa significantly potentiated the TGF response (P = 0.001), while in nNOS -/- mice, this potentiation was absent. In rabbits on three different sodium diets, TGF responses were similar and were potentiated equally by 7-NI. However, in JGAs from rabbits on a low-NaCl diet, adding 7-NI to the macula densa while perfusing it with low-NaCl fluid caused Af-Art vasoconstriction, decreasing the diameter by 14% (from 21.7 +/- 1.3 to 18.6 +/- 1.5 microm; P < 0.001). This effect was not observed in JGAs from rabbits fed a normal- (19.0 +/- 0.5 vs. 19.3 +/- 0.8 microm after 7-NI) or high-NaCl diet (18.6 +/- 0.7 vs. 18.4 +/- 0.7 microm)., Conclusions: First, in this in vitro preparation, chronic changes in macula densa nNOS do not play a major role in the regulation of TGF. Compensatory mechanisms may develop during chronic alteration of nNOS that keep TGF relatively constant. Second, nNOS regulates TGF response acutely. Third, the results obtained in the +/+ and -/- mice also confirm that the effect of 7-NI is due to inhibition of macula densa nNOS. Finally, during low sodium intake (without induction of TGF), the regulation of basal Af-Art resistance by macula densa nNOS suggests that NO in the macula densa helps maintain renal blood flow during the high renin secretion caused by low sodium intake.

Background: Recent studies have revealed angiotensin II subtype 1 (AT1) receptors on macula densa cells, raising the possibility that angiotensin II (Ang II) could enhance tubuloglomerular feedback (TGF) by affecting macula densa cell function. We hypothesized that Ang II enhances TGF via activation of AT1 receptors on the luminal membrane of the macula densa., Methods: Rabbit afferent arterioles and the attached macula densa were simultaneously microperfused in vitro, keeping pressure in the afferent arteriole at 60 mm Hg., Results: The afferent arteriole diameter was measured while the macula densa was perfused with low NaCl (Na+, 5 mmol/L; Cl-, 3 mmol/L) and then with high NaCl (Na+, 79 mmol/L; Cl-, 77 mmol/L) to induce a TGF response. When TGF was induced in the absence of Ang II, the afferent arteriole diameter decreased by 2.4 +/- 0.5 microm (from 17.3 +/- 1.0 to 14.9 +/- 1.2 microm). With Ang II (0.1 nmol/L) present in the lumen of the macula densa, the diameter decreased by 3.8 +/- 0.7 microm (from 17.3 +/- 1.0 to 13.5 +/- 1.2 microm, P < 0.05 vs. TGF with no Ang II, N = 8). To test whether Ang II enhances TGF via activation of AT1 receptors on the luminal membrane of the macula densa, Ang II plus losartan (1 micromol/L) was added to the lumen. Losartan itself did not alter TGF. When TGF was induced in the absence of Ang II and losartan, the afferent arteriole diameter decreased by 2.3 +/- 0.3 microm (from 15.9 +/- 1.0 to 13.6 +/- 1.2 microm). When Ang II and losartan were both present in the macula densa perfusate, the diameter decreased by 2.4 +/- 0.4 microm (from 15.8 +/- 0.9 to 13.4 +/- 0.7 microm, P> 0.8 vs. TGF with no Ang II and losartan, N = 7). We then examined whether AT2 receptors on the macula densa influence the effect of luminal Ang II on TGF. When TGF was induced in the absence of Ang II plus PD 0123319-0121B (1 micromol/L), the afferent arteriole diameter decreased by 2.4 +/- 0.2 microm (from 17.0 +/- 0.9 to 14.6 +/- 0.8 microm). When Ang II and PD 0123319-0121B were both present in the macula densa lumen, the diameter decreased by 3.9 +/- 0.2 microm (from 16.8 +/- 0.9 to 12.9 +/- 0.9 microm, P < 0.001 vs. TGF with no Ang II and PD 0123319-0121B, N = 8). PD 0123319-0121B itself had no effect on TGF. To assure that this effect of Ang II was not due to leakage into the bath, losartan was added to the bath. When TGF was induced in the absence of Ang II with losartan in the bath, the afferent arteriole diameter decreased by 2.8 +/- 0.5 microm (from 19.3 +/- 1.2 to 16.5 +/- 0.8 microm). After Ang II was added to the macula densa perfusate and losartan to the bath, the diameter decreased by 4.0 +/- 0.7 microm (from 18.9 +/- 1.1 to 14.9 +/- 0.5 microm, P < 0.01 vs. TGF with no Ang II in the lumen and losartan in the bath, N = 8)., Conclusions: These results demonstrate that Ang II enhances TGF via activation of AT1 receptors on the luminal membrane of the macula densa.

Background: Afferent and efferent arteriole resistance exerts critical and opposite actions in the regulation of glomerular capillary pressure (PGC) and glomerular filtration rate (GFR). Tubuloglomerular feedback (TGF) plays an important role in the regulation of afferent arteriole resistance; however, the role of TGF in the regulation of efferent arteriole resistance is less well established. We hypothesized that TGF caused by increased NaCl in the tubular fluid stimulates the macula densa to initiate a cascade of events resulting in efferent arteriole vasodilation, mediated by adenosine via its A2 receptor., Methods: Rabbit efferent arterioles and adherent tubular segments with macula densa were simultaneously microperfused in vitro while changing NaCl concentration at the macula densa. To study whether autacoids produced by the glomerulus participate in the effect of TGF on efferent arterioles, they were perfused orthograde or retrograde. To eliminate the hemodynamic influence of the afferent arteriole during orthograde perfusion, the perfusion pipette was advanced to the distal end of the afferent arteriole, and the tip of the pressure pipette was placed beyond the afferent arteriole; for retrograde perfusion, the efferent arteriole was perfused from its distal end., Results: In efferent arterioles perfused orthograde and preconstricted with norepinephrine (NE), increasing NaCl concentration at the macula densa increased the diameter by 33%. In preconstricted efferent arterioles perfused retrograde, increasing NaCl at the macula densa increased the diameter by 33%. Efferent arteriole vasodilation was completely blocked by a selective adenosine A2 receptor antagonist (3, 7-dimethyl-1-propargylxanthine) but not by an adenosine A1 receptor antagonist (FK838)., Conclusions: Our data show that in vitro, preconstricted efferent arterioles dilate in response to increased macula densa NaCl, and this process is mediated by activation of adenosine A2 receptors. Thus, TGF changes efferent arteriole resistance in the opposite direction from the afferent arteriole, possibly amplifying TGF regulation of PGC and GFR. In vivo efferent arteriole TGF may only buffer the signals that cause efferent arteriole resistance to parallel changes in afferent arteriole resistance. Effects of TGF on efferent arterioles perfused orthograde or retrograde were similar, suggesting that glomerular autacoids do not participate in this process.

Background: In vivo and in vitro studies have shown that nitric oxide (NO) is an important modulator of transport processes along the nephron. The thick ascending limb (TAL) plays a significant role in the urine-concentrating mechanism and in the maintenance of acid/base balance., Methods: TALs from male Sprague-Dawley rats were isolated and perfused, and net bicarbonate flux (J(HCO3)(-) was determined., Results: In perfused TALs, 0.5 mmol/L L-arginine (L-Arg), the substrate for NO synthase, significantly lowered J(HCO3)(-) from 35.4 +/- 4.6 to 23.2 +/- 2.9 pmol. mm(-1). min(-1), a decrease of 36.9 +/- 11.6% (P < 0.025). D-Arg (0.5 mmol/L) had no effect on J(HCO3)(-) (N = 7). In the presence of 5 mmol/L L-NAME, an NO synthase (NOS) inhibitor, the addition of L-Arg did not affect TAL J(HCO3)(-) (43.4 +/- 4.4 vs. 44.6 +/- 5.0 pmol. mm(-1). min(-1)). L-NAME alone (5 mmol/L) did not affect TAL J(HCO3)(-). After removing L-Arg from the bath, J(HCO3)(-) increased from 26.2 +/- 3.9 to 34.8 +/- 3.2 pmol. mm(-1). min(-1) (P < 0.01), indicating no cytotoxicity of NO. We next investigated the effect of cGMP analogues on TAL J(HCO3)(-). 8-Br-cGMP (50 micromol/L) and db-cGMP (50 micromol/L) significantly decreased J(HCO3)(-) by 26.3 +/- 9.1% and 35.1 +/- 11.6%, respectively. In the presence of cGMP (50 micromol/L), the addition of L-Arg had no effect on J(HCO3)(-). In the presence of KT-5823 (2 mircromol/L), a protein kinase G inhibitor, the addition of L-Arg did not change TAL J(HCO3)(-) (N = 5)., Conclusions: We conclude that (1) endogenously produced NO inhibits TAL J(HCO3)(-) in an autocrine manner, (2) cGMP mediates all the effects of NO, and (3) this effect is mediated by protein kinase G activation.

Background: Previous studies have suggested that nitric oxide (NO) produced within cells of the macula densa (MD) modulates tubuloglomerular feedback (TGF). We tested the hypothesis that NO produced in the MD acts locally as an autacoid to activate soluble guanylate cyclase and cGMP-dependent protein kinase in the MD itself., Methods: Rabbit afferent arterioles (Af-Arts) and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring the Af-Art diameter before and after increasing NaCl in the MD perfusate (from 17 mmol/L of Na and 2 of Cl to 65 mmol/L of Na and 50 of Cl). TGF was studied before (control TGF) and after inhibiting components of the NO-cGMP-dependent cascade in the tubular or vascular compartment., Results: Increasing NaCl concentration in the MD perfusate decreased the Af-Art diameter by 3.2 +/- 0.5 microm (from 18.5 +/- 1.3 to 15.4 +/- 1.3 microm, P < 0.001). Adding a soluble guanylate cyclase inhibitor (LY83583) to the MD increased TGF response to 6.3 +/- 1.1 microm (P < 0.031 vs. control TGF). Similarly, when cGMP-dependent protein kinase was inhibited with KT5823, TGF was augmented from 2.6 +/- 0.3 to 4.0 +/- 0.7 microm (P < 0.023). An analogue of cGMP in the MD reversed the TGF-potentiating effect of both 7-nitroindazole (7NI; an nNOS inhibitor) and LY83583. Inhibition of MD guanylate cyclase did not alter the effect of acetylcholine (a NO-cGMP-dependent vasodilator) on the Af-Art. Perfusing the Af-Art with the guanylate cyclase inhibitor did not potentiate TGF, suggesting that the effect of NO occurred at the MD via a cGMP-dependent mechanism. To determine whether the effect of NO in the MD was entirely mediated by cGMP, TGF was studied after giving (1) LY83583 or (2) LY83583 plus 7NI. Adding the nNOS inhibitor to the MD did not potentiate the TGF response further., Conclusions: We concluded the following: (1) NO produced by the MD inhibits TGF via stimulation of soluble guanylate cyclase, generating cGMP and activating cGMP-dependent protein kinase; (2) NO acts on the MD itself rather than by diffusing to the Af-Art; and (3) most, if not all, of the effect of NO in the MD is due to a cGMP-dependent mechanism rather than to other NO mediators.

Background: Sulfonylurea receptors are classified as either high-affinity type 1 (SUR1) or low-affinity type 2 receptors (SUR2), and the gene expression of SURs has recently been demonstrated in kidney. However, functional data regarding a renal SUR are lacking. We previously demonstrated that mesangial cell (MC) gene and protein expression of extracellular matrix components were up-regulated by the sulfonylurea, tolazamide. After noting this biological response, we next sought to investigate the presence of a sulfonylurea receptor in rat MCs., Methods: Equilibrium binding studies employing [3H]glibenclamide as a ligand were performed on crude MC membrane preparations. Gene expression for SUR was explored by Northern analysis of cultured MCs and whole kidney tissue. The effect of sulfonylurea on intracellular Ca2+ in MCs was assayed by spectrofluorometry, and glibenclamide-induced changes in the contractility of MCs were assessed., Results: MCs bound [3H]glibenclamide with a KD of 2.6 microM and a Bmax of 30.4 pmol/mg protein as determined by Scatchard analysis. Three SUR2 transcripts were detected in MCs. A major transcript was detected at 5.5 kb and minor transcripts at 7.5 and 8.6 kb. Following sulfonylurea treatment of MCs, real-time videomicroscopy revealed intense MC contraction, coinciding with oscillatory increments of intracellular Ca2+ concentration. Further evidence of sulfonylurea-induced MC contraction was demonstrated by glibenclamide-induced deformation of a silicone rubber substrate., Conclusions: These results demonstrate that SUR2 resides on MCs. Functional activation of this receptor by sulfonylurea induces Ca2+ transients that result in MC contraction.

Background: Measurements of Cl- in nanoliter samples, such as those collected during isolated, perfused tubule experiments, have been difficult, somewhat insensitive, and/or require custom-made equipment. We developed a technique using a fluorescent Cl- indicator, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), to make these measurements simple and reliable., Methods: This is a simple procedure that relies on the selectivity of the dye and the fact that Cl-quenches its fluorescence. To measure millimolar quantities of Cl- in nanoliter samples, we prepared a solution of 0.25 mm SPQ and loaded it into the reservoir of a continuous-flow ultramicrofluorometer, which can be constructed from commercially available components. Samples were injected with a calibrated pipette via an injection port, and the resultant peak fluorescent deflections were recorded. The deflections represent a decrease in fluorescence caused by the quenching effect of the Cl- injected., Results: The method yielded a linear response with Cl- concentrations from 5 to 200 mm NaCl. The minimum detectable Cl- concentration was approximately 5 mm. The coefficient of variation between 5 and 200 mm was 1.7%. Resolution, defined as two times the standard error divided by the slope, between 10 and 50 mm and between 50 and 200 mm was 1 mm and 2.6 mm, respectively. Furosemide, diisothiocyanostilbene-2,2'-disulfonic acid and other nonchloride anions (HEPES, HCO3, SO4, and PO4) did not interfere with the assay, whereas 150 mm NaBr resulted in a peak height greater than 150 NaCl. In addition, the ability to measure Cl- did not vary with pH within the physiological range., Conclusion: We developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.

Measurement of sodium in the nanoliter samples obtained from in vitro studies of isolated, perfused nephron segments has traditionally been associated with time-consuming sample preparation and/or expensive instrumentation. A new instrument is described which can measure picomole quantities of sodium without the need for special processing. The instrument is based on the continuous flow devices developed by Vurek and a sodium-sensitive glass detector. The response to varying concentrations of NaCl was logarithmic as predicted from the Nernst equation. The response to 50 mM NaCl was 22.1 +/- 0.2 mV; 100 mM, 29.9 +/- 0.2 mV; 150 mM, 35.2 +/- 0.2 mV; and 200 mM, 39.3 +/- 0.6 mV (mean +/- SD). The slope of the standard curve was 27.8 mV/log unit change in sodium concentration. Potassium, calcium and ammonium, possible interfering ions, had no effect on the response to 100 mM NaCl. When the response to 100 mM NaCl was measured in buffered solutions of varying pH, it was 29.1 +/- 0.2 mV at pH 6.21, 28.9 +/- 0.4 mV at pH 7.04 and 28.7 +/- 0.4 mV at pH 8.54, indicating that pH did not alter the response to 100 mM NaCl. By treating the detector with NaOH and HCl, the response to 50, 100 and 150 mM improved to the point that the slope of the standard curve was 51.2 mV/log unit change in sodium concentration. The calculated resolution after treatment was 1.0 mM in the 50 to 100 mM range and 1.3 mM in the 100 to 150 mM range using a 10.9 nl pipet.(ABSTRACT TRUNCATED AT 250 WORDS)