Role of mesangial cells and gap junctions in tubuloglomerular feedback

Role of mesangial cells and gap junctions in tubuloglomerular feedback.BackgroundTubuloglomerular feedback (TGF) is a process whereby the resistance of the afferent arterioles delivering blood to the glomeruli is regulated by the NaCl concentration of the forming urine in the lumen of the macula densa. Intraglomerular mesangial cells are located between capillaries within the glomerulus, while extraglomerular mesangial cells are located between the macula densa and the afferent arteriole. They are electrically and chemically coupled via gap junctions. The purpose of this study was to investigate the role of mesangial cells and gap junctions in TGF using the isolated, perfused juxtaglomerular apparatus.MethodJuxtaglomerular apparatuses were dissected from male New Zealand white rabbits and perfused in vitro. The NaCl concentration at the macula densa was changed from 17/2 to 65/50 Na/Cl to initiate a TGF response. Afferent arterioles were perfused at 60 mm Hg throughout the experiment. Changes in luminal diameter caused by increasing the NaCl concentration at the macula densa were taken as the TGF response. TGF was measured before and after disrupting the gap junctions or damaging the mesangial cells in paired experiments.ResultsDuring the control period, TGF decreased afferent arteriole diameter by 2.9 ± 0.2 μm. After mesangial cells were damaged by perfusing Thy 1-1 antibody and complement into the afferent arteriole, the TGF response was completely eliminated. Separate experiments showed no statistically significant change in TGF response with time, or when antibody and complement were perfused into the macula densa lumen. The presence of Thy 1-1 antibody and complement in the afferent arteriole perfusate did not alter the ability of norepinephrine to constrict or acetylcholine to dilate the afferent arteriole. To investigate the role of gap junctions in TGF, we used heptanol to disrupt them. During the control period, TGF decreased afferent arteriole diameter by 2.9 ± 0.4 μm. After perfusing heptanol into the lumen of the afferent arteriole, the TGF response was completely eliminated. When heptanol was added to the bath, it had no significant effect on TGF response.DiscussionThe data show that after mesangial cells were selectively damaged, the constriction of the afferent arteriole induced by increasing the NaCl concentration at the macula densa was eliminated. However, such treatment had no effect when Thy 1-1 was perfused into the macula densa lumen, and did not alter the response of the afferent arteriole to norepinephrine or acetylcholine. Disruption of the gap junctions also eliminated the TGF response. These data indicate that the mesangial cells play a key role in mediating the TGF response, and that gap junctions among mesangial cells and between mesangial cells and vascular smooth muscle cells communicate the TGF signal to the afferent arteriole

Possible role of adenosine in macula densa control of glomerular hemodynamics

Possible role of adenosine in macula densa control of glomerular hemodynamics.BackgroundThe macula densa (MD), a plaque of specialized tubular epithelial cells, senses changes in tubular NaCl concentration and sends a signal(s) that controls the resistance of the glomerular afferent arteriole (Af-Art). This mechanism, called tubuloglomerular feedback (TGF), is thought to be important in the homeostasis of body fluids and electrolytes. Our aim was to determine the range of NaCl concentrations in tubular fluid at the MD that would elicit the Af-Art response. In addition, we examined the possible involvement of adenosine in transmitting the signal from the MD to the Af-Art.MethodsRabbit Af-Arts and attached MD were simultaneously microperfused in vitro, keeping pressure in the Af-Art at 60mm Hg.ResultsIncreasing the Na+/Cl- concentration of the MD perfusate from 26/7 to 41/22 mEq/L decreased the luminal diameter of the terminal Af-Art segment by 10 ± 4% (N = 9; P < 0.01). The response was maximal at 55/36 mEq/L (18 ± 6%), so that further elevation of NaCl concentration had no additional effect (20 ± 6% at 84/65 mEq/L). When FK838 (10-6 mol/L), a specific adenosine A1 receptor antagonist, was added to both Af-Art perfusate and bath, Af-Art constriction was completely abolished. The maximum response was 20 ± 3% before FK838 and 0.6 ± 1% afterward (N = 12). Adding adenosine at 10-8 mol/L to both bath and perfusate significantly augmented Af-Art constriction induced by increased NaCl at the MD (P < 0.01); however, adding 10-8 to 10-6 mol/L adenosine to the MD perfusate had no effect regardless of the NaCl concentration at the MD.ConclusionsThese results demonstrate that MD control of Af-Art resistance is induced by relatively low NaCl concentrations at the MD, and that activation of the adenosine A1 receptor in the vascular and interstitial space (but not the tubular lumen) may be essential for signal transmission from the MD to the Af-Art

The Involvement of Urinary Kallikrein in the Renal Escape from the Sodium Retaining Effect of Mineralocorticoids

It is well known that the normal kidney escapes the sodium retaining effect of mineralocorticoids. However, the mechanism that mediates this escape is not understood. The possible role of kallikrein in this escape phenomenon was investigated by placing seven dogs in metabolic cages and giving them a constant sodium diet. After they had been on this diet three days, urine was collected for two 24-hour periods. DOCA (25 mg/day) was then given intramuscularly for five days. Urine was collected daily during this DOCA period and for two additional 24- hour periods. Urine volume, sodium, potassium, protein, and kallikrein excretion were then measured. Urinary kallikrein increased from 251.9 ± 34.8 (mean ± SE) in the second day of the control period to 639.8 ± 110.1 IJ-g/day (P \u3c .01) by the third day of treatment. It remained elevated two days after DOCA was discontinued. Sodium excretion decreased significantly on the first day of DOCA treatment, returning to the previous values thereafter. Urine protein excretion remained constant. The enhanced urinary kallikrein during the escape suggests that the kallikrein system could be involved in the regulation of sodium metabolism by acting as a natriuretic factor, or perhaps by regulating the renal blood flow

Increased intracellular Ca++ in the macula densa regulates tubuloglomerular feedback

Increased intracellular Ca++ in the macula densa regulates tubuloglomerular feedback.BackgroundTubuloglomerular feedback is initiated by an increase in NaCl at the macula densa lumen, which in turn increases intracellular Ca++. In the present study, we examined the role of increased intracellular Ca++ in tubuloglomerular feedback and the source of the increased Ca++. We hypothesized that an increase in intracellular Ca++ at the macula densa via the basolateral Na+/Ca++ exchanger, caused by an increase in luminal NaCl, initiates Ca++-mediated Ca++ release from intracellular stores, which is essential for tubuloglomerular feedback.MethodsRabbit afferent arterioles and attached macula densas were simultaneously microperfused in vitro. Tubuloglomerular feedback was induced by increasing macula densa Na+/Cl- from 11/10mmol/L (low) to 81/80mmol/L (high) and was measured before and after treatment.ResultsTo investigate whether elevations in intracellular Ca++ are required for tubuloglomerular feedback, the calcium ionophore A23187 or the Ca++ chelator BAPTA-AM was added to the macula densa lumen. During the control period, tubuloglomerular feedback decreased afferent arteriole diameter from 18.1 ± 1.1 μm to 15.3 ± 0.8 μm. Adding 2 × 10-6 mol/L A23187 to the low NaCl macula densa perfusate induced tubuloglomerular feedback; diameter decreased from 18.0 ± 1.0 μm to 15.4 ± 0.9 μm (N = 6; P < 0.01). After adding BAPTA-AM (25 μmol/L) to the macula densa lumen, tubuloglomerular feedback response was completely eliminated. We next studied the source of increased macula densa Ca++ in response to increased NaCl concentration. During the control period, tubuloglomerular feedback decreased afferent arteriole diameter from 18.5 ± 1.6 μm to 15.3 ± 1.2 μm (N = 6; P < 0.01). After adding the Na+/Ca++ exchanger inhibitor 2′4′-dichlorobenzamil (10 μmol/L) or KB-R7943 (30 μmol/L) to the bath, the tubuloglomerular feedback response was blocked; however, the afferent arteriole response to angiotensin II or adenosine was not altered. Next, we tested the Ca++-adenosine triphosphatase (ATPase) inhibitor thapsigargin (0.1 μmol/L), which has been reported to inhibit sarcoplasmic reticulum Ca++-ATPase activity and prevent restoration of intracellular Ca++ stores. When thapsigargin was added to the macula densa lumen, it reduced the first tubuloglomerular feedback response by 33% and completely eliminated the second and third tubuloglomerular feedback responses. In the absence of thapsigargin, there was no significant decrease in the tubuloglomerular feedback responses (N = 6). Neither the L-type Ca++ channel blocker nifedipine (25 μmol/L), nor the T-type Ca++ channel blocker pimozide (10 μmol/L), inhibited tubuloglomerular feedback when added to the macula densa lumen.ConclusionWe concluded that (1) increased intracellular Ca++ at the macula densa is required for the tubuloglomerular feedback response; (2) Na+/Ca++ exchange appears to initiate Ca++-mediated Ca++ release from intracellular stores; and (3) luminal L-type or T-type Ca++ channels are not involved in tubuloglomerular feedback

Role of neuronal nitric oxide synthase in the macula densa

Role of neuronal nitric oxide synthase in the macula densa.BackgroundThere 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).MethodsAf-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.ResultsTGF 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 μm; P < 0.001). This effect was not observed in JGAs from rabbits fed a normal- (19.0 ± 0.5 vs. 19.3 ± 0.8 μm after 7-NI) or high-NaCl diet (18.6 ± 0.7 vs. 18.4 ± 0.7 μm).ConclusionsFirst, 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

Characterization of a mouse cortical collecting duct cell line

Characterization of a mouse cortical collecting duct cell line. A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepi-thelial resistance (885.7 ± 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 ± 3.5mV. The associated short-circuit current (SCC) averaged 71.8 ± 10.3 µA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 ± 0.9 and B = 127.4 ± 0.4; [K+], L = 8.6 ± 0.6 and B = 2.1 ± 0.3; [Cl], L = 68.6 ± 5.8 and B = 101.8 ± 6.6; [HCO3], L = 15.5 ± 1.5 and B = 8.6 ± 1.2; while pH was 7.16 ± 0.03 at the luminal and 6.94 ± 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+. Lactate accumulated predominantly at the basolateral side (L = 7.1 ± 0.44 and B = 17.5 ± 0.52 mM); osmotic concentration was 272 ± 1.4 at the luminal and 290 ±3.0 mOsm/kg at the basolateral side. These data demonstrate that the M-1 cell line retains many phenotypic properties of the CCD epithelium and thus should prove useful as a model in studying mechanisms of ion transport in this segment

Role of angiotensin II type 2 receptors and kinins in the cardioprotective effect of angiotensin II type 1 receptor antagonists in rats with heart failure

AbstractObjectivesWe studied the role of angiotensin II type 2 (AT2) receptors and kinins in the cardioprotective effect of angiotensin II type 1 antagonists (AT1-ant) in rats with heart failure (HF) after myocardial infarction.BackgroundThe AT1-ant is as effective as angiotensin-converting enzyme inhibitors in treating HF, but the mechanisms whereby AT1-ant exert their benefits on HF in vivo are more complex than previously understood.MethodsBrown Norway Katholiek rats (BNK), which are deficient in kinins because of a mutation in the kininogen gene, and their wild-type control (Brown Norway [BN]) underwent myocardial infarction. Two months later, they were treated for two months with: 1) vehicle; 2) AT1-ant (L158809, Merck, Rahway, New Jersey); 3) AT1-ant + AT2-ant (PD-123319, Parke Davis, Ann Arbor, Michigan); or 4) AT1-ant + kinin B2receptor antagonist (B2-ant) (icatibant) (only BN). We measured left ventricular weight (LVW) gravimetrically, myocyte cross-sectional area (MCSA) and interstitial collagen fraction (ICF) histologically, and ejection fraction by ventriculography.ResultsDevelopment of HF was comparable in BN and BNK rats. The AT1-ant reduced LVW and MCSA and the AT2-ant blocked these effects in BN rats, but the B2-ant did not. The AT1-ant reduced LVW and MCSA in BNK rats, and this effect was reversed by the AT2-ant. In BN rats, ICF was reduced and LVEF increased by AT1-ant, and both AT2-ant and B2-ant reversed these effects. In BNK rats, the AT1-ant failed to reduce ICF, and its therapeutic effect on LVEF was significantly blunted.ConclusionsIn HF, the AT2receptor plays an important role in the therapeutic effects of AT1-ant, and this effect may be mediated partly through kinins; however, kinins appear to play a lesser role in the antihypertrophic effect of AT1-ant

Role of neuropeptide Y in the development of two-kidney, one-clip renovascular hypertension in the rat

AbstractObjective: Along with the renin-angiotensin system, sympathetic stimulation may contribute to renovascular hypertension. The vasoactive peptide neuropeptide Y (NPY) is co-released with and potentiates the pressor effects of norepinephrine through the Y-1 receptor. NPY, by exaggerating sympathetic activity, may contribute to renovascular hypertension, possibly by augmenting adrenergic-mediated renin release. This was studied by determining the effect of continuous Y-1 blockade on the development of two-kidney, one-clip renovascular hypertension and the effect of NPY on in vitro renin release. Methods: Mean arterial pressure and renal blood flow responses to NPY (10 μg/kg, administered intravenously) were measured in five anesthetized Sprague-Dawley rats before and after BIBO3304TF administration to test the Y-1 antagonist BIBO3304TF. In hypertension studies, 28 rats underwent left renal artery clipping. Of these, 13 were implanted with a mini-osmotic pump for continuous BIBO3304TF infusion (0.3 μg/h, administered intravenously); the other 15 underwent sham implantation. Systolic blood pressure was then monitored for 4 weeks. Finally, in vitro renin release was measured from renal cortical slices (n = 6-12) incubated with NPY (10–8 to 10–6 mol/L) or NPY plus the adrenergic agonist isoproterenol (10–4 mol/L). Results: BIBO3304TF attenuated the NPY-induced increase in mean arterial pressure by 54% (P <.02) and the NPY-induced decrease in renal blood flow by 38% (P <.05). In 4-week hypertension studies, systolic blood pressure in clipped controls increased from 130 ± 3 mm Hg to 167 ± 6 mm Hg (P <.01), whereas BIBO3304TF-treated rats had no significant increase (125 ± 3 mm Hg to 141 ± 8 mm Hg). Final systolic blood pressure was 26 mm Hg lower in BIBO3304TF-treated rats than in controls (P <.01). In renal cortical slices, no NPY effect was observed in basal or isoproterenol-stimulated renin release. Conclusions: The Y-1 receptor antagonist BIBO3304TF attenuated acute pressor responses to NPY and blunted the development of two-kidney, one-clip renovascular hypertension in rats. NPY may contribute to the hypertensive response in this renovascular hypertension model. Our in vitro data do not suggest that this is due to NPY enhancement of renin release. (J Vasc Surg 2000;32:1015-21.

The Role of High Molecular Weight Kininogen (Fitzgerald Factor) in the Activation of Various Plasma Proteolytic Enzyme Systems

Bovine high molecular weight kininogen (bHMWK) partially corrects the aPTT of Fitzgerald-trait plasma, which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied, all in equimolar concentrations, were lys-bradykinin-free HMWK, bradyk In In-fragment 1-2-tree HMWK, heavy chain, fragment 1 -2-Hght chain, and light chain. Bradykinin- fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Eitzgeraldtrait plasma aPTT. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lysbradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. On a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. Bovine plasma kallikrein released kinins from both bHMWK and hHMWK; however, while the correcting activity of bHMWK was completely destroyed after sixty minutes of incubation, that of hHMWK was fully retained. These data suggest that: (1) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (2) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (3) bovine plasma kallikrein releases kinins but probably does not cause the release of fragment 1-2 from hHMWK

非麻酔下正常血圧ラットにおける腎Kallikrein-Kinin系抑制時の腎水・ナトリウム代謝

Recent findings have suggested that the endogenous renal kallikrein-kinin system acts on the renal water-sodium metabolism at the distal nephron as a paracrine hormone system. The present study was designed to determine the acting site, and secondarily, to evaluate the role of the kallikrein-kinin system on renal hemodynamics and prostaglandin E? (PGE?) production. IgG (anti-KAL-IgG) and Fab fragment (anti-KAL-Fab) were isolated from monoclonal antibody against rat glandular kallikrein ; both anti-KAL-IgG and anti-KAL-Fab were found to inhibit glandular kallikrein activity in vitro. Next, purified IgG (anti-ricin-IgG) and Fab fragment (anti-ricin-Fab) from monoclonal antibody to ricin, which is a vegetable protein and does not exist in mammals, were tested ; neither anti-ricin-IgG nor anti-ricin-Fab blocked kallkrein activity in vitro. The anti-KAL-Fab were observed to pass through the glomeruli and reach the luminal site of the distal nephron, but anti-KAL-IgG did not pass through the glomerulus. In this study, both anti-KAL-Fab and anti- KAL-IgG were administered to unanesthetized normotensive rats. When 1.0 mg anti-KAL-Fab was administered, urinary kallikrein activity (kininogenase activity) and the excretion of immunoreactive kinin were significantly decreased (-73.4±4.3% and -84.2± 5.3% change from control values, respectively). At the same time, the urine volume and urinary sodium excretion significantly decreased by 27.0±7.1% and 21.4±6.1%, respectively. In the rats receiving anti-ricin-IgG or anti-ricin-Fab, the renal function revealed no change. Bolus injection of 2.0 mg anti-KAL-Fab caused antidiuretic and antinatriuretic actions without change in GFR, renal blood flow or mean blood pressure. On the other hand, urinary PGE? fell by 39.4±18.4% following the injection of 2.0mg anti-KAL-Fab. Anti-ricin-Fab demonstrated no change in any of these parameters. The change in PGE? correlated significantly with that of urine volume (r=0.73 ; p< 0.001) and urinary sodium excretion (r=0.61 ; p<0.01). Thus, these findings show that 1) anti-KAL-Fab probably suppresses the renal kallikrein activity in the luminal site of the nephron ; 2) inhibition of kinin generation causes decreases in the water and sodium excretions in normotensive conscious rats ; 3) these renal changes are not dependent on renal hemodynamic alteration ; and 4) luminal kinin partially controls PGE? formation in the kid- neys. From these results, it was concluded that kinin on the luminal site of the distal nephron plays a role in the regulation of water and sodium excretion acting as a paracrine hormone, either directly or via PGE?