Biomedical subjects
Publications and source records attributed to J Schnermann.
As the transgenic and gene-targeting technology has become an invaluable experimental approach to study the function of gene products, the need has been expanded to assess the physiology in the mouse, which is virtually the only animal species to which that new genetic technology can apply. In this regard, renal physiologists have also received fruits of success from modern technology in several key areas, and areas are expanding in both depth and scope.
To examine the role of aquaporin-1 (AQP1) in near-isosmolar fluid reabsorption in the proximal tubule, we compared osmolalities in micropuncture samples of late proximal tubular fluid and plasma in wild-type (+/+) and AQP1-knockout (-/-) mice. Compared with matched wild-type mice, the -/- animals produce a relatively hypotonic urine (607 +/- 42 vs. 1,856 +/- 101 mosmol/kgH(2)O) and have a higher plasma osmolality under micropuncture conditions (346 +/- 11 vs. 318 +/- 5 mosmol/kgH(2)O; P < 0.05). Measurements of tubular fluid osmolality were done in three groups of mice, +/+, -/-, and hydrated -/- mice in which plasma osmolality was reduced to 323 +/- 1 mosmol/kgH(2)O. Late proximal tubular fluid osmolalities were 309 +/- 5 (+/+, n = 21), 309 +/- 4 (-/-, n = 24), and 284 +/- 3 mosmol/kgH(2)O (hydrated -/-, n = 19). Tubular fluid chloride concentration averaged 152 +/- 1 (+/+), 154 +/- 1 (-/-), and 140 +/- 1 mM (hydrated -/-). Transtubular osmotic gradients in untreated and hydrated AQP1 -/- mice were 39 +/- 4 (n = 25) and 39 +/- 3 mosmol/kgH(2)O (n = 19), values significantly higher than in +/+ mice (12 +/- 2 mosmol/kgH(2)O; n = 24; both P < 0.001). AQP1 deficiency in mice generates marked luminal hypotonicity in proximal tubules, resulting from the retrieval of a hypertonic absorbate and indicating that near-isosmolar fluid absorption requires functional AQP1.
Experiments were performed in mice to investigate whether cyclooxygenase-2 (COX-2) in epithelial cells near the tubulovascular contact point (macula densa and TAL cells) may regulate renin gene expression in juxtaglomerular granular cells. Renin activity, afferent arteriolar granularity, and renin mRNA were determined in wild-type mice and in COX-2-knockout mice on control and low-NaCl diets. Renin activity in microdissected glomeruli assessed as angiotensin I formation in the presence of excess substrate and afferent arteriolar granularity determined by direct visualization and immunostaining were significantly reduced in COX-2 -/- compared with wild-type animals. Similarly, renal cortical mRNA levels were lower in COX-2 -/- than in wild-type mice. Maintaining mice on a low-salt diet for 14 days induced an increase in renin mRNA, afferent arteriolar granularity, and renin activity in wild-type mice. In contrast, renin mRNA and renin granularity did not significantly increase in low-salt-treated COX-2 -/- mice, whereas the increase in juxtaglomerular renin enzyme activity was markedly attenuated, but not fully blocked. In additional experiments we found that COX-2 mRNA was increased in angiotensin type 1A receptor-knockout mice compared with wild-type mice. We conclude that COX-2 in the tubulovascular contact region is a critical determinant of renin synthesis in granular cells under resting conditions and that it participates in the stimulation of renin expression caused by a low-NaCl intake.
In these experiments, we used a strain of angiotensin converting enzyme (ACE) germline null mutant mice, generated by J. H. Krege and co-workers (J. H. Krege, S. W. M. John, L. L. Langenbach, J. B. Hodgin, J. R. Hagaman, E. S. Bachman, J. C. Jennette, D. A. O'Brien, and O. Smithies. Nature 375: 146-148, 1995), to examine the effect of chronic ACE deficiency on the magnitude of tubuloglomerular feedback (TGF) responses. The genotype was determined by PCR on DNA extracted from the tail and was verified after each experiment by assessment of the blood pressure response to an injection of ANG I. To assess TGF responsiveness, we determined the change in stop-flow pressure (PSF) caused by increasing NaCl concentration at the macula densa by using micropuncture techniques. When loop of Henle flow rate was increased from 0 to 40 nl/min, PSF fell from a mean of 42.3 +/- 1.95 to 33.6 +/- 2.09 mmHg (n = 6, P = 0.005) in wild-type mice (+/+), fell from 40.6 +/- 2.35 to 38.6 +/- 1.93 mmHg in heterozygous (+/-) mice (n = 7, P = 0.014), and did not change in homozygous ACE (-/-) mice [36.7 +/- 2.02 mmHg vs. 36.4 +/- 2.01 mmHg; n = 4, P = not significant (NS)]. During an infusion of ANG II at a dose that did not significantly elevate blood pressure (70 ng. kg-1. min-1), TGF response magnitude (PSF 0 - PSF 40) increased from 6.5 +/- 1.4 to 9.8 +/- 1.19 mmHg in +/+ (P = 0.006), from 1.14 +/- 0.42 to 4.6 +/- 1.3 mmHg in +/- (P = 0.016), and from 0.42 +/- 0.25 to 4.02 +/- 1.06 in -/- mice (P = 0.05). Absence of TGF responses in ACE null mutant mice and restoration of near-normal responses during an acute infusion of ANG II supports previous conclusions that ANG II is an essential component in the signal transmission pathway that links the macula densa with the glomerular vascular pole.
The Na/H exchanger isoform 3 (NHE3) is expressed in the proximal tubule and thick ascending limb and contributes to the reabsorption of fluid and electrolytes in these segments. The contribution of NHE3 to fluid reabsorption was assessed by micropuncture in homozygous (Nhe3-/-) and heterozygous (Nhe3+/-) knockout mice, and in their wild-type (WT, Nhe3+/+) littermates. Arterial pressure was lower in the Nhe3-/- mice (89 +/- 6 mmHg) compared with Nhe3+/+ (118 +/- 4) and Nhe3+/- (108 +/- 5). Collections from proximal and distal tubules demonstrated that proximal fluid reabsorption was blunted in both Nhe3+/- and Nhe3-/- mice (WT, 4. 2 +/- 0.3; Nhe3+/-, 3.4 +/- 0.2; and Nhe3-/-, 2.6 +/- 0.3 nl/min; P < 0.05). However, distal delivery of fluid was not different among the three groups of mice (WT, 3.3 +/- 0.4 nl/min; Nhe3+/-, 3.3 +/- 0.2 nl/min; and Nhe3-/-, 3.0 +/- 0.4 nl/min; P < 0.05). In Nhe3-/- mice, this compensation was largely attributable to decreased single-nephron glomerular filtration rate (SNGFR): 10.7 +/- 0.9 nl/min in the Nhe3+/+ vs. 6.6 +/- 0.8 nl/min in the Nhe3-/-, measured distally. Proximal-distal SNGFR differences in Nhe3-/- mice indicated that much of the decrease in SNGFR was due to activation of tubuloglomerular feedback (TGF), and measurements of stop-flow pressure confirmed that TGF is intact in Nhe3-/- animals. In contrast to Nhe3-/- mice, normalization of early distal flow rate in Nhe3+/- mice was not related to decreased SNGFR (9.9 +/- 0.7 nl/min), but rather, to increased fluid reabsorption in the loop segment (Nhe3+/+, 2.6 +/- 0.2; Nhe3+/-, 3.6 +/- 0.5 nl/min). We conclude that NHE3 is a major Na/H exchanger isoform mediating Na+ and fluid reabsorption in the proximal tubule. In animals with NHE3 deficiency, normalization of fluid delivery to the distal tubule is achieved through alterations in filtration rate and/or downstream transport processes.
Previous results from our laboratory have shown that in the isolated perfused juxtaglomerular apparatus, nonselective inhibitors of cyclooxygenase (COX) activity prevent the stimulation of renin secretion by a reduction in luminal NaCl concentration at the macula densa. The present studies were performed to examine which COX isoform is involved in NaCl-dependent renin secretion. In the absence of COX inhibitors, a reduction in luminal NaCl (from Na 141/Cl 120 mM to Na 26/Cl 7 mM) caused an increase in renin secretion rate from 4.5 +/- 1.8 to 26.1 +/- 7.4 nGU/min (P < 0.01, n = 19). The presence of the COX-1 inhibitor valerylsalicylate (500 microM) in lumen and bath did not affect the stimulation of renin secretion by a reduction in luminal NaCl concentration (5 +/- 1.8 nGU/min at high NaCl, and 30.5 +/- 9.4 nGU/min at low NaCl; P < 0.01, n = 8). In contrast, the specific COX-2 inhibitor NS-398 (50 microM) in lumen and bath abolished the stimulating effect of low luminal NaCl (12.8 +/- 3.9 nGU/min at high NaCl, and 10.7 +/- 3.1 nGU/min at low NaCl; NS, n = 15). The finding that COX-2 is critically involved in macula densa control of renin secretion indicates that the COX-2-expressing epithelial cells in the tubuloglomerular contact area are a likely source of prostaglandins participating in the signaling pathway between the macula densa and renin-producing granular cells.
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Micropuncture methods have been used widely as a means to define the function of single tubules and study the functional connection between tubules and afferent arterioles (so-called tubuloglomerular feedback [TGF]). Transgenic mouse strains have become a new research tool with the potential of shedding new light on the role of specific gene products in renal tubular and vascular function. The micropuncture approach has therefore been adapted to studies in the mouse kidney. Although the data presented here support the feasibility of using this technique in the mouse, technical improvements are desirable in the areas of anesthesia, ureteral urine collections, blood collections, volume replacement, and functional stability for extended time periods. During ketamine/inactin anesthesia, TGF responses could regularly be elicited in wild-type mice. In contrast, changes in loop flow did not alter stop-flow pressure in angiotensin II type 1A receptor and angiotensin-converting enzyme knockout mice. Infusion of angiotensin II in subpressor doses partially restored TGF responsiveness in angiotensin-converting enzyme knockout animals. Normal TGF responses compared to wild type were found in nitric oxide synthase I and thromboxane receptor knockout mice. Using free-flow micropuncture techniques, the proximal-distal single-nephron GFR difference was found to be augmented in aquaporin-1 and Na/H exchanger-3 knockout mice, suggesting TGF activation in these strains of mice. These results support an essential role of angiotensin II in TGF regulation mediated through the angiotensin II type 1A receptor. Chronic nitric oxide synthase I and thromboxane receptor deficiency did not change TGF responsiveness. Aquaporin-1 and Na/H exchanger-3 deficiency enhances TGF suppression of TGF probably by volume depletion-mediated TGF sensitization. The use of micropuncture methodology in transgenic mice combines old and new research tools in a way that promises to yield important new insights into single-nephron function in physiologic and pathophysiologic conditions.
Renin secretion from isolated, perfused, thick ascending limb/glomerulus preparations from mice, under baseline conditions, has been found to be approximately 10-fold higher than that observed with the same preparations from rabbits. Higher renin secretion rates appear to be accompanied by higher plasma renin activities in mice, compared with rats, rabbits, or humans. Experiments were performed to determine the extent of the renin-angiotensin system dependence of renal hemodynamics in mice. Administration of the type 1 angiotensin II (AT1) receptor blocker candesartan (10 mg/kg) to untreated control mice increased renal blood flow by 55% (from 1.8+/-0.2 to 2.8+/-0.2 ml/min) and decreased renal vascular resistance by 42% (from 55+/-7.5 to 31.8+/-2.3 mmHg x min/ml). Similarly, acute extracellular volume expansion increased renal blood flow by 84% and reduced renal vascular resistance by 48%. In mice with null mutations in either the AT1 receptor or the angiotensin-converting enzyme gene, renal vascular resistance was significantly lower than in wild-type mice. Tubuloglomerular feedback, which is an angiotensin II-dependent vasoconstrictor response, was found to be abolished in both strains of knockout mice. Acute AT1 receptor blockade by candesartan reduced tubuloglomerular feedback responses to a flow rate step change of 0 to 30 nl/min by approximately 80% (from 6.1+/-1.4 to 1.3+/-0.4 mmHg). Candesartan increased the steady-state autoregulatory index from 0.19 to 0.55 (in a pressure interval of 90 to 100 mmHg), suggesting reduced efficiency of steady-state autoregulation. These results indicate that the renin-angiotensin system exerts tonic control over renal vascular resistance in mice to a greater extent than previously observed in other mammalian species.
To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 +/- 40 vs. 1081 +/- 68 milliosmolar). Transepithelial osmotic water permeability (Pf) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [-/-] mice was 0.033 +/- 0.005 cm/s (SE, n = 6 mice, 37 degreesC), much lower than that of 0.15 +/- 0.03 cm/s (n = 8) in tubules from wild-type [+/+] mice (P < 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 +/- 0.12 (-/-, n = 5) and 0.64 +/- 0.15 (+/+, n = 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 +/- 0. 05 (-/-, n = 8 mice [53 tubules]) and 1.95 +/- 0.09 (+/+, n = 7 mice [40 tubules]) (P < 0.001), corresponding to 26 +/- 3% [-/-] and 48 +/- 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 +/- 0.3 [-/-] and 4.4 +/- 0.5 [+/+], corresponding to 62 +/- 4% [-/-] and 76 +/- 3% [+/+] absorption (P < 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.
Luminal [NaCl] at the macula densa (MD) has two established effects: regulation of glomerular arteriolar resistance through tubuloglomerular feedback (TGF) and control of renin secretion. TGF acts as a minute-to-minute stabilizer of distal salt delivery, thereby minimizing the impact of random perturbations in filtration and absorption forces on NaCl excretion. During long-lasting perturbations of MD [NaCl], control of renin secretion becomes the dominant function of the MD. The potentially maladaptive effect of TGF under chronic conditions is prevented by TGF adaptations permitting adjustments in glomerular filtration rate to occur. TGF adaptation is mechanistically coupled to the endpoint targeted by chronic deviations in MD [NaCl], the rate of local and systemic angiotensin II generation. Studies of TGF in transgenic mice are expected to provide further insights into the mechanisms mediating between luminal [NaCl] and afferent arterioles. TGF responses are virtually abolished in mice in which either the AT1A gene or the angiotensin converting enzyme gene is rendered nonfunctional by homologous recombination. In contrast, TGF responses are unaltered in nitric oxide synthase I knockout mice. Thus, an intact renin-angiotensin system appears to be critical for the TGF signaling pathway.
Luminal NaCl concentration at the macula densa (MD) has the two established effects of regulating glomerular arteriolar resistance and renin secretion. Tubuloglomerular feedback (TGF), the inverse relationship between MD NaCl concentration and glomerular filtration rate (GFR), stabilizes distal salt delivery and thereby NaCl excretion in response to random perturbations unrelated to changes in body salt balance. Control of vasomotor tone by TGF is exerted primarily by NaCl transport-dependent changes in local adenosine concentrations. During long-lasting perturbations of MD NaCl concentration, control of renin secretion becomes the dominant function of the MD. The potentially maladaptive effect of TGF under chronic conditions is prevented by TGF adaptations, permitting adjustments in GFR to occur. TGF adaptation is mechanistically coupled to the end point targeted by chronic deviations in MD NaCl, the rate of local and systemic angiotensin II generation. MD control of renin secretion is the result of the coordinated action of local mediators that include nitric oxide synthase (NOS) and cyclooxygenase (COX) products. Thus vascular smooth muscle cell activation during high MD transport and granular cell activation during low MD transport is achieved by different extracellular mediators. The coordinated regulation of NOS I and COX-2 expression in MD cells and of renin expression in granular cells suggests that control of juxtaglomerular regulation of gene transcription or mRNA metabolism may be another consequence of a chronic alteration in MD NaCl concentration.
The effect of the adenosine type 1 receptor agonist N6-cyclohexyladenosine (CHA) on glomerular vascular reactivity was studied in male angiotensin II type 1A (AT1A) receptor knockout mice (9). Vascular reactivity was assessed as the response of stop-flow pressure (PSF) to infusion of CHA into loops of Henle using micropuncture techniques. In AT1A +/+ mice at ambient arterial blood pressure (96.7 +/- 2.8 mmHg), the presence of CHA (10 (-5) M) in the perfusate increased PSF responses from 6.8 +/- 0.6 to 14.3 +/- 0.9 mmHg when the loop of Henle of the index nephron was perfused and from 0.7 +/- 0.3 to 12.3 +/- 1.0 mmHg when the loop of an adjacent nephron was perfused. At reduced arterial blood pressure (82.8 +/- 1. 3 mmHg), index nephron perfusion with CHA increased PSF responses from 4.5 +/- 0.3 to 9.4 +/- 0.4 mmHg. In AT1A -/- mice with a mean arterial blood pressure of 80 +/- 1.9 mmHg, CHA increased PSF responses only from 0.1 +/- 0.3 to 3.6 +/- 0.54 mmHg during index nephron perfusion and from 0.25 +/- 0.2 to 2.7 +/- 0.55 mmHg during adjacent nephron perfusion, significantly less than in wild-type animals (P < 0.001). Responses to CHA were intermediate in AT1A +/- mice. Thus AT1A receptor knockout mice show a markedly reduced constrictor response to CHA both in the presence and absence of simultaneous activation of the tubuloglomerular feedback system. These data support the notion of a functional interaction between adenosine and angiotensin II in the regulation of afferent arteriolar tone.
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Experiments were performed to examine the effect of changes in dietary salt intake on the neuronal form of the constitutive nitric oxide synthase (ncNOS, type I NOS), renin, and angiotensinogen mRNA expression in the kidney. Three groups of Sprague-Dawley rats were studied as follows: rats maintained on a 3% Na diet plus 0.45% NaCl in the drinking fluid for 7 days (high salt), rats given a single injection of furosemide (2 mg/kg i.p.) and a 0.03% Na diet for 7 days (low salt), and rats on a diet containing 0.2% Na (control). mRNA expression was assessed with reverse transcription-polymerase chain reaction (RT-PCR) methods using cDNA prepared from samples of renal cortex and microdissected tubular segments. ncNOS PCR products were quantified by comparison with a dilution series of a mutant deletion template. Compared with their respective control, ncNOS mRNA levels in renal cortical tissue were elevated in rats on a low-salt diet and reduced in rats on a high-salt diet. Similar changes were seen in the expression of renin and angiotensinogen mRNA. Dietary salt intake did not alter the mRNA levels for ncNOS from the inner medulla or for endothelial constitutive NOS (ecNOS, type III NOS) and inducible NOS (iNOS, type II NOS) in the renal cortex. ncNOS mRNA was found in glomeruli dissected with the macula densa-containing segment (MDCS), but only at marginal levels in glomeruli without MDCS. Furthermore, a low-salt diet stimulated ncNOS mRNA in glomeruli with MDCS by 6.2-fold compared with a high-salt diet. There was no effect of salt diet on ncNOS mRNA in glomeruli without MDCS or in inner medullary collecting ducts. These results suggest that ncNOS expression in macula densa cells is inversely regulated by salt intake, thus following the known response of the renin-angiotensin system to changes in salt balance.
The present study was undertaken to investigate the mRNA localization of the two major kidney-specific Na-K-Cl transport proteins, the bumetanide-sensitive cotransporter (NKCC2 in rabbit and BSC1 in rat) and the thiazide-sensitive cotransporter (TSC). NKCC2 from rabbit and mouse has been shown to exist in three isoforms (designated A, B, and F) that differ only in a 96-bp region. The divergent region of each of the three NKCC2 isoforms was cloned from rat kidney by a polymerase chain reaction (PCR)-based strategy, and isoform-specific primers were chosen. RNA and cDNA were prepared from renal cortex and medulla and from microdissected nephron segments. Using reverse transcription (RT)-PCR, the B isoform was detected only in cortex and the F isoform only in medulla, whereas the A from was found in both. In dissected nephron segments, the B form was found exclusively in cortical thick ascending limb (CTAL) and macula densa-containing segment (MDCS), the F form only in medullary thick ascending limb (MTAL) and outer medullary collecting duct, and the A form in CTAL, MDCS, and MTAL. An additional isoform including both A and F exons was identified by direct sequencing of a 592-bp product from medulla. The AF product was found only in the medulla and was localized exclusively in MTAL. TSC mRNA was detected exclusively in the distal convoluted tubule. Differential nephron localization of NKCC2 isoforms suggests that Na-K-Cl cotransporters may differ in their transport characteristics to explain regulation of salt transport along the nephron.
The furosemide sensitivity of the tubuloglomerular feedback (TGF) response has suggested an important role for the Na-2Cl-K cotransporter in the mechanism by which increased luminal NaCl concentration causes afferent arteriolar vasoconstriction. The present experiments in anesthetized rats were performed to evaluate the effect of K channel blockade with Ba on TGF, since Ba has been shown to inhibit NaCl transport in the thick ascending limb. The presence of either 1.5 or 2 mM BaCl2 during retrograde perfusion with a 135 mM NaCl solution reduced the decrease of early proximal flow rate (VEP) by 2.7 +/- 0.76 (P < 0.02) and 4.2 +/- 0.8 nl/min (P < 0.01) compared with perfusion without BaCl2. Retrograde perfusion with 38 mM NaCl + 5 mM KCl reduced VEP by 10.4 +/- 1.3 nl/min, whereas 40 mM NaCl + 1.5 mM BaCl2 caused a reduction by only 6.1 +/- 1.4 nl/min (P < 0.001). In contrast to the inhibition caused by retrograde perfusion with low concentrations of BaCl2, increased vasoconstriction was seen during retrograde perfusion with 5 mM BaCl2 or during orthograde perfusion with 10 mM BaCl2. The addition of 10(-4) M furosemide to a solution containing 5 mM BaCl2 largely blocked the increased vasoconstrictor response. Peritubular perfusion with a solution containing 5 mM BaCl2 caused a fall in stop-flow pressure in an adjacent nephron by 10.7 +/- 1.5 mmHg (P < 0.001). These results indicate that under our experimental conditions Ba ions exert a dual effect on vascular responses to changes in luminal NaCl concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
The presence of NO synthase (NOS) in cells of the macula densa (MD) suggests a role for arginine-derived NO in tubulovascular information transfer. To investigate the postnatal development of the neuronal isoform of NOS and of renin in the kidney, the cellular distribution of these enzymes was examined in perfusion-fixed kidneys of 2-, 6-, and 15-day-old rats at both the protein and mRNA level (n = 4 rats/group). NOS and renin and their mRNAs were localized by immunohistochemical and in situ hybridization methods. In addition, NOS levels were assessed by using NADPH diaphorase (NADPH-d) histochemistry. For quantification, the fraction of NOS- and renin-positive glomeruli as well as the number of NOS-positive MD cells was evaluated at all stages. Presence of NOS in single cells of the developing distal tubule was encountered already in the S-shaped body. Full expression of a NOS signal in MD cells was seen as soon as a glomerular urinary space was developed. Double labeling with NADPH-d and antibody to Tamm-Horsfall protein (THP) indicated mutual exclusiveness of NADPH-d-positive MD cells and neighboring THP-positive distal tubule cells at all levels of development. The relative intensity of renin status was 2 day > 6 day > 15 day, whereas NOS expression was maximal on postnatal day 6. Our data are consistent with an involvement of MD NO synthesis in the early organization of the juxtaglomerular apparatus during nephrogenesis and suggest an interdependent relation with renin-producing cells.