Russell D Brown

Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1

While macro- and microscopic kidney development appear to proceed normally in mice that lack Foxi1, electron microscopy reveals an altered ultrastructure of cells lining the distal nephron. Northern blot analyses, cRNA in situ hybridizations, and immunohistochemistry demonstrate a complete loss of expression of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells of the collecting ducts, many of which have been implicated in hereditary forms of distal renal tubular acidosis (dRTA). In Foxi1-null mutants the normal epithelium with its two major cell types - principal and intercalated cells - has been replaced by a single cell type positive for both principal and intercalated cell markers. To test the functional consequences of these alterations, Foxi1(-/-) mice were compared with WT littermates in their response to an acidic load. This revealed an inability to acidify the urine as well as a lowered systemic buffer capacity and overt acidosis in null mutants. Thus, Foxi1(-/-) mice seem to develop dRTA due to altered cellular composition of the distal nephron epithelium, thereby denying this epithelium the proper gene expression pattern needed for maintaining adequate acid-base homeostasis.

Proinsulin C-peptide reduces diabetes-induced glomerular hyperfiltration via efferent arteriole dilation and inhibition of tubular sodium reabsorption

C-peptide reduces diabetes-induced glomerular hyperfiltration in diabetic patients and experimental animal models. However, the mechanisms mediating the beneficial effect of C-peptide remain unclear. We investigated whether altered renal afferent-efferent arteriole tonus or alterations in tubular Na+ transport (T(Na)) in response to C-peptide administration mediate the reduction of diabetes-induced glomerular hyperfiltration. Glomerular filtration rate, filtration fraction, total and cortical renal blood flow, total kidney O2 consumption (QO2), T(Na), fractional Na+ and Li+ excretions, and tubular free-flow and stop-flow pressures were measured in anesthetized adult male normoglycemic and streptozotocin-diabetic Sprague-Dawley rats. The specific effect of C-peptide on transport-dependent QO2 was investigated in vitro in freshly isolated proximal tubular cells. C-peptide reduced glomerular filtration rate (-24%), stop-flow pressure (-8%), and filtration fraction (-17%) exclusively in diabetic rats without altering renal blood flow. Diabetic rats had higher baseline T(Na) (+40%), which was reduced by C-peptide. Similarly, C-peptide increased fractional Na+ (+80%) and Li+ (+47%) excretions only in the diabetic rats. None of these parameters was affected by vehicle treatments in either group. Baseline QO2 was 37% higher in proximal tubular cells from diabetic rats than controls and was normalized by C-peptide. C-peptide had no effect on ouabain-pretreated diabetic cells from diabetic rats. C-peptide reduced diabetes-induced hyperfiltration via a net dilation of the efferent arteriole and inhibition of tubular Na+ reabsorption, both potent regulators of the glomerular net filtration pressure. These findings provide new mechanistic insight into the beneficial effects of C-peptide on diabetic kidney function.

Role of NOX2 in the regulation of afferent arteriole responsiveness

NADPH oxidases (NOX) are the major source of reactive oxygen species (ROS) in the vasculature and contribute to the control of renal perfusion. The role of NOX2 in the regulation of blood pressure and afferent arteriole responsiveness was investigated in NOX2(-/-) and wild-type mice. Arteriole constrictions to ANG II (10(-14)-10(-6) mol/l) were weaker in NOX2(-/-) compared with wild types. N(omega)-nitro-l-arginine methyl ester (l-NAME; 10(-4) mol/l) treatment reduced basal diameters significantly more in NOX2(-/-) (-18%) than in wild types (-6%) and augmented ANG II responses. Adenosine (10(-11)-10(-4) mol/l) constricted arterioles of wild types but not of NOX2(-/-). However, simultaneous inhibition of adenosine type-2 receptors induced vasoconstriction, which was stronger in NOX2(-/-). Adenosine (10(-8) mol/l) enhanced the ANG II response in wild type, but not in NOX2(-/-). This sensitizing effect by adenosine was abolished by apocynin. Chronic ANG II pretreatment (14 days) did not change the ANG II responses in NOX2(-/-), but strengthened the response in wild types. ANG II pretreatment augmented the l-NAME response in NOX2(-/-) (-33%), but not in wild types. Simultaneous application of l-NAME and ANG II caused a stronger constriction in the NOX2(-/-) (-64%) than in wild types (-46%). Basal blood pressures were similar in both genotypes, however, chronic ANG II infusion elevated blood pressure to a greater extent in wild-type (15 +/- 1%) than in NOX2(-/-) (8 +/- 1%) mice. In conclusion, NOX2 plays an important role in the control of afferent arteriole tone and is involved in the contractile responses to ANG II and/or adenosine. NOX2 can be activated by elevated ANG II and may play an important role in ANG II-induced hypertension. NOX2-derived ROS scavenges nitric oxide, causing subsequent nitric oxide-deficiency.

Hydronephrosis causes salt-sensitive hypertension in rats

Background Hypertension is a common disease in the Western world and approximately 5% of all cases are secondary to kidney malfunction. It is not clear whether unilateral hydronephrosis due to partial obstruction affects blood pressure. Aim The aim of this study was to determine whether hypertension develops and to investigate the effects of different salt diets on the blood pressure in hydronephrotic animals. Methods Unilateral partial ureteral obstruction was created in 3-week-old Sprague–Dawley rats. A telemetric device was implanted 4–6 weeks later and blood pressure was measured on normal, low- and high-salt diets. Plasma samples were collected on all diets for renin analysis. Results All hydronephrotic animals developed hypertension that correlated to the degree of hydronephrosis. The blood pressure increased slowly with time and was salt sensitive. In severe hydronephrosis, blood pressure increased from 118 ± 5 mmHg on low salt to 140 ± 6 mmHg on high salt intake, compared to control levels of 82 ± 2 and 84 ± 2 mmHg, respectively. Plasma renin concentration was increased in the hydronephrotic group of animals compared to controls on all diets, but the difference was only significant on a normal salt diet, 165 ± 15 versus 86 ± 12 μGU/ml respectively. In animals with severe hydronephrosis the plasma renin levels were lower, and the changes less, than in those with mild and moderate hydronephrosis. Conclusion This study demonstrates the presence of a salt-sensitive hypertension in hydronephrosis. A systemic effect of the renin–angiotensin system alone cannot be responsible for the hypertension.

SOD1 deficiency causes salt sensitivity and aggravates hypertension in hydronephrosis

Hydronephrosis causes renal dysfunction and salt-sensitive hypertension, which is associated with nitric oxide deficiency and abnormal tubuloglomerular feedback (TGF) response. We investigated the role of oxidative stress for salt sensitivity and for hypertension in hydronephrosis. Hydronephrosis was induced in superoxide dismutase 1-transgenic (SOD1-tg), SOD1-deficient (SOD1-ko), and wild-type mice and in rats. In mice, telemetric measurements were performed during normal (0.7% NaCl) and high-sodium (4% NaCl) diets and with chronic tempol supplementation. The 8-iso-prostaglandin-F(2alpha) (F2-IsoPs) and protein excretion profiles and renal histology were investigated. The acute effects of tempol on blood pressure and TGF were studied in rats. In hydronephrosis, wild-type mice developed salt-sensitive hypertension (114 +/- 1 to 120 +/- 2 mmHg), which was augmented in SOD1-ko (125 +/- 3 to 135 +/- 4 mmHg) but abolished in SOD1-tg (109 +/- 3 to 108 +/- 3 mmHg). SOD1-ko controls displayed salt-sensitive blood pressure (108 +/- 1 to 115 +/- 2 mmHg), which was not found in wild types or SOD1-tg. Chronic tempol treatment reduced blood pressure in SOD1-ko controls (-7 mmHg) and in hydronephrotic wild-type (-8 mmHg) and SOD1-ko mice (-16 mmHg), but had no effect on blood pressure in wild-type or SOD1-tg controls. SOD1-ko controls and hydronephrotic wild-type and SOD1-ko mice exhibited increased fluid excretion associated with increased F2-IsoPs and protein excretion. The renal histopathological changes found in hydronephrotic wild-type were augmented in SOD1-ko and diminished in SOD-tg mice. Tempol attenuated blood pressure and normalized TGF response in hydronephrosis [DeltaP(SF): 15.2 +/- 1.2 to 9.1 +/- 0.6 mmHg, turning point: 14.3 +/- 0.8 to 19.7 +/- 1.4 nl/min]. Oxidative stress due to SOD1 deficiency causes salt sensitivity and plays a pivotal role for the development of hypertension in hydronephrosis. Increased superoxide formation may enhance TGF response and thereby contribute to hypertension.

Mechanisms of neonatal increase in glomerular filtration rate

To investigate the mechanisms responsible for the neonatal increase in glomerular filtration rate (GFR), renal function studies (whole kidney and micropuncture) were carried out in anesthesized fetal sheep (133-140 days gestation; term = 150 days) and lambs (12-18 days). Fetuses were delivered and placed in a water bath (39.5 degrees C), keeping the umbilical cord moist and intact. Lambs were studied on a thermostatically controlled heating pad. Animals were prepared for either blood flow studies or micropuncture measurements. Expected differences in blood composition and cardiovascular and renal function were observed between fetuses and lambs, and values obtained for most variables were similar to those measured in chronically catheterized unanesthetized animals. Fetal GFR was much lower than that of lambs (0.20 vs. 0.62 ml.min(-1).g kidney(-1), P < 0.001). Free-flow, stop-flow, and net filtration pressures (NFP) were lower in the fetuses than the lambs (NFP 20.8 vs. 23.8 mmHg, P < 0.001), as was the calculated ultrafiltration coefficient (0.014 vs. 0.022 ml.min(-1).g(-1).mmHg(-1), P < 0.001). Thus, we conclude that rises in both net filtration pressure and the ultrafiltration coefficient contribute to the large increase in GFR between fetal life and approximately 2 wk after birth.

Tubuloglomerular feedback response in the prenatal and postnatal ovine kidney

The tubuloglomerular feedback mechanism (TGF) plays an important role in regulating single-nephron glomerular filtration rate (GFR) by coupling distal tubular flow to arteriolar tone. It is not known whether TGF is active in the developing kidney or whether it can regulate renal vascular tone and thus GFR during intrauterine life. TGF characteristics were examined in late-gestation ovine fetuses and lambs under normovolemic and volume-expanded (VE) conditions. Lambs and pregnant ewes were anesthetized and the fetuses were delivered via a caesarean incision into a heated water bath, with the umbilical cord intact. Under normovolemic conditions, mean arterial pressure of the fetuses was lower than lambs (51 ± 1 vs. 64 ± 3 mmHg). The maximum TGF response (ΔP(SFmax)) was found to be lower in fetuses than lambs when tubular perfusion was increased from 0 to 40 nl/min (5.4 ± 0.7 vs. 10.6 ± 0.4 mmHg). Furthermore, the flow rate eliciting half-maximal response [turning point (TP)] was 15.7 ± 0.9 nl/min in fetuses compared with 19.3 ± 1.0 nl/min in lambs, indicating a greater TGF sensitivity of the prenatal kidney. VE decreased ΔP(SFmax) (4.2 ± 0.4 mmHg) and increased TP to 23.7 ± 1.3 nl/min in lambs. In fetuses, VE increased stop-flow pressure from 26.6 ± 1.5 to 30.3 ± 0.8 mmHg, and reset TGF sensitivity so that TP increased to 21.3 ± 0.7 nl/min, but it had no effect on ΔP(SFmax). This study provides direct evidence that the TGF mechanism is active during fetal life and responds to physiological stimuli. Moreover, reductions in TGF sensitivity may contribute to the increase in GFR at birth.

Fetal tubuloglomerular feedback in an ovine model of mild maternal renal disease

Fetuses of pregnant ewes, which were subtotally nephrectomized prior to mating, were studied to assess whether mild maternal renal impairment would affect fetal tubuloglomerular feedback (TGF) under control conditions and after the inhibition of macula densa‐derived nitric oxide (NO). Based on previous observations we hypothesized that, the TGF curve of fetuses of subtotally nephrectomized (STNx) ewes would resemble that of a volume expanded fetus with a high production rate of NO and that inhibition of neuronal nitric oxide synthase (nNOS) would increase the sensitivity of the TGF system in these fetuses. Renal function studies were performed on anaesthetized fetal sheep (133–140 days gestation; term ~150 days; Isoflurane 2–4% in oxygen). Fetuses were removed from the uterus and placed in a water bath (39.5°C) while maintaining umbilical blood flow. Glomerular filtration rate (GFR) and urine flow rate were markedly increased in fetuses of STNx ewes compared to fetuses of untreated ewes. Interestingly, and contrary to our hypothesis, the fetuses of STNx ewes exhibited no difference in TGF sensitivity in the presence or absence of 7‐nitroindazole (7NI; nNOS inhibitor), compared to fetuses of untreated ewes, although sensitivity and reactivity increased in both groups after 7NI. There was however, a decrease in the stop flow pressure and net filtration pressure with an increase in the filtration coefficient (Kf). These factors suggest that maternal renal impairment drives the glomerular hypertrophy which has previously been found to be present in the neonatal period. Thus, we conclude that at ~138 days gestation, the fetal kidney has matured functionally and fetuses of STNx ewes are able to maintain fluid and electrolyte homeostasis even in the presence of increased transplacental flux.

Tubuloglomerular feedback responses in offspring of dexamethasone-treated ewes

Via developmental programming, prenatal perturbations, such as exposure to glucocorticoids and maternal malnutrition alter kidney development and contribute to the development of hypertension. To examine the possibility that alterations in tubuloglomerular feedback (TGF) contribute to the development of hypertension in offspring following maternal dexamethasone treatment (Dex) in early gestation, studies were conducted in fetal sheep and lambs. Pregnant ewes were infused with dexamethasone (0.48 mg/h) at 26-28 days gestation. No differences were observed in mean arterial pressure, glomerular filtration rate. or electrolyte excretion rates between the Dex and Untreated fetuses or lambs. Gestational exposure to Dex markedly enhanced TGF sensitivity, as the turning point in Dex-treated fetuses was significantly lower (12.9 ± 0.9 nl/min; P < 0.05) compared with Untreated fetuses (17.0 ± 1.0 nl/min). This resetting of TGF sensitivity persisted after birth (P < 0.01). TGF reactivity did not differ between the groups in fetuses or lambs. In response to nitric oxide inhibition, TGF sensitivity increased (the turning point decreased) and reactivity increased in Untreated fetuses and lambs, but these effects were blunted in the Dex-treated fetuses and lambs. Our data suggest that an altered TGF response may be an underlying renal mechanism contributing to the development of hypertension in the Dex model of fetal programming. The lower tonic level of NO production in these dexamethasone-exposed offspring may contribute to the development of hypertension as adults.

1008 ANGIOTENSIN TYPE-2 RECEPTOR CONTRIBUTES TO REDUCED VASCULAR RESPONSE TO ANGIOTENSIN-II IN YOUNG RATS

Background: The angiotensin type-2 receptor (AT2R) is known to oppose the vasoconstrictive actions of angiotensin II (AngII). During fetal development AT2R expression is very high, which upon maturation regresses. The present study investigates the sensitivity to acute AngII in young rats and the contribution of the AT2R in attenuating the vasoconstrictor response to AngII. Methods: Male Sprague-Dawley rats at 3 and 6 weeks of age were anesthetized. Mean arterial pressure (MAP) and renal blood flow (RBF; transonic flowmeter), was measured in response to i.v. administration of AngII at 0 (basal) 10 (10AngII) and 100ng/kg/hr (100AngII). The AT2R inhibitor, PD-123319, was given and responses to 100AngII were repeated. Results: In the 6wk rats, 10 and 100AngII increased MAP (89 ± 4 mmHg) by 10 ± 7 and 18 ± 5 mmHg, respectively. Likewise, RBF (4.0 ± 0.6 ml/min/gkw) was reduced by 8 ± 3% and 34 ± 3%. In the 3wk rats basal MAP and RBF were 72 ± 4 mmHg and 2.9 ± 0.3 ml/min/gkwt, The 10AngII dose had no effect on MAP, while 100AngII increased MAP by 6 ± 2 mmHg. RBF in the 3wk rats was not was not affected by the 10ng AngII dose (-2 ± 1% change), while the higher dose produced a 10 ± 3% decrease. PD-123319 potentiated the change in RBF to 100AngII in the 3wk animals (23 ± 4% reduction), but had no additional effect in the 6wk rats. Conclusions: These data indicate that during development, the presence of the AT2R in young rodents contributes to the attenuated vasoconstrictor response to AngII. These findings may have implication for the use of drugs that inhibit the renin-angiotensin system for the treatment of pediatric hypertension and renal failure.