NEPHRIN EXPRESSION IN THE POST-NATAL DEVELOPING KIDNEY IN NORMOTENSIVE AND HYPERTENSIVE RATS

Nephrin is a slit diaphragm protein and its expression in the developing kidney is largely unknown. In this study, we explored the expression of nephrin in the developmental kidney in spontaneously hypertensive (SHR) and in Wistar-Kyoto (WKY) rats at different time points, from day 5 after birth to adulthood. Real time RT-PCR, in situ hybridization and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin in the kidney. SHR had hypertension at week 10 and albuminuria at week 20. Nephrin expression in both SHR and WKY increased from day 5 to adulthood. Furthermore, both gene and protein expression of nephrin were significantly lower in SHR after birth when compared to WKY at the same age. These findings suggest that both in normotensive and hypertensive rats, nephrin expression increased from birth to the adult age and that down-regulation of nephrin in SHR evident from the early developmental kidney to adulthood may contribute to the development of albuminuria in adult SHR.     

Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension

Aims/hypothesis. The location of nephrin has been identified as the slit-diaphragm of the glomerular podocyte. Recent evidence suggests that nephrin could play a key role in the function of the glomerular filtration barrier and the development of proteinuria but its status in long-term diabetes is still not understood. We studied the expression of nephrin in a hypertensive model of diabetic nephropathy and investigated the potential influence of angiotensin II blockade on nephrin gene and protein expression. Methods. Streptozotocin-diabetic spontaneously hypertensive rats were given either no treatment or the angiotensin II antagonist, irbesartan, at a dose of 15 mg/kg per day by gavage for 32 weeks. Non-diabetic spontaneously hypertensive rats were used as a control group. Real time RT-PCR and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin. Results. Diabetic spontaneously hypertensive rats developed albuminuria and had a reduction in both gene and protein expression of nephrin when compared with control rats. Irbesartan treatment prevented the development of albuminuria and completely abrogated the down regulation of nephrin in diabetic rats. Conclusion/interpretation. Long-term diabetes in spontaneously hypertensive rats is associated with a reduction in both gene and protein expression of nephrin within the kidney. These changes in nephrin levels were completely prevented by angiotensin II antagonist treatment, suggesting a potential novel mechanism to explain the antiproteinuric effect of agents which interrupt the renin-angiotensin system. [Diabetologia (2001) 44: 874–877] Received: 19 January 2001 and in revised form: 16 March 2001     

Kidney renin gene expression in spontaneously hypertensive rats

We studied the expression of kidney renin gene in hypertensive animals by measuring the kidney renin messenger (m) RNA. The kidney renin mRNA was quantified by densitometric Northern blot analysis using a 32P-labelled rat renin genomic DNA fragment as a hybridization probe. Spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) were treated with a low-sodium diet plus furosemide, captopril or propranolol for a week. Plasma renin activity (PRA) in SHR and WKY was increased similarly by sodium depletion and by treatment with captopril. PRA in both strains was not decreased significantly by treatment with propranolol. Both sodium depletion and captopril treatment caused significant increases in the kidney renin mRNA in SHR and WKY. However, the increases in the kidney renin mRNA of SHR were greater than those in the corresponding WKY (SHR, 10.0- and 22.1-fold increases; WKY, 6.2- and 7.8-fold increases, respectively). Propranolol had no effect on the kidney renin gene expression in either WKY or SHR. These results indicate that SHR show an enhanced expression of the renin gene in the kidney compared with WKY in response to stimuli that increase renin release.     

Expression of the Na+/H+ exchanger regulatory protein family in genetically hypertensive rats

OBJECTIVE: To examine a possible involvement of a regulatory protein of Na+/H+ exchanger (NHE) in the increased renal NHE activity in spontaneously hypertensive rats (SHR), we investigated mRNA expression of inhibitory members of the NHE regulatory protein family, NHERF1 and NHERF2, in the kidney. DESIGN: Prehypertensive 4-week-old and hypertensive 11-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used to determine the changes in NHE activity and NHERF family expression in the kidney. Dahl salt sensitive (DS) and resistant rats were also used to examine whether these changes are specific for SHR. METHODS: mRNA expression in the kidney was quantified by RNase protection assay. The NHE activity in primary cultured proximal tubular cells was measured as Na-dependent pHi recovery rate by the NH4Cl prepulse technique with 2'7'-bis-(2-carboxyethyl)-5.6-carboxyfluorescein (BCECF). RESULTS: NHERF1 mRNA expression was significantly decreased in both prehypertensive and hypertensive SHR in comparison with age-matched WKY rats, whereas NHERF2 mRNA expression was significantly increased in SHR only in the hypertensive period. Antihypertensive treatment did not abolish these changes seen in control SHR. On the other hand, hypertensive DS rats fed a high-salt diet showed significant decreases in NHE activity and NHE3 mRNA expression compared with normotensive DS rats fed a low-salt diet, without significant changes in NHERF1 and NHERF2 mRNA expression. CONCLUSION: These results suggest that decreased expression of NHERF1 may be related to the enhanced NHE activity in SHR and that these changes are likely to be genetically determined, whereas the increased NHERF2 expression may be induced as a compensatory mechanism.     

Renal renin-angiotensin system activity in naturally reared and cross-fostered spontaneously hypertensive rats

BACKGROUND: Young (4 week) spontaneously hypertensive rats (SHR) exhibit greater renal responses to angiotensin II (Ang II) than normotensive Wistar Kyoto (WKY) rats. SHR pups cross-fostering to a WKY dam at birth (SHRX) are less sensitive to Ang II and have lower adult blood pressure. The aim of this study was to compare renal renin-angiotensin system activity in young naturally reared and cross-fostered SHR pups. METHODS: SHR and WKY rats were reared either by their natural mothers or by a foster mother of the opposite strain. At 5, 10, and 15 days of age, renal tissue renin activity and Ang II concentration were measured by radioimmunoassay. Renin-secreting cells were identified by in situ hybridization and AT(1) receptor expression was compared using Western blots. Ang II-mediated cAMP generation was measured in isolated proximal tubules. CONCLUSIONS: Tissue renin activity and numbers of renin-secreting cells did not differ, but Ang II was higher in SHRX. The AT(1) receptor expression was significantly lower in SHRX compared with SHR. Basal and Ang II-stimulated cAMP was lower in SHR tubules compared with WKY and SHRX tubules.Cross-fostering reversed the increased renal sensitivity of the SHR to Ang II. These data suggest that renal AT(1) receptor expression can be manipulated during the postnatal period and that this may affect adult blood pressure.     

通心络对高血压大鼠血管内皮功能的保护作用

目的探讨通心络对高血压大鼠血管内皮的保护作用。方法实验动物分为3组,分别为自发性高血压大鼠组(SHR组),SHR服用通心络组(TXL组)和正常血压大鼠组(WKY组)。通过放射免疫及聚合酶链反应检测主动脉一氧化氮(NO)、内皮素(ET)、血管内皮生长因子(VEGF)基因表达。结果SHR组NO水平低于WKY组,TXL组NO水平高于SHR组。SHR组ET-1含量及mRNA均高于WKY组,TXL组ET-1含量及mRNA表达低于SHR组,SHR组及TXL组mRNA表达均高于WKY组,以SHR组为最高。结论通心络可能对高血压大鼠血管内皮功能有保护作用。     

Vascular Pressure-Flow Analysis in Normal and Hypoxemic Spontaneously Hypertensive Rats

The pressure-flowurelationship of the autoperfused subclavian vascular bed was compared in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after spinal cord transection. Studies were performed under normoxemic and hypoxemic conditions. In adult SHR, vascular resistance was greater relative to WKY under both conditions. In young (8 week old) SHR vascular resistance was consistently greater over a wide range of perfusion pressures compared to young WKY rats when blood oxygen content was normal. Vascular resistance was not different between young SHR and WKY when the animals were hypoxemic. The results demonstrated that elevated vascular resistance in adult SHR was independent of oxygen availability and supraspinal nerve function; however, in young SHR elevated vascular resistance was dependent upon oxygen availability, although independent of supraspinal nerve function.     

Role of oxidative stress in the sex differences in blood pressure in spontaneously hypertensive rats

OBJECTIVE: The hypothesis was tested that differences in oxidative stress play a role in the sex differences in the development and maintenance of hypertension in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: Male and female SHR [and Wistar-Kyoto (WKY) rats in the long-term study] (n = 6-12 per group) received tempol (30 mg/kg per day) or tap water for 6 weeks from 9 to 15 weeks of age or from birth until 15 weeks of age. Blood pressure [mean arterial pressure (MAP)] and kidney tissue F2-isoprostane (IsoP) were measured at 15 weeks of age. RESULTS: In SHR given tempol for 6 weeks, blood pressure and IsoP were reduced in males, but not in females. In SHR given tempol from birth, MAP was higher in SHR than WKY rats (SHR males, 181 +/- 2 mmHg; SHR females, 172 +/- 3 mmHg; WKY males, 100 +/- 2 mmHg; WKY females, 101 +/- 2 mmHg, P < 0.01), and tempol reduced MAP by 14% (156 +/- 3) and 26% (127 +/- 4) in male and female SHR, respectively, but had no effect on WKY rats. IsoP was higher in SHR than WKY rats and higher in male SHR than female SHR (SHR males, 5.18 +/- 0.23 ng/mg; SHR females, 3.71 +/- 0.19 ng/mg, P < 0.01; WKY males, 1.72 +/- 0.45 ng/mg; WKY females, 2.21 +/- 0.08 ng/mg, P < 0.05, compared with SHR). Tempol reduced IsoP in SHR to levels found in WKY rats, but had no effect on IsoP in WKY rats. CONCLUSIONS: Development of hypertension in SHR is mediated in part by oxidative stress independent of sex. Also, tempol is effective in reducing blood pressure in females only when given prior to the onset of hypertension.     

自发性高血压大鼠肾脏血管紧张素转换酶2 mRNA转录及其蛋白表达

目的观察不同月龄自发性高血压大鼠(SHR)肾脏血管紧张素转换酶2(ACE2) mRNA转录及其蛋白表达,初步探讨ACE2在高血压发生、发展过程中的可能作用。方法雄性SHR1月龄组(S1)、2月龄组(S2)、3月龄组(S3)、6月龄组(S6)和9月龄组(S9)共5组,每组各6只,各组均有相应月龄匹配的Wistar-Kyoto(WKY)大鼠作对照。采用RBP-Ⅰ型大鼠血压心率测定仪测量大鼠尾动脉收缩压(SBP);逆转录聚合酶链式反应(RT-PCR)法检测肾脏ACE2 mRNA的转录水平;免疫组化染色结合计算机图像分析方法测定肾脏ACE2蛋白的表达水平。结果1)SHR的SBP随着月龄的增加而上升,6月龄后趋于稳定。2)SHR和WKY肾脏ACE2蛋白和 mRNA水平均随着月份的增加而增加,3月龄时达高峰,6月龄后趋于稳定;且SHR肾脏ACE2蛋白和 mRNA水平均低于同龄的WKY。S1肾脏髓质内侧部ACE2免疫染色阳性面积百分比较皮质和髓质外侧部高,与1月后的分布相反。结论1)SHR肾脏ACE2 mRNA和蛋白的表达水平比WKY大鼠低。2)大鼠肾脏ACE2 mRNA和蛋白的表达具有时间和部位分布上的差异。     

Role of prostanoids in the increased vascular responsiveness and delayed tachyphylaxis to serotonin in the kidney of spontaneously hypertensive rats.

The isolated and perfused kidney of the spontaneously hypertensive rat (SHR) exhibits an increased vascular reactivity and a delayed tachyphylaxis to serotonin (5-hydroxytryptamine) when compared with normotensive Wistar-Kyoto (WKY) rats. Experiments were designed to determine the involvement of products of cyclooxygenase in the augmented response and delayed tachyphylaxis to serotonin in the SHR kidney. Kidneys taken from male, 4-month-old SHR and WKY rats were studied in parallel and perfused with Tyrode's solution at constant, optimal flow rates. Vasoconstrictor responses were recorded as increases in perfusion pressure. The vasoconstrictor responses to serotonin, norepinephrine and angiotensin II were exaggerated in the SHR kidney compared with that of the WKY rat. Indomethacin did not affect the responsiveness to serotonin in the kidney of the SHR but increased the responses to the higher doses of the monoamine in the kidney of the WKY rats. Indomethacin accelerated the tachyphylaxis to serotonin in the SHR but delayed it in the WKY rats. Dazoxiben did not alter the responses to serotonin in the SHR. Responses to norepinephrine in the kidneys from both strains were not affected by indomethacin. The inhibitor of cyclooxygenase reduced the responses to angiotensin II in the kidneys from both hypertensive and normotensive animals. The basal and stimulated (serotonin, norepinephrine and angiotensin II) release of prostaglandins were measured by radioimmunoassay. The basal release of prostacyclin was lower, but that of thromboxane A2 higher, in the kidneys of SHR compared with those of WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced DNA synthesis in heart and kidney of newborn spontaneously hypertensive rats

Studies comparing relative organ weight (per gram of body weight) in spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto rats (WKY), have shown that the heart and kidney of SHR are enlarged at birth. In addition, higher DNA concentrations in these organs provide evidence of cardiac and renal cellular hyperplasia in newborn SHR. The rate of DNA synthesis was estimated with [methyl-3H]thymidine labeling in vivo for heart, kidney, liver, adrenals, and aorta of newborn WKY and SHR, and significantly higher relative organ weights, protein levels, and DNA contents were observed only for heart and kidney from SHR. In contrast, relative weight was significantly lower for liver from SHR and the same for adrenals from WKY and SHR despite lower adrenal protein and DNA contents for SHR. Relative weight and protein content of aorta were higher in SHR, possibly indicating hypertrophy of this organ. Several differences in [methyl-3H]thymidine labeling were observed in organs from SHR. Significantly higher organ accumulation (cpm/mg organ weight) was observed for heart from SHR (p less than 0.01), and higher incorporation into DNA (cpm/cpm) was observed for kidney and liver from SHR (p less than 0.001). Most significant was the finding of higher DNA specific activity for heart (+33%; p less than 0.05) and kidney (+39%; p less than 0.001) from SHR. These data provide evidence of enhanced DNA synthesis in the heart and kidney of newborn SHR.     

Ventricular cells in culture from adult spontaneously hypertensive rats exhibit decreased growth

We have investigated the growth characteristics of adult rat ventricular cells (ARVC) in culture from 8-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Protein synthesis in ARVC estimated from tritiated leucine uptake was augmented in both strains after exposure of cells to either norepinephrine or fetal bovine serum (FBS), but the response was significantly smaller in SHR than in WKY. A relative increase in the protein content of ARVC incubated with norepinephrine or FBS was also smaller in SHR than in WKY. These results demonstrate that SHR cardiac myocytes are not genetically growth-accelerated and show a decreased growth response to growth stimuli such as norepinephrine and serum during the adult period.     

Enhanced depressor response to endothelial nitric oxide synthase gene transfer into the nucleus tractus solitarii of spontaneously hypertensive rats.

Previously, we demonstrated that endothelial nitric oxide synthase (eNOS) gene transfer into the nucleus tractus solitarii (NTS) decreased blood pressure, heart rate and sympathetic nerve activity in conscious normotensive Wistar-Kyoto rats (WKY). In order to determine whether overexpression of eNOS in the NTS causes different effects on blood pressure and heart rate between spontaneously hypertensive rats (SHR) and WKY, we transfected adenovirus vectors encoding either eNOS (AdeNOS) or beta-galactosidase (Ad beta gal) into the NTS of SHR and WKY in vivo. The local expression of eNOS in the NTS was confirmed by Western blot analysis for eNOS protein, and the magnitude of expression did not differ between SHR and WKY. Blood pressure and heart rate were monitored by the use of a radio-telemetry system in a conscious state before and 7 days after the gene transfer. Systolic blood pressure (SBP) and heart rate decreased on day 7 in both AdeNOS-transfected SHR and WKY. However, the magnitude of decreases in SBP of AdeNOS-transfected SHR was greater than that of AdeNOS-transfected WKY (-24.1 +/- 2.9 vs. -15.9 +/- 2.1 mmHg, p < 0.05). Transfection of Ad beta gal into the NTS did not alter SBP in either group. A depressor response evoked by microinjection of L-glutamate into the NTS did not differ between the two strains. These results suggest that overexpression of eNOS in the NTS causes a greater depressor response in SHR than in WKY in a conscious state. An abnormality of the L-arginine-NO pathway in the NTS may be related to the hypertensive mechanism(s) of SHR.     

Age dependent changes in myosin ATPase activity in the myocardium of spontaneously hypertensive rats

Male spontaneously hypertensive rats (SHR) and age matched Wistar Kyoto normotensive (WKY) rats of 5 weeks, 16 weeks, and 52 weeks of age were used to determine whether duration of hypertension has any effect on contractile protein ATPase and myosin isoenzyme distribution. Myofibrils, actomyosin, and myosin were isolated from the left ventricles of WKY rats and SHR and assayed for myosin ATPase activity and myosin isoenzyme distribution. Myofibrillar ATPase activity was assayed at various free [Ca++] ranging from 10(-7) to 10(-5) mol X litre-1. Ca++ stimulated actomyosin ATPase activity was determined at several Ca++ concentrations both at low ionic strength, which favours actin-myosin interaction, and at high ionic strength, which diminishes actin interaction with myosin. Purified myosin ATPase activity was assayed in the presence of K+-EDTA and in the presence of several concentrations of Ca++. Actin activated myosin ATPase activity was assayed using 26 mumol X litre-1 skeletal muscle actin. Under all these assay conditions no differences were observed in the contractile protein ATPase activity between SHR and WKY rats in any age group. On the other hand, in both SHR and WKY rats the contractile protein ATPase activity under all assay conditions was significantly decreased in 52 week old rats compared with 5 week old rats. The predominant myosin isoenzyme was Vi in 5 week and 16 week old WKY rats and SHR. In 52 week old WKY rats and SHR, however, significant amounts of isoenzymes V2 and V3 were present along with V1. Percentage distribution of V1, V2, V3 isoenzymes calculated from densitometric scans of gels did not show any differences between WKY rats and SHR in any age group. These results suggest that neither myosin ATPase activity nor myosin isoenzyme distribution is altered in the moderately hypertrophied left ventricles of SHR. Moreover, the data indicate that the myocardium of SHR, despite the persistence of pressure overload, undergoes a similar decrease in myosin ATPase activity and an increase in myosin isoenzyme V3 to age matched normotensive WKY rats.