The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Satoshi Umemura - One of the best experts on this subject based on the ideXlab platform.
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expression of endothelial nitric oxide synthase is suppressed in the renal vasculature of angiotensinogen Gene Knockout mice
Cell and Tissue Research, 2006Co-Authors: Minoru Kihara, Keiko Sato, Tatsuo Hashimoto, Nozomi Imai, Yoshiyuki Toya, Satoshi UmemuraAbstract:We have attempted to elucidate the mechanism by which endothelial-type nitric oxide synthase (eNOS) is regulated in the kidney, with special reference to the role of renal hemodynamics and angiotensin II (Ang II). We compared angiotensinogen Gene Knockout (Atg−/−) mice, which lacked Ang II (resulting in sodium/water depletion and severe hypotension), with wild-type (Atg+/+) mice. Using Western blot analysis and the NADPH diaphorase histochemical reaction, we found that the expression and activity of eNOS were markedly lower in the renal vessels of Atg−/− mice compared with wild-type (Atg+/+) mice. Dietary salt loading significantly enhanced renal eNOS levels and increased blood pressure in Atg−/− mice, but severe hypotension almost abolished the effects of salt loading. In contrast, in Atg+/+ mice, altered salt intake or hydralazine had no effect on renal eNOS levels. These results suggest that perfusion pressure plays an essential role in maintaining renal vascular eNOS activity, whereas Ang II plays a supportive role, especially when renal circulation is impaired.
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alterations in renal endothelial nitric oxide synthase expression by salt diet in angiotensin type 1a receptor Gene Knockout mice
Journal of The American Society of Nephrology, 2004Co-Authors: Keiko Sato, Minoru Kihara, Tatsuo Hashimoto, Yoshiyuki Toya, K Matsushita, Yuichi Koide, Koichi Tamura, Nobuhito Hirawa, Akiyoshi Fukamizu, Satoshi UmemuraAbstract:ABSTRACT. The effects of altered dietary salt intake and/or hydralazine-induced hypotension on renal endothelial nitric oxide synthase (eNOS) expression were determined in angiotensin type-1a receptor Gene Knockout (At1a−/−) and wild-type (At1a+/+) mice. In At1a−/− mice, the levels of renal cortical eNOS mRNA and protein were 5 times and 3.5 times higher, respectively, in the high-salt (4% NaCl) group than in the low-salt group (0.3% NaCl). Systemic BP of the high-salt group (105 ± 4.4 mmHg) was significantly higher than that of the low-salt group (77.0 ± 4.7 mmHg). When hydralazine was administered to the mutant mice fed a high-salt diet, BP was reduced to 72.5 ± 1.3 mmHg, with decreases in the levels of renal eNOS mRNA and protein expression to about half of those found in nontreated group. Consistent with the results for eNOS mRNA and protein expression, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and eNOS immunoreactivity localized in the endothelium of the renal vasculature changed parallel with the amount of salt intake. In contrast to mutant mice, At1a+/+ mice did not show any changes in renal eNOS expression during the manipulation of salt intake and/or hydralazine-induced hypotension. These results suggest that At1a receptor-mediated inputs play critical roles in maintaining renal vascular eNOS expression and activity during changes in salt-water balance and systemic BP.
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expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type 1a receptor Gene Knockout mice
Kidney International, 1998Co-Authors: Minoru Kihara, Yoshiyuki Toya, Satoshi Umemura, Takeshi Sugaya, Machiko Yabana, Shunichi Kobayashi, Kovichi Tamura, Tetsuo Kadota, Reiji Kishida, Kazuo MurakamiAbstract:Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor Gene-Knockout mice. Angiotensin type-1a (AT1a) receptor Gene-Knockout ( AT1a -/- ) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a -/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a -/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a -/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a -/- mice (205.5 ± 26.1 ng/ml/hr) than in wild-type mice (8.0 ± 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin Gene expression were 12-times and 2.6-times higher in AT1a -/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a -/- mice compared with angiotensinogen-Knockout mice. When AT1a -/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a -/- mice.
Qilong Ying - One of the best experts on this subject based on the ideXlab platform.
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Generating Gene Knockout rats by homologous recombination in embryonic stem cells
Nature Protocols, 2011Co-Authors: Chang Tong, Guanyi Huang, Charles Ashton, Ping Li, Qilong YingAbstract:We describe here a detailed protocol for Generating Gene Knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of Gene-targeting vectors, Generation of Gene-targeted rat ES cells and, finally, production of Gene-targeted rats. The major differences between this protocol and the classical mouse Gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based Gene-targeting technique allows sophisticated Genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to Generate Tp53 (also known as p53) Gene Knockout rats. The entire process requires ∼1 year to complete, from derivation of ES cells to Generation of Knockout rats.
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production of p53 Gene Knockout rats by homologous recombination in embryonic stem cells
Nature, 2010Co-Authors: Chang Tong, Ping Li, Nancy L Wu, Qilong YingAbstract:The rat is a widely used animal model for studying human physiology and disease, but functional genomics and Genetic research have been stifled by the limited availability of Gene-targeting tools. Qi-Long Ying and colleagues have now established Gene targeting by homologous recombination in rat embryonic stem cells, and have Generated for the first time p53 Gene-Knockout rats, suitable for physiological and pharmacological studies of the ubiquitous tumour suppressor p53. The rat is a animal model widely used for studying human physiology and disease, but functional genomics and Genetic research have been stifled by the limited availability of Gene targeting tools. These authors have established Gene targeting by homologous recombination in rat embryonic stem cells, and have Generated p53 Gene Knockout rats for the first time. The use of homologous recombination to modify Genes in embryonic stem (ES) cells provides a powerful means to elucidate Gene function and create disease models1. Application of this technology to engineer Genes in rats has not previously been possible because of the absence of germline-competent ES cells in this species. We have recently established authentic rat ES cells2,3. Here we report the Generation of Gene Knockout rats using the ES-cell-based Gene targeting technology. We designed a targeting vector to disrupt the tumour suppressor Gene p53 (also known as Tp53) in rat ES cells by means of homologous recombination. p53 Gene-targeted rat ES cells can be routinely Generated. Furthermore, the p53 Gene-targeted mutation in the rat ES-cell genome can transmit through the germ line via ES-cell rat chimaeras to create p53 Gene Knockout rats. The rat is the most widely used animal model in biological research4,5,6,7. The establishment of Gene targeting technology in rat ES cells, in combination with advances in genomics and the vast amount of research data on physiology and pharmacology in this species, now provide a powerful new platform for the study of human disease.
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production of p53 Gene Knockout rats by homologous recombination in embryonic stem cells
Nature, 2010Co-Authors: Chang Tong, Youzhen Yan, Qilong YingAbstract:The use of homologous recombination to modify Genes in embryonic stem (ES) cells provides a powerful means to elucidate Gene function and create disease models. Application of this technology to engineer Genes in rats has not previously been possible because of the absence of germline-competent ES cells in this species. We have recently established authentic rat ES cells. Here we report the Generation of Gene Knockout rats using the ES-cell-based Gene targeting technology. We designed a targeting vector to disrupt the tumour suppressor Gene p53 (also known as Tp53) in rat ES cells by means of homologous recombination. p53 Gene-targeted rat ES cells can be routinely Generated. Furthermore, the p53 Gene-targeted mutation in the rat ES-cell genome can transmit through the germ line via ES-cell rat chimaeras to create p53 Gene Knockout rats. The rat is the most widely used animal model in biological research. The establishment of Gene targeting technology in rat ES cells, in combination with advances in genomics and the vast amount of research data on physiology and pharmacology in this species, now provide a powerful new platform for the study of human disease.
Minoru Kihara - One of the best experts on this subject based on the ideXlab platform.
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expression of endothelial nitric oxide synthase is suppressed in the renal vasculature of angiotensinogen Gene Knockout mice
Cell and Tissue Research, 2006Co-Authors: Minoru Kihara, Keiko Sato, Tatsuo Hashimoto, Nozomi Imai, Yoshiyuki Toya, Satoshi UmemuraAbstract:We have attempted to elucidate the mechanism by which endothelial-type nitric oxide synthase (eNOS) is regulated in the kidney, with special reference to the role of renal hemodynamics and angiotensin II (Ang II). We compared angiotensinogen Gene Knockout (Atg−/−) mice, which lacked Ang II (resulting in sodium/water depletion and severe hypotension), with wild-type (Atg+/+) mice. Using Western blot analysis and the NADPH diaphorase histochemical reaction, we found that the expression and activity of eNOS were markedly lower in the renal vessels of Atg−/− mice compared with wild-type (Atg+/+) mice. Dietary salt loading significantly enhanced renal eNOS levels and increased blood pressure in Atg−/− mice, but severe hypotension almost abolished the effects of salt loading. In contrast, in Atg+/+ mice, altered salt intake or hydralazine had no effect on renal eNOS levels. These results suggest that perfusion pressure plays an essential role in maintaining renal vascular eNOS activity, whereas Ang II plays a supportive role, especially when renal circulation is impaired.
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alterations in renal endothelial nitric oxide synthase expression by salt diet in angiotensin type 1a receptor Gene Knockout mice
Journal of The American Society of Nephrology, 2004Co-Authors: Keiko Sato, Minoru Kihara, Tatsuo Hashimoto, Yoshiyuki Toya, K Matsushita, Yuichi Koide, Koichi Tamura, Nobuhito Hirawa, Akiyoshi Fukamizu, Satoshi UmemuraAbstract:ABSTRACT. The effects of altered dietary salt intake and/or hydralazine-induced hypotension on renal endothelial nitric oxide synthase (eNOS) expression were determined in angiotensin type-1a receptor Gene Knockout (At1a−/−) and wild-type (At1a+/+) mice. In At1a−/− mice, the levels of renal cortical eNOS mRNA and protein were 5 times and 3.5 times higher, respectively, in the high-salt (4% NaCl) group than in the low-salt group (0.3% NaCl). Systemic BP of the high-salt group (105 ± 4.4 mmHg) was significantly higher than that of the low-salt group (77.0 ± 4.7 mmHg). When hydralazine was administered to the mutant mice fed a high-salt diet, BP was reduced to 72.5 ± 1.3 mmHg, with decreases in the levels of renal eNOS mRNA and protein expression to about half of those found in nontreated group. Consistent with the results for eNOS mRNA and protein expression, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and eNOS immunoreactivity localized in the endothelium of the renal vasculature changed parallel with the amount of salt intake. In contrast to mutant mice, At1a+/+ mice did not show any changes in renal eNOS expression during the manipulation of salt intake and/or hydralazine-induced hypotension. These results suggest that At1a receptor-mediated inputs play critical roles in maintaining renal vascular eNOS expression and activity during changes in salt-water balance and systemic BP.
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expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type 1a receptor Gene Knockout mice
Kidney International, 1998Co-Authors: Minoru Kihara, Yoshiyuki Toya, Satoshi Umemura, Takeshi Sugaya, Machiko Yabana, Shunichi Kobayashi, Kovichi Tamura, Tetsuo Kadota, Reiji Kishida, Kazuo MurakamiAbstract:Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor Gene-Knockout mice. Angiotensin type-1a (AT1a) receptor Gene-Knockout ( AT1a -/- ) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a -/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a -/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a -/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a -/- mice (205.5 ± 26.1 ng/ml/hr) than in wild-type mice (8.0 ± 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin Gene expression were 12-times and 2.6-times higher in AT1a -/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a -/- mice compared with angiotensinogen-Knockout mice. When AT1a -/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a -/- mice.
Norbert Perrimon - One of the best experts on this subject based on the ideXlab platform.
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crispr based engineering of Gene Knockout cells by homology directed insertion in polyploid drosophila s2r cells
Nature Protocols, 2020Co-Authors: Baolong Xia, Gabriel Amador, Raghuvir Viswanatha, Jonathan Zirin, Stephanie E Mohr, Norbert PerrimonAbstract:Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR Gene Knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for Genes with high copy number due to polyploidy or Gene amplification because frameshifts in all alleles can be difficult to Generate and detect. Here we describe a homology-directed insertion method to Knockout Genes in the polyploid Drosophila S2R+ cell line. This protocol allows Generation of homozygous mutant cell lines using an insertion cassette which autocatalytically Generates insertion mutations in all alleles. Knockout cells Generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number Genes.
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crispr based engineering of Gene Knockout cells by homology directed insertion in polyploid drosophila s2r cells
Nature Protocols, 2020Co-Authors: Baolong Xia, Gabriel Amador, Raghuvir Viswanatha, Jonathan Zirin, Stephanie E Mohr, Norbert PerrimonAbstract:Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR Gene Knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for Genes with high copy number due to polyploidy or Gene amplification because frameshifts in all alleles can be difficult to Generate and detect. Here we describe a homology-directed insertion method to Knockout Genes in the polyploid Drosophila S2R+ cell line. This protocol allows Generation of homozygous mutant cell lines using an insertion cassette which autocatalytically Generates insertion mutations in all alleles. Knockout cells Generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2–3 months and can be applied to other polyploid cell lines or high-copy-number Genes. This homology-directed insertion-based CRISPR Gene-editing protocol enables Knockout of all alleles of a target Gene in the polyploid Drosophila S2R+ cell line, using either two sequential rounds of homology-directed insertion or a single round with a donor vector containing four different sgRNAs.
Yoshiyuki Toya - One of the best experts on this subject based on the ideXlab platform.
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expression of endothelial nitric oxide synthase is suppressed in the renal vasculature of angiotensinogen Gene Knockout mice
Cell and Tissue Research, 2006Co-Authors: Minoru Kihara, Keiko Sato, Tatsuo Hashimoto, Nozomi Imai, Yoshiyuki Toya, Satoshi UmemuraAbstract:We have attempted to elucidate the mechanism by which endothelial-type nitric oxide synthase (eNOS) is regulated in the kidney, with special reference to the role of renal hemodynamics and angiotensin II (Ang II). We compared angiotensinogen Gene Knockout (Atg−/−) mice, which lacked Ang II (resulting in sodium/water depletion and severe hypotension), with wild-type (Atg+/+) mice. Using Western blot analysis and the NADPH diaphorase histochemical reaction, we found that the expression and activity of eNOS were markedly lower in the renal vessels of Atg−/− mice compared with wild-type (Atg+/+) mice. Dietary salt loading significantly enhanced renal eNOS levels and increased blood pressure in Atg−/− mice, but severe hypotension almost abolished the effects of salt loading. In contrast, in Atg+/+ mice, altered salt intake or hydralazine had no effect on renal eNOS levels. These results suggest that perfusion pressure plays an essential role in maintaining renal vascular eNOS activity, whereas Ang II plays a supportive role, especially when renal circulation is impaired.
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alterations in renal endothelial nitric oxide synthase expression by salt diet in angiotensin type 1a receptor Gene Knockout mice
Journal of The American Society of Nephrology, 2004Co-Authors: Keiko Sato, Minoru Kihara, Tatsuo Hashimoto, Yoshiyuki Toya, K Matsushita, Yuichi Koide, Koichi Tamura, Nobuhito Hirawa, Akiyoshi Fukamizu, Satoshi UmemuraAbstract:ABSTRACT. The effects of altered dietary salt intake and/or hydralazine-induced hypotension on renal endothelial nitric oxide synthase (eNOS) expression were determined in angiotensin type-1a receptor Gene Knockout (At1a−/−) and wild-type (At1a+/+) mice. In At1a−/− mice, the levels of renal cortical eNOS mRNA and protein were 5 times and 3.5 times higher, respectively, in the high-salt (4% NaCl) group than in the low-salt group (0.3% NaCl). Systemic BP of the high-salt group (105 ± 4.4 mmHg) was significantly higher than that of the low-salt group (77.0 ± 4.7 mmHg). When hydralazine was administered to the mutant mice fed a high-salt diet, BP was reduced to 72.5 ± 1.3 mmHg, with decreases in the levels of renal eNOS mRNA and protein expression to about half of those found in nontreated group. Consistent with the results for eNOS mRNA and protein expression, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and eNOS immunoreactivity localized in the endothelium of the renal vasculature changed parallel with the amount of salt intake. In contrast to mutant mice, At1a+/+ mice did not show any changes in renal eNOS expression during the manipulation of salt intake and/or hydralazine-induced hypotension. These results suggest that At1a receptor-mediated inputs play critical roles in maintaining renal vascular eNOS expression and activity during changes in salt-water balance and systemic BP.
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expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type 1a receptor Gene Knockout mice
Kidney International, 1998Co-Authors: Minoru Kihara, Yoshiyuki Toya, Satoshi Umemura, Takeshi Sugaya, Machiko Yabana, Shunichi Kobayashi, Kovichi Tamura, Tetsuo Kadota, Reiji Kishida, Kazuo MurakamiAbstract:Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor Gene-Knockout mice. Angiotensin type-1a (AT1a) receptor Gene-Knockout ( AT1a -/- ) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a -/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a -/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a -/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a -/- mice (205.5 ± 26.1 ng/ml/hr) than in wild-type mice (8.0 ± 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin Gene expression were 12-times and 2.6-times higher in AT1a -/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a -/- mice compared with angiotensinogen-Knockout mice. When AT1a -/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a -/- mice.
