Development of the kidney vasculature.

Renal vascularization and nephrogenesis occur simultaneously following a tightly regulated developmental program influenced by growth factors, extracellular matrix components and cell membrane receptors. Both processes of angiogenesis and vasculogenesis probably participate in the formation of renal vessels. The origin and fate of the various renal vascular cells and the molecular mechanisms that initiate and guide intrarenal vascularization are fundamental questions that remain to be answered.

Oxygen regulates vascular endothelial growth factor-mediated vasculogenesis and tubulogenesis.

To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O2) or low oxygen (1-3% O2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O2-treated explants (2.5 +/- 0.17 microgram/kidney, n = 9) than in 20% O2 explants (1.5 +/- 0.09 microgram/kidney, n = 9), P < 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2-treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2-treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.

Developmental regulation of angiotensin type 1 and 2 receptor gene expression and heart growth.

Little is known regarding the developmental regulation of the cardiac angiotensin type 1 (AT1) and type 2 (AT2) receptor genes or their role in normal cardiac growth. Regulation of AT1 and AT2 receptor genes were examined using total and poly A + RNA isolated from whole Sprague-Dawley rat hearts. AT1 mRNA levels were 3.5-fold higher in the 19-day-old fetal heart compared to the 90-day-old adult as detected with 2 or 5 microg of poly A + RNA. AT2 mRNA was only detectable with 20 microg of poly A + RNA. AT2 mRNA levels were highest in the 19-day-old fetal heart with no detectable message in the 90-day-old adult heart. Qualitative PCR for AT2 mRNA also could not detect AT2 mRNA in the adult heart. Treatment with the AT1 receptor antagonist losartan for 3 weeks in the 21-day-old rat or for 4 days in the 38-day-old rat resulted in a significant decrease in heart/body weight in both groups and body weight in the 3-week treatment group. AT2 blockade for 4 days with PD123319 or beta-receptor blockade with propranolol for 3 weeks did not alter heart/body weights. Losartan treatment also resulted in a three-fold increase in cardiac AT1 mRNA levels in both the 4-day and 3-week treatment groups compared to controls. We conclude that Ang II, acting primarily, if not exclusively via the AT1 receptor plays a significant role in the regulation of normal cardiac growth in the young rat.

Accumulation of acidic renin isoforms in kidneys of cyclosporine-A-treated rats.

Chronic cyclosporin A (CsA) treatment results in major hemodynamic changes in the renal microvasculature and in expression of the intrarenal renin angiotensin system. Changes in renin expression in kidneys of CsA-treated rats include the recruitment of immunoreactive renin in afferent arterioles and in the juxtaglomerular apparatus. This study presents evidence that an acidic isoform of renin is increased in kidneys of CsA-treated rats. Immunoblots of rat kidney homogenate separated by polyacrylamide-gel electrophoresis and also by isoelectric focusing demonstrate the presence of an acidic isoform (pl 5.5 and estimated molecular weight of approximately 32 to 36 kd) seen in increased amounts in kidney homogenate from CsA-treated rats. Silver-stained two-dimensional gels of renin separated from kidney homogenate with pepstatin agarose confirm the presence of an acidic renin isoform in CsA-treated rats. In rats that received CsA for varied intervals of 1, 3, 5, and 8 wk, this acidic isoform is shown to significantly accumulate relative to duration of treatment with CsA when immunoreactive bands are analyzed by densitometric scanning (r2 = 0.90, P < 0.001). Renin enzymatic activity also increased in kidney homogenate of CsA-treated rats relative to duration of treatment with CsA (r2 = 0.486, P < 0.001). Prorenin in these same samples was significantly decreased compared with controls. The acidic renin isoform identified in kidney homogenate of CsA-treated rats may be involved in the vascular changes that are seen in this model.

Angiotensin II regulates nephrogenesis and renal vascular development.

To test the hypothesis that angiotensin II (ANG II) is necessary for normal embryonic and postnatal kidney development, the effect of angiotensin receptor blockade or angiotensin converting enzyme inhibition on nephrovascular development was studied in newborn Sprague-Dawley rats and in Rana catesbeiana tadpoles undergoing prometamorphosis. Blockade of ANG II type 1 receptor (AT1) in newborn rats induced an arrest in nephrovascular maturation and renal growth, resulting in altered kidney architecture, characterized by fewer, thicker, and shorter afferent arterioles, reduced glomerular size and number, and tubular dilatation. Inhibition of ANG II generation in tadpoles induced even more marked developmental renal abnormalities. Blockade of ANG II type 2 receptor (AT2) in newborn rats did not alter renal growth or morphology. Results indicate that ANG II regulates nephrovascular development, a role that is conserved across species.

Angiotensin II type 1 receptor: role in renal growth and gene expression during normal development.

To determine whether angiotensin II (ANG II) modulates renal growth and renin and angiotensin type 1 (AT1) gene expression via AT1 during development, weanling rats were given ANG II antagonist losartan (DuP 753) for 3 wk. Body weight (g), kidney weight (g), and kidney weight-to-body weight ratio were lower in losartan-treated rats (162 +/- 7, 1.6 +/- 0.06, and 9.5 +/- 0.1 x 10(-3)) than in control rats (184 +/- 5, 1.8 +/- 0.07, and 10.1 +/- 0.1 x 10(-3); P < 0.05). Renal DNA content (mg/kidney) was lower in losartan-treated (2.4 +/- 0.17) than in control rats (3.3 +/- 0.31; P < 0.05), whereas protein-to-DNA and RNA-to-DNA ratios were similar in losartan-treated and control rats. Renin mRNA levels were sevenfold higher in losartan-treated than in control rats, as determined by quantitative standardized dot blot analysis. In addition, blockade of AT1 with losartan induced recruitment of renin-synthesizing and renin-containing cells in the renal vasculature, as determined by immunocytochemistry and in situ hybridization. To establish whether AT1 blockade has a direct effect on renin gene expression, freshly isolated renin-producing cells were exposed in vitro to losartan (10(-6) M) or culture media (control). Losartan induced a twofold increase in steady-state renin mRNA levels above control (P < 0.05). Intrarenal AT1 mRNA levels were not altered by losartan given either in vivo or in vitro to freshly dispersed cells. To define whether immature renin-secreting cells are responsive to ANG II, renin release was determined by reverse hemolytic plaque assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin receptor regulates cardiac hypertrophy and transforming growth factor-beta 1 expression.

The role of angiotensin II via the angiotensin type 1 or type 2 receptor in the development of cardiac hypertrophy was determined in adult male Sprague-Dawley rats subjected to coarctation of the abdominal aorta. Five groups of animals were studied: coarctation, coarctation plus DuP 753, coarctation plus PD 123319, sham plus DuP 753, or sham operation. Type 1 receptor blockade was accomplished with DuP 753 given in the drinking water and type 2 blockade with PD 123319 delivered by osmotic minipumps beginning with the day of surgery until 72 hours after aortic coarctation. Mean carotid blood pressures and the carotid-femoral artery blood pressure gradients were not different among coarctation, coarctation plus DuP 753, and coarctation plus PD 123319 animals. However, ratios of heart weight to body weight were higher in coarctation (4.95 +/- 0.8) or coarctation plus PD 123319 (4.52 +/- 0.5) than in sham animals (3.6 +/- 0.4; P < .005 and .05, respectively). In coarctation plus DuP 753-treated animals heart weight-body weight ratios were not different from sham or sham plus DuP 753 animals (3.9 +/- 0.4 versus 3.6 +/- 0.4 or 3.3 +/- 0.08, respectively). Type 1 receptor mRNA levels were significantly increased in the coarctation group, with the highest levels in the coarctation plus DuP 753 and sham plus DuP 753 groups. To determine whether growth factors were involved in the hypertrophic process, we measured transforming growth factor-beta 1 mRNA levels. Northern analysis demonstrated a twofold increase in coarctation animals compared with sham or coarctation plus DuP 753-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of renin and AT1 receptor in the rat.

The enzyme renin and the angiotensin II (Ang II), subtype I receptor (ATI) are developmentally regulated in a tissue-specific manner. In early life, renin is expressed widely along the renal vasculature. As maturation progresses, there is a decrease in renin mRNA levels and a shift in the localization of renin close to the glomerulus. In addition, in the newborn rat, the number of renin-secreting cells is higher than in the adult rat. Exposure of neonatal and adult cells to Ang II results in a decrease of similar magnitude in the number of renin-secreting cells. These findings suggest that the high levels of renin observed in immature animals are due to increased renin synthesis and release rather than to a blunted response to Ang II. Expression of the ATI gene is also developmentally regulated in a tissue-specific manner. With maturation, ATI mRNA levels decrease in the kidney while they increase in the liver. The localization of ATI transcripts in precursor cells of the nephrogenic cortex suggests a role for this receptor in nephron growth and development. Inhibition of ATI with DUP753 results in delayed kidney and somatic growth and in increased renin mRNA levels and recruitment of renin-containing cells. These observations suggest that Ang II exerts a tonic negative feedback on renin gene expression via the ATI receptor subtype. Further studies are necessary to delineate the molecular and cellular signals mediating these developmental changes.

Decreased perfusion pressure modulates renin and ANG II type 1 receptor gene expression in the rat kidney.

To determine whether decreased perfusion pressure affects the abundance and distribution of renin and its mRNA and the expression of the angiotensin II type 1 (AT1) receptor gene within the kidney, adult male Sprague-Dawley rats were subjected to aortic coarctation proximal to the renal arteries (Coarc, n = 8) and compared with sham-operated rats (Sham, n = 6). Renal renin distribution was determined by immunocytochemistry using a specific polyclonal antibody against rat renin. Renin mRNA was assessed by in situ hybridization to a 35S-labeled oligonucleotide complementary to rat renin mRNA. Kidney AT1 mRNA levels were determined by Northern analysis using a 1,133-base pair rat AT1 cDNA. Femoral arterial blood pressure, measured 24 h after surgery, was lower in Coarc than in Sham rats (75 +/- 5.4 vs. 122 +/- 2.3 mmHg, P < 0.05). Aortic coarctation increased the percent of juxtaglomerular apparatuses (%JGA) containing renin and its mRNA (85 +/- 2.5 and 66 +/- 2.8 vs. 49 +/- 5.3 and 36 +/- 1.7%, Coarc vs. Sham, P < 0.05) and the intensity of hybridization signals (497 +/- 89 vs. 71 +/- 12 grains/JGA, Coarc vs. Sham, P < 0.05). In addition, recruitment of renin gene expressing cells was observed along afferent arterioles in Coarc rats, whereas renin and its mRNA were limited to the JGAs in Sham rats. Renal AT1 receptor gene expression was threefold lower in Coarc than in Sham rats. We conclude that reduction of perfusion pressure after abdominal aortic coarctation acutely enhances renin gene expression and downregulates AT1 receptor gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of CsA on the expression of renin and angiotensin type 1 receptor genes in the rat kidney.

To determine whether Cyclosporine A (CsA) alters the intrarenal expression of the renin and type 1 angiotensin II receptor genes, male adult Sprague-Dawley rats were given 25 mg/kg/day CsA s.c. for three weeks (CsA, N = 20) and were compared to pair-fed vehicle treated rats (Con, N = 20). The intrarenal distribution of renin and its mRNA was assessed by immunocytochemistry and in situ hybridization. In addition, kidney renin and type 1 angiotensin II (AT1) receptor mRNA levels were determined by Northern blot analysis. The percentage of juxtaglomerular apparatuses containing renin was higher in the CsA (84 +/- 5.5%) than in the Con (61 +/- 6.7%) group, (P < 0.05). The length of renin immunostaining along afferent arterioles was higher in the CsA (74 +/- 4.5 microns) than in the Con (37 +/- 5.1 microns) group, (P < 0.05). In contrast, neither renin mRNA levels nor its intrarenal distribution were altered by chronic CsA administration. Kidney AT1 receptor mRNA levels were lower in the CsA group than in the Con group. We conclude that chronic CsA: (1) induces recruitment of renin containing cells along the afferent arteriole, (2) causes no changes in intrarenal renin mRNA levels or distribution, suggesting that post-transcriptional events may be responsible for the persistence and/or uptake of renin by the preglomerular vasculature, (3) promotes a downregulation of AT1 receptor gene in the kidney, suggesting that local angiotensin II may control AT1 receptor gene expression by a negative feedback.

Ontogeny of type 1 angiotensin II receptor gene expression in the rat.

To determine whether the expression of the type 1 angiotensin II receptor (AT1) gene is developmentally regulated and whether the regulation is tissue specific, AT1 mRNA levels were determined by Northern blot analysis in livers and kidneys from fetal, newborn, and adult rats, using a 1133-bp rat AT1 cDNA. In the liver, AT1 mRNA levels increased fivefold from 15 d gestation to 5 d of age. Liver AT1 mRNA levels at 5 d of age were similar to those of adult rats. In the kidney, AT1 mRNA levels were higher in immature than in adult animals. The intrarenal distribution of AT1 mRNA was assessed by in situ hybridization to a 35S-labeled 24 residues oligonucleotide complementary to rat AT1 mRNA. In the adult, AT1 mRNA was present in glomeruli, arteries, and vasa recta, whereas in the newborn AT1 mRNA was observed also over the nephrogenic area of the cortex. We conclude that: (a) fetal kidney and liver express the AT1 gene; (b) the AT1 gene expression is developmentally regulated in a tissue-specific manner; (c) during maturation, localization of AT1 mRNA in the kidney shifts from a widespread distribution in the nephrogenic cortex to specific sites in glomeruli, arteries, and vasa recta, suggesting a role for the angiotensin receptor in nephron growth and development.

Dietary protein modulates intrarenal distribution of renin and its mRNA during development.

To determine whether high protein feeding throughout development affects renal growth, renal hemodynamics, and the intrarenal distribution of renin and its mRNA in the adult animal, male Wistar rats were fed diets containing either 20% protein [normal (NP), n = 12] or 40% protein [high (HP), n = 12] from weaning until studied at 6 or 12 wk of age. Kidney weight, kidney weight-to-body weight ratio, cortical DNA content, and cortical protein-to-DNA ratio were higher in HP- than in NP-fed rats at 6 and 12 wk of age. Somatic and kidney growth response to HP was blunted by angiotensin II type 1 receptor antagonist Dup 753. Glomerular filtration rate and renal plasma flow were higher in HP- than in NP-fed rats at 6 and 12 wk of age. The intrarenal distribution of renin and renin mRNA, assessed by immunocytochemistry and in situ hybridization, respectively, were markedly different between the two groups. In NP-fed rats, renin and renin mRNA were confined to a juxtaglomerular location. In HP-fed rats, renin and its mRNA extended proximally along the afferent arterioles. The percentage of visible afferent arteriolar length containing renin or renin mRNA was higher in HP-fed rats (60 +/- 3.2 and 61 +/- 3.9%, respectively) than in NP-fed rats (39 +/- 2.5 and 33 +/- 0.6%; P less than 0.05). Also, the percentage of juxtaglomerular apparatuses (JGAs) containing renin or renin mRNA was higher in HP-fed rats (80 +/- 1.6 and 72 +/- 2%, respectively), than in NP-fed rats (46 +/- 2.2 and 40 +/- 4%; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Focal glomerulosclerosis in children: an Argentinian experience.

Twenty-six children presenting with idiopathic nephrotic syndrome and a histological diagnosis of focal glomerulosclerosis were studied retrospectively to evaluate their response to treatment, outcome and clinicopathological correlations. Twenty-two patients (84.6%) were steroid resistant; of these, 8 of the 19 with focal segmental glomerulosclerosis and 2 of the 3 with focal global glomerulosclerosis responded to cyclophosphamide (CY) within 16 weeks of starting therapy. Seven patients relapsed after a CY-induced remission, but 5 of them became steroid responsive. After an average follow-up of 83 months, 17 patients are in remission with normal renal function, 3 patients have persistent nephrotic range proteinuria and 6 patients are in chronic renal failure. Persistence of proteinuria, a high percentage of segmentally sclerotic glomeruli and diffuse mesangial proliferation were indicators of poor prognosis. We believe longer courses of CY therapy than those traditionally utilized are responsible for the relatively good results in our patients.