Distal nephron renin may provide a possible pathway for angiotensin (Ang) I generation from proximally delivered angiotensinogen. To examine the effects of Ang II on distal nephron renin, we compared renin protein and mRNA expression in control and Ang II–infused rats. Kidneys from sham (n=9) and Ang II–infused (80 ng/kg per minute, 13 days, n=10) Sprague-Dawley rats were processed by immunohistochemistry, Western blot, reverse transcriptase–polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Ang II infusion increased systolic blood pressure (181±4 versus 115±5 mm Hg) and suppressed plasma and kidney cortex renin activity. Renin immunoreactivity was suppressed in juxtaglomerular apparatus (JGA) cells in Ang II–infused rats compared with sham (0.1±0.1 versus 1.0±0.1 relative ratio) but increased in distal nephron segments (6.4±1.4 versus 1.0±0.1 cortex; 2.5±0.3 versus 1.0±0.2 medulla). Tubular renin immunostaining was apically distributed in principal cells colocalizing with aquaporin-2 in connecting tubules and cortical and medullary collecting ducts. Renin protein levels were decreased in the kidney cortex of Ang II–infused rats compared with that of sham (0.4±0.2 versus 1.0±0.4) rats but higher in the kidney medulla (1.2±0.4 versus 1.0±0.1). In kidney medulla, RT-PCR and quantitative real-time PCR showed similar levels of renin transcript in both groups. In summary, the detection of renin mRNA in the renal medulla, which is devoid of JGA, indicates local synthesis rather than an uptake of JGA renin. In contrast to the inhibitory effect of Ang II on JGA renin, Ang II infusion stimulates renin protein expression in collecting ducts and maintains renin transcriptional levels in the medulla, which may contribute to the increased intrarenal Ang II levels in Ang II–dependent hypertension.

Renin synthesis and secretion by principal cells of the collecting duct are enhanced in angiotensin (Ang) II–dependent hypertension. The presence of renin/(pro)renin and its receptor, the (pro)renin receptor ([P]RR), in the collecting duct may provide a pathway for Ang I generation with further conversion to Ang II. To assess whether (P)RR activation occurs during Ang II–dependent hypertension, we examined renal (P)RR levels and soluble (P)RR excretion in the urine of chronic Ang II–infused rats (80 ng/min; for 2 weeks; n=10) and sham-operated rats (n=10). Systolic blood pressure and Ang II levels in the plasma and kidney were increased whereas plasma renin activity was suppressed in Ang II–infused rats. Renal (P)RR transcripts were upregulated in the cortex and medulla of Ang II–infused rats. (P)RR immunoreactivity in collecting duct cells and the protein levels of the full-length form (37-kDa band) were significantly decreased in the medulla of Ang II–infused rats. The soluble (P)RR (28-kDa band) was detected in the renal medulla and urine samples of Ang II–infused rats, which also showed increases in urinary renin content. To determine whether the soluble (P)RR could stimulate Ang I formation, urine samples were incubated with recombinant human (pro)renin. Urine samples of Ang II–infused rats exhibited increased Ang I formation compared with sham-operated rats. Thus, in chronic Ang II–infused rats, the catalytic activity of the augmented renin produced in the collecting duct may be enhanced by the intraluminal soluble (P)RR and cell-surface located (P)RR, thus contributing to enhanced intratubular Ang II formation.

Angiotensin II (ANG II)-infused rats exhibit increases in distal nephron renin expressed in principal cells of connecting tubules and collecting ducts. This study was performed to determine whether the augmentation of distal nephron renin involves ANG II type 1 (AT₁) receptor activation. Male Sprague-Dawley rats (200–220 g) were divided into three groups: 1) sham operated (n = 8); 2) ANG II infused (80 ng/min, 13 days, n = 8); and 3) ANG II infused plus AT₁ receptor blocker (ARB), olmesartan (5 mg/days, n = 8). ANG II infusion increased systolic blood pressure (BP; 178 ± 4 vs. 122 ± 1 mmHg; P < 0.001) and suppressed plasma renin activity (PRA; 0.08 ± 0.1 vs. 5.3 ± 0.8 ng ANG I·ml⁻¹·h⁻¹). ARB treatment prevented the increase in BP (113 ± 6 mmHg) and led to increases in PRA (15.8 ± 1.5 ng ANG I·ml⁻¹·h⁻¹). Renin protein levels measured in the kidney medulla, to avoid contribution from juxtaglomerular appartus cells, were higher in ANG II-infused rats [1.64 ± 0.3 vs. 1.00 ± 0.1 densitometric units (DU) compared with sham-operated rats; P < 0.05], and ARB treatment prevented this increase (1.01 ± 0.1). Similarly, renin immunoreactivity increased in medullary collecting ducts of ANG II-infused compared with sham-operated rats (2.5 ± 0.3 vs. 1.0 ± 0.2 DU; P < 0.001), which was also prevented by ARB (1.01 ± 0.06). Renin qRTPCR in ANG II-infused rats showed higher mRNA levels in the kidney medulla compared with sham-operated rats (5.5 ± 2.3 vs. 0.04 ± 0.02 ratio to GAPDH mRNA levels; P < 0.001); however, renin transcript levels were normalized in the ARB-treated rats. These data demonstrate that the augmentation of distal nephron renin in ANG II-infused hypertensive rats is AT₁ receptor mediated. The augmented distal tubular renin may contribute to increased intratubular ANG II levels and distal nephron sodium reabsorption in ANG II-dependent hypertension.

Renin in collecting duct cells is upregulated in chronic angiotensin II–infused rats via angiotensin II type 1 receptors. To determine whether stimulation of collecting duct renin is a blood pressure–dependent effect; changes in collecting duct renin and associated parameters were assessed in both kidneys of 2-kidney, 1-clip Goldblatt hypertensive (2K1C) rats. Renal medullary tissues were used to avoid the contribution of renin from juxtaglomerular cells. Systolic blood pressure increased to 184±9 mm Hg in 2K1C rats (n=19) compared with sham rats (121±6 mm Hg; n=12). Although renin immunoreactivity markedly decreased in juxtaglomerular cells of nonclipped kidneys (NCK: 0.2±0.0 versus 1.0±0.0 relative ratio) and was augmented in clipped kidneys (CK: 1.7±1.0 versus 1.0±0.0 relative ratio), its immunoreactivity increased in cortical and medullary collecting ducts of both kidneys of 2K1C rats (CK: 2.8±1.0 cortex; 2.1±1.0 medulla; NCK: 4.6±2.0 cortex, 3.2±1.0 medulla versus 1.0±0.0 in sham kidneys). Renal medullary tissues of 2K1C rats showed greater levels of renin protein (CK: 1.4±0.2; NCK: 1.5±0.3), renin mRNA (CK: 5.8±2.0; NCK: 4.9±2.0), angiotensin I (CK: 120±18 pg/g; NCK: 129±13 pg/g versus sham: 67±6 pg/g), angiotensin II (CK: 150±32 pg/g; NCK: 123±21 pg/g versus sham: 91±12 pg/g; P <0.05), and renin activity (CK: 8.6 μg of angiotensin I per microgram of protein; NCK: 8.3 μg of angiotensin I per microgram of protein; sham: 3.4 μg of angiotensin I per microgram of protein) than sham rats. These data indicate that enhanced collecting duct renin in 2K1C rats occurs independently of blood pressure. Upregulation of distal tubular renin helps to explain how sustained intrarenal angiotensin II formation occurs even during juxtaglomerular renin suppression, thus allowing maintained effects on tubular sodium reabsorption that contribute to the hypertension.

During renin–angiotensin system activation, cyclooxygenase-2 (COX-2)-derived prostaglandins attenuate the pressor and antinatriuretic effects of angiotensin II (AngII) in the renal medulla. The (pro)renin receptor (PRR) is abundantly expressed in the collecting ducts (CD) and its expression is augmented by AngII. PRR overexpression upregulates COX-2 via mitogen-activated kinases/extracellular regulated kinases 1/2 in renal tissues; however, it is not clear whether this effect occurs independently or in concert with AngII type 1 receptor (AT1R) activation. We hypothesized that PRR activation stimulates COX-2 expression independently of AT ₁ R in primary cultures of rat renal inner medullary cells. The use of different cell-specific immunomarkers (aquaporin-2 for principal cells, anion exchanger type 1 for intercalated type-A cells, and tenascin C for interstitial cells) and costaining for AT ₁ R, COX-2, and PRR revealed that PRR and COX-2 were colocalized in intercalated and interstitial cells whereas principal cells did not express PRR or COX-2. In normal rat kidney sections, PRR and COX-2 were colocalized in intercalated and interstitial cells. In rat renal inner medullary cultured cells, treatment with AngII (100 nmol/L) increased COX-2 expression via AT ₁ R. In addition, AngII and rat recombinant prorenin (100 nmol/L) treatments increased extracellular regulated kinases 1/2 phosphorylation, independently. Importantly, rat recombinant prorenin upregulated COX-2 expression in the presence of AT ₁ R blockade. Inhibition of mitogen-activated kinases/extracellular regulated kinases 1/2 suppressed COX-2 upregulation mediated by either AngII or rat recombinant prorenin. Furthermore, PRR knockdown using PRR-short hairpin RNA blunted the rat recombinant prorenin-mediated upregulation of COX-2. These results indicate that COX-2 expression is upregulated by activation of either PRR or AT ₁ R via mitogen-activated kinases/extracellular regulated kinases 1/2 in rat renal inner medullary cells.

Collecting duct (CD) renin is stimulated by angiotensin (Ang) II, providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates the epithelial sodium channel via the Ang II type 1 receptor and increases mineralocorticoid receptor activity attributed to increased aldosterone release. Our objective was to determine whether CD renin augmentation is mediated directly by Ang II type 1 receptor or via the epithelial sodium channel and mineralocorticoid receptor. In vivo studies examined the effects of epithelial sodium channel blockade (amiloride; 5 mg/kg per day) on CD renin expression and urinary renin content in Ang II–infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure, medullary renin mRNA, urinary renin content, and intrarenal Ang II levels. Amiloride cotreatment did not alter these responses despite a reduction in the rate of progression of systolic blood pressure. In primary cultures of inner medullary CD cells, renin mRNA and (pro)renin protein levels increased with Ang II (100 nmol/L), and candesartan (Ang II type 1 receptor antagonist) prevented this effect. Aldosterone (10 ⁻¹⁰ to 10 ⁻⁷ mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II–treated cells. However, inhibition of protein kinase C with calphostin C prevented the Ang II–mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, protein kinase C activation with phorbol 12-myristate 13-acetate increased renin expression to the same extent as Ang II. These data indicate that an Ang II type 1 receptor–mediated increase in CD renin is induced directly by Ang II via the protein kinase C pathway and that this regulation is independent of mineralocorticoid receptor activation or epithelial sodium channel activity.