Glomerular type 1 angiotensin receptors augment kidney injury and inflammation in murine autoimmune nephritis.

Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.

Collecting Duct Renin: A major player in Angiotensin II-dependent Hypertension.

Recently, the focus of interest on the role of the renin angiotensin system in the pathophysiology of hypertension has shifted towards greater emphasis on new developments in local renin angiotensin systems in specific tissues. We have focused our recent investigations on the role of the intrarenal-intratubular RAS in hypertension. All of the components needed for angiotensin II generation are present within the various compartments in the kidney. This brief review is focused on recent evidence that inappropriate activation of renin in distal nephron segments, by acting on angiotensinogen generated in the proximal tubule cells and delivered to the distal nephron may contribute to increased distal intrarenal angiotensin II formation, sodium retention and development and progression of hypertension.

Heme oxygenase induction attenuates afferent arteriolar autoregulatory responses.

Heme oxygenases (HO-1, HO-2) catalyze conversion of heme to iron, carbon monoxide (CO), and biliverdin/bilirubin. We studied the effects of renal HO-1 induction on afferent arteriole (Aff-Art) autoregulatory responses to increases in renal perfusion pressure (RPP). Rats were treated with hemin and SnCl2 to induce HO-1, and Aff-Art autoregulatory responses were evaluated using the rat blood-perfused juxtamedullary nephron preparation. Renal HO-1 expression was significantly increased in hemin- and SnCl2-treated rats, while HO-2 was not altered. Aff-Art autoregulatory constrictor responses to increases in RPP from 100 to 150 mmHg were attenuated in hemin- and SnCl2-treated rats compared with control rats (+1.1+/-3.3, n=9 and +4.4+/-5.3, n=9 vs. -14.2+/-1.5%, n=10, respectively) (P<0.05). Acute HO inhibition with chromium mesoporphyrin (CrMP; 15 micromol/l) restored Aff-Art autoregulatory responses in hemin- and SnCl2-treated rats. Superfusing Aff-Arts from control rats with 100 micromol/l biliverdin did not alter autoregulatory responses; however, superfusion with 1 mmol/l CO significantly attenuated autoregulatory responses to increases in RPP from 100 to 150 mmHg (+3.3+/-5.4 vs. -16.6+/-3.8%, n=6) (P<0.05). Acute soluble guanylate cyclase inhibition with 10 micromol/l ODQ restored Aff-Art autoregulatory responses in hemin-treated rats. Immunohistochemistry shows HO-2 to be expressed mainly in epithelial cells with weak staining in proximal tubules, interlobular arteries, and Aff-Arts. In hemin- and SnCl2-treated rats, HO-1 was induced in tubular epithelial cells but not interlobular arteries and Aff-Arts. We conclude that induction of renal HO-1 attenuates Aff-Art constrictor responses to increases in RPP via increasing CO production from tubular epithelial cells, suggesting that an augmented HO system in pathophysiological conditions modulates renal autoregulation.

Collecting duct renin is upregulated in both kidneys of 2-kidney, 1-clip goldblatt hypertensive rats.

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 microg of angiotensin I per microgram of protein; NCK: 8.3 microg of angiotensin I per microgram of protein; sham: 3.4 microg 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.

Purinergic receptors contribute to early mesangial cell transformation and renal vessel hypertrophy during angiotensin II-induced hypertension.

Chronic ANG II infusions lead to increases in intrarenal ANG II levels, hypertension, and tissue injury. Increased blood pressure also elicits increases in renal interstitial fluid (RIF) ATP concentrations that stimulate cell proliferation. We evaluated the contribution of purinergic receptor activation to ANG II-induced renal injury in rats by treating with clopidogrel, a P2Y12 receptor blocker, or with PPADS, a nonselective P2 receptor blocker. alpha-Actin expression in mesangial cells, afferent arteriolar wall thickness (AAWT), cortical cell proliferation, and macrophage infiltration were used as early markers of renal injury. Clopidogrel and PPADS did not alter blood pressure, renin or kidney ANG II content. alpha-Actin expression increased from control of 0.6 +/- 0.4% of mesangial area to 6.3 +/- 1.9% in ANG II-infused rats and this response was prevented by clopidogrel (0.4 +/- 0.2%) and PPADS. The increase in AAWT from 4.7 +/- 0.1 to 6.0 +/- 0.1 mm in ANG II rats was also prevented by clopidogrel (4.8 +/- 0.1 mm) and PPADS. ANG II infusion led to interstitial macrophage infiltration (105 +/- 16 vs. 62 +/- 4 cell/mm(2)) and tubular proliferation (71 +/- 15 vs. 20 +/- 4 cell/mm(2)) and these effects were prevented by clopidogrel (52 +/- 4 and 36 +/- 3 cell/mm(2)) and PPADS. RIF ATP levels were higher in ANG II-infused rats than in control rats (11.8 +/- 1.9 vs. 5.6 +/- 0.6 nmol/l, P < 0.05). The results suggest that activation of vascular and glomerular purinergic P2 receptors may contribute to the mesangial cell transformation, renal inflammation, and vascular hypertrophy observed in ANG II-dependent hypertension.

Young Scholars Award Lecture: Intratubular angiotensinogen in hypertension and kidney diseases.

Recent findings related to the renin-angiotensin system have provided a more elaborated understanding of the pathophysiology of hypertension and kidney diseases. These findings have led to unique concepts and issues regarding the intrarenal renin-angiotensin system. Angiotensinogen is the only known substrate for renin that is the rate-limiting enzyme of the renin-angiotensin system. Because the level of angiotensinogen in human beings is close to the Michaelis-Menten constant value for renin, changes in angiotensinogen levels can control the activity of the renin-angiotensin system, and its upregulation may lead to elevated angiotensin peptide levels and increases in blood pressure. Enhanced intrarenal angiotensinogen mRNA or protein levels or both have been observed in multiple models of hypertension including angiotensin II-dependent hypertensive rats, Dahl salt-sensitive hypertensive rats, and spontaneously hypertensive rats, as well as in kidney diseases including diabetic nephropathy, immunoglobulin A (IgA) nephropathy, and radiation nephropathy. Renal angiotensinogen is formed primarily in proximal tubular cells and is secreted into the tubular fluid. Urinary angiotensinogen excretion rates show a clear relationship to kidney angiotensin II contents and kidney angiotensinogen levels, suggesting that urinary angiotensinogen may serve as an index of the intrarenal renin-angiotensin system status. Establishment of concise and accurate methods to measure human angiotensinogen may allow clinical studies that would provide important information regarding the roles of intrarenal angiotensinogen in the development and progression of hypertension and kidney diseases.

Intratubular Renin-Angiotensin System in Hypertension.

It is well recognized that the renin-angiotensin system plays an important role in the regulation of arterial pressure and sodium homeostasis. Recent years, many studies have shown that local tissue angiotensin II levels are differentially regulated and cannot be explained on the basis of circulating concentrations. All of the components needed for angiotensin II generation are present within the various compartments in the kidney including the renal interstitium and the tubular network. The cascade of the renin-angiotensin system demonstrates three major possible sites for the pharmacological interruption of the renin-angiotensin system: the interaction of renin with its substrate, angiotensinogen, the angiotensin converting enzyme, and angiotensin II type 1 receptors. This brief article will focus on the role of the intratubular renin-angiotensin system in the pathophysiology of hypertension and the responses to the renin-angiotensin system blockade by renin inhibitors, angiotensin converting enzyme inhibitors and angiotensin II type 1 receptor blockers.

AT1 receptor-mediated enhancement of collecting duct renin in angiotensin II-dependent hypertensive rats.

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 x ml(-1) x 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 x ml(-1) x h(-1)). Renin protein levels measured in the kidney medulla, to avoid contribution from juxtaglomerular apparatus 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.

Enhancement of collecting duct renin in angiotensin II-dependent hypertensive rats.

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.

AT1 receptor mediated augmentation of intrarenal angiotensinogen in angiotensin II-dependent hypertension.

Angiotensin (Ang) II-infused hypertensive rats exhibit increases in renal angiotensinogen mRNA and protein, as well as urinary angiotensinogen excretion in association with increased intrarenal Ang II content. The present study was performed to determine if the augmentation of intrarenal angiotensinogen requires activation of Ang II type 1 (AT1) receptors. Male Sprague-Dawley rats (200 to 220 g) were divided into 3 groups: sham surgery (n=10), subcutaneous infusion of Ang II (80 ng/min, n=11), and Ang II infusion plus AT1 blocker (ARB), olmesartan (5 mg/d, n=12). Ang II infusion progressively increased systolic blood pressure (SBP) compared with sham (178+/-8 mm Hg versus 119+/-4 at day 11). ARB treatment prevented hypertension (113+/-6 at day 11). Twenty-four-hour urine collections were taken at day 12, and plasma and tissue samples were harvested at day 13. The Ang II+ARB group had a significant increase in plasma Ang II compared with Ang II and sham groups (365+/-46 fmol/mL versus 76+/-9 and 45+/-14, respectively). Nevertheless, ARB treatment markedly limited the enhancement of kidney Ang II by Ang II infusion (65+/-17 fmol/g in sham, 606+/-147 in Ang II group, and 288+/-28 in Ang II+ARB group). Ang II infusion significantly increased kidney angiotensinogen compared with sham (1.69+/-0.21 densitometric units versus 1.00+/-0.17). This change was reflected by increased angiotensinogen immunostaining in proximal tubules. ARB treatment prevented this increase (1.14+/-0.12). Urinary angiotensinogen excretion rates were enhanced 4.7x in Ang II group (4.67+/-0.41 densitometric units versus 1.00+/-0.21) but ARB treatment prevented the augmentation of urinary angiotensinogen (0.96+/-0.23). These data demonstrate that augmentation of intrarenal angiotensinogen in Ang II-infused rats is AT1-dependent and provide further evidence that urinary angiotensinogen is closely linked to intrarenal Ang II in Ang II-dependent hypertension.