Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats

Abstract

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ∼10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.

Fig. 1.

ANG II levels in chronic ANG II-infused rats and sham-operated rats. Plasma ANG II levels (A), kidney cortex ANG II levels (B), and medullary ANG II levels (C) from ANG II-infused (n = 10), and sham-operated rats (n = 10). Values are means ± SE. *P < 0.05 compared with sham.

Fig. 2.

Urinary ANG II and urinary AGT excretion rates in ANG II-infused rats and sham-operated rats. uANG II (A) and uAGT (B) excretion rates were augmented in ANG II-infused rats (n = 10) compared with controls (n = 10). uANG II and uAGT excretion rates were measured from urine collected at day–1 and day 12 of ANG II infusion. *P < 0.001 vs. sham.

Fig. 3.

Renal medulla renin content and urine renin content. Kidney medullary renin content was measured in inner renal medullary samples from chronic ANG II-infused rats and sham-operated rats (A). Urinary renin content (uRen; B) was measured in 24-h urine samples collected on day −1 of ANG II infusions (n = 20) and in collections from day 13 of sham-operated rats (n = 10), ANG II-infused rats (n = 10), ANG II-infused rats fed a high-salt (HS; 8% NaCl; n = 5) diet, and ANG II + HS + candesartan (26 mg/l in drinking water for 14 days; n = 5). Values are means ± SE. *P < 0.05 ANG II rats vs. sham rats.

Fig. 4.

Active renin (A) and prorenin measured in the presence of trypsin (B) in the urine of chronic ANG II-infused rats and sham-operated rats. Notice that the amount of prorenin expressed in enzymatic units excreted (EUE) per day of urinary renin (uRen) after trypsinization (B) is 1,000 times higher than renin (A). Values are means ± SE. *P < 0.01 ANG II rats vs. sham rats.

Fig. 5.

Renin and prorenin protein in the urine in chronic ANG II-infused rats and sham-operated rats. Renin and prorenin protein were visualized by Western blot in 3 independent experiments using urine samples concentrated ×30. The identity of renin (38 kDa) and prorenin (48 kDa) bands was based on comparison with positive controls using human recombinant renin and prorenin proteins (2.5 μl/lane; A). The specificity of the antibody and the identities of renin bands were confirmed by preadsorption using a polyclonal rabbit antirenin antibody (sc-22752, Santa Cruz Biotechnology) preadsorbed with excess (×3) human recombinant prorenin for 72 h (B). hPR, human recombinant prorenin; hRen, human recombinant renin; MW, molecular weight; Ang, angiotensin.