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

Abstract

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.

Figure 1

Systolic blood pressure and PRA: comparison of systolic blood pressure (A) and PRA (B) in sham-operated rats (n=14) and 2K1C rats (n=16). Values are means ± SEs; *P<0.001 Goldblatt rats vs sham rats.

Figure 2

Angiotensin peptide contents in rat kidney cortex and medulla. The levels of Ang I and Ang II were measured by HPLC in kidney cortex and medulla samples from Goldblatt (n=8) rats and sham (n=8) rats. Values are expressed in picograms per gram of tissue. Values are means ± SEs; *P<0.05 vs sham; ¶P<0.05 sham cortex vs sham medulla; ¥P<0.05 NCK cortex vs NCK medulla.

Figure 3

Renin and prorenin content in kidney cortex and medulla: renin content was determined in the absence of trypsin and by incubating the kidney tissue samples with excess renin substrate (A). To measure prorenin content, total renin was activated with 10 μL of trypsin (50 μg/mL) for 16 to 18 hours at 37°C (B). Values were determined from the amount of Ang I product measured by HPLC and expressed as means ± SEs; *P<0.05 vs sham rats; #P<0.05 CK cortex vs CK medulla.

Figure 4

Renin protein expression in JG cells and cortical and medullary CD cells: renin immunoreactivity by using the immunoperoxidase technique in kidney cortex and medulla. A through C, cortex of kidney sections (3 μm) with specific renin immunostaining in sham rats (A), and CK (B) and NCK (C) of Goldblatt rats. Arrows show positive specific JG renin immunoreactivity (DAB chromogen) in a sham (A) and in the CK (B) and NCK (C) from Goldblatt rats. Higher renin immunoreactivity (*DAB chromogen) are shown in the CDs of the renal cortexes of both CKs (B) and NCKs (panel C) relative to the sham kidney section (A). D and E, Renin immunoreactivity (*DAB chromogen) in the collecting ducts of the renal medulla of sham (D), CK (E), and NCK (F) sections. In addition, we show the densitometric analyses of the renin intensity immunoreactivity in JG cells (JG renin; G) and cortical (H) and medullary (I) CDs of sham rats and CKs and NCKs of Goldblatt rats performed in 4 kidney sections per animal (10 microscopic fields per kidney section at the renal cortex and medulla regions) and compared with sham kidneys. Sham rats, n=5; Goldblatt rats, n=6. Glom indicates glomerulus; IDV, integrated densitometric values. Values are means ± SEs; *P<0.0001 vs sham rats. Renin antibody concentration is 1:4000. *P<0.05 vs sham rats; #P<0.05 CK vs NCK.

Figure 5

Renin protein and mRNA expression in the rat renal medulla. Renin Western blot analysis in kidney medulla from Goldblatt and sham-operated rats. A representative autoradiograph of renin protein and densitometric analysis of the immunoreactive band showed that the CKs and NCKs of Goldblatt rats exhibited significantly higher renin protein levels in the renal medulla relative to sham-operated rat kidneys (A). Quantification of mRNAs levels (B) were measured by real-time quantitative RT-PCR in rat kidney cortex and medullary tissues. Renin quantitative RT-PCR in CKs and NCKs of Goldblatt (n=9) rats showed higher mRNA levels in the kidney medulla compared with sham-operated (n=9) rats. The mRNAs levels for the target genes were determined in each sample per triplicate and expressed relative to GAPDH in arbitrary units. *P<0.05 vs control.