Prorenin Receptor in Distal Nephron Segments of 2-Kidney, 1-Clip Goldblatt Hypertensive Rats

INTRODUCTION

Intrarenal angiotensin II (AngII) is formed by tissue renin acting on angiotensinogen (AGT) delivered to the kidney as well by AGT produced intrarenally by the proximal tubule cells. In addition to their localization in juxtaglomerular cells, renin messenger RNA (mRNA) and protein are present in connecting tubules and in cortical and medullary collecting ducts.^1-3 Renin in the distal nephron segments is specifically expressed by principal cells^2,3 and seems to be differentially regulated from that produced by juxtaglomerular cells.⁴ In response to chronic AngII infusions, renin mRNA and protein levels increase in principal cells from connecting tubules and collecting ducts, indicating stimulation of tubular renin during AngII-dependent hypertension.³ This stimulatory effect of collecting duct renin in AngII-dependent hypertension is mediated by an AngII type 1 receptor (AT₁R) mechanism because treatment with AT₁R blockers (ARBs) prevents the stimulation of distal nephron renin.⁴ Furthermore, the upregulation of renin expression in both kidneys of 2-kidney, 1-clip (2K1C) Goldblatt hypertensive rats indicates that renin stimulation occurs independently of blood pressure.⁵ These recent studies support the hypothesis that inappropriate activation of renin in distal nephron segments—by acting on AGT originating from the proximal tubular segments—leads to increases in distal intrarenal AngII formation and contributes to sodium retention and the development and progression of hypertension.^3,6

Nguyen et al⁷ cloned the prorenin receptor (PRR), a 350–amino acid protein with a single transmembrane domain that binds renin or prorenin, increasing the catalytic activity of renin and fully activating prorenin. The PRR activation also elicits intracellular signals via the extracellular signal-regulated kinase 1 and the extracellular signal-regulated kinase 2 mitogen-activated protein kinase. The PRR is expressed in the heart, brain, placenta, kidney, and liver. In the human kidney, the PRR has been localized to glomerular mesangial cells and the subendothelium of renal arteries.⁷ Immunohistochemistry studies have also identified the PRR in tubular segments in normal human and rat kidneys at the basolateral side of distal tubular cells, as well as in macula densa cells.^8,9

The coordinated presence of renin and the PRR in the distal nephron segments may provide a pathway for distal nephron angiotensin I (AngI) generation and, with further conversion by angiotensin-converting enzyme (ACE) activity present in the collecting ducts,^10,11 lead to subsequent generation of AngII in distal nephron segments. However, the exact cellular localization of the PRR in distal nephron segments and the alterations occurring in hypertension models remain unclear. In the present study, we examined the cell type localization, regional distribution, and gene expression of the PRR in normal rat kidneys and in both clipped and nonclipped kidneys from hypertensive 2K1C Goldblatt rats.

RESULTS

PRR Immunohistochemistry in the Distal Nephron Segments

As shown in Figure 1, co-immunofluorescence staining at high-power magnification (oil immersion, 100×) demonstrates the positive PRR immunoreactivity in a collecting duct (Figures 1A-1C). Positive PRR immunofluorescence is mainly observed in the apical aspects of some cells of the collecting duct (color green, Figure 1A). Co-immunofluorescence with AQP2 (red, Figure 1B) identifies that cells stained with AQP2 (red, Figure 1B) are a different cell type than cells stained positive for the PRR (green, Figure 1A). The absence of colocalization after merging the 2 immunofluorescence images (Figure 1C) and the lack of co-staining of the same cell type by using the PRR (brown, Figure 1D) and AQP2 (red, Figure 1E) on consecutive sections indicate that the PRR is expressed by the intercalated cells and not the principal cells (AQP2-positive cells). Figure 1F provides a lower magnification (20×) view of the PRR immunoreactivity in cortical and medullary collecting ducts of sham kidneys, CKs, and NCKs and shows a similar pattern of expression: positive cells labeled at the apical side of the cell alternate with negative cells.

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Figure 1.

Prorenin receptor (PRR) immunoreactivity in rat kidneys. A: Specific PRR immunofluorenscence (Alexa Fluor 488, green) is shown on the apical aspect of some cells of a distal tubular segment. The absence of colocalization of PRR and anti-aquaporin 2 (AQP2) by using (A) immunofluorescence (PRR, Alexa Fluor 488, green) and (B) AQP2 (Alexa Fluor 594, red) on the same kidney section indicates that PRR and AQP2 are located in different cell types. Consecutive kidney sections stained by immunoperoxidase technique show (D) PRR positive staining (DAB chromogen, brown) in those cells that are (E) AQP2 negative (Vulcan Fast Red), indicating that PRR is expressed by intercalated cells in the distal nephron segment of rat kidneys. (F) Specific PRR immunoreactivity (DAB, brown chromogen) in the renal cortex (upper panels) and medulla (lower panels) from sham rats and clipped kidney and nonclipped kidney of 2-kidney, 1-clip rats is shown. Glom, glomerulus.

Densitometric analysis of positive PRR immunoreactivities in cortical and medullary collecting duct cells from kidney sections of sham rats and the CKs and NCKs of 2K1C rats is shown in Figure 2. PRR immunoreactivity was determined in 10 different microscopic fields/tissue section/animal using a Nikon Eclipse 50i microscope, 40× objective, and an integrated Nikon Digital Sight DS-U2 Camera System for image processing. The intensity of PRR immunoreactivity in both kidneys of Goldblatt rats and in sham-rat kidney sections was analyzed using NIS-Elements AR Software (version 3.0 for Windows; Nikon Instruments, Inc., Melville, NY), allowing a computerized determination of the area of positive staining (μm) and the intensity of immunoreactivity (sum of density of positive tubules in an analyzed area). For this analysis, the immunoreactivity of PRR was analyzed separately from the cortical and medullary collecting ducts. The results are expressed in IDUs of the relative intensity normalized to the PRR immunostaining average of the sham kidney. CKs of 2K1C rats exhibited higher PRR immunoreactivity in cortical and medullary collecting duct cells than sham rats and NCK rats (CK: 2.3 ± 1.0 IDU; P<0.05 vs sham: 1.0 ± 0.0 IDU and NCK: 1.3 ± 0.0 IDU). Immunoreactivity was also detected in mesangial cells, but its intensity was not analyzed in this study. In addition, control experiments were performed using consecutive sections incubated with normal blocking goat serum to confirm the specificity of the PRR antibody.

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Figure 2.

Semiquantitation of prorenin receptor (PRR) immunoreactivity in collecting duct cells. Densitometric analyses of the PRR intensity immunoreactivity in collecting duct cells of sham rats and clipped (CKs) and nonclipped kidneys (NCKs) of Goldblatt rats performed in 4 kidney sections/animal (10 microscopic fields/kidney sections at the renal cortex and medulla regions). Sham rats, n=5; 2-kidney, 1-clip rats, n=6. Values are mean ± standard error of the mean. Values are expressed in intensity densitometric units (IDUs); *P<0.05 versus sham rats. PRR antibody concentration 1:200. ^#P<0.05 CK versus NCK.

mRNA Levels of PRR in Renal Medullary Tissues

To obtain an index of the PRR expression in the distal nephron segments, the quantitation of its mRNA levels was performed only in renal medullary tissues to avoid contribution of PRR from glomeruli, mesangial cells, podocytes, and macula densa cells from the renal cortex. Specific rat PRR transcript levels were slightly but significantly higher in the medullary tissues of CK rats (1.3 ± 0.1 arbitrary unit [au]; P<0.05) but not of NCK rats (0.9 ± 0.1 au) compared to sham rats (1.0 ± 0.0 au) (Figure 3).

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Figure 3.

Kidney medullary prorenin receptor (PRR) messenger RNA (mRNA), measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). PRR qRT-PCR showed higher mRNA levels in the medullas of clipped kidneys (CKs) compared to nonclipped kidneys (NCKs) and sham kidneys. The mRNA levels for the target gene were determined in each sample in triplicate and expressed relative to β-actin in arbitrary units (au). *P<0.05 versus sham rats.

DISCUSSION

This study demonstrates that the PRR is immunoexpressed in cortical and medullary collecting ducts of normal rat kidneys and in both CKs and NCKs of hypertensive 2K1C Goldblatt rats. The cell-type immunolocalization of the PRR appears to be restricted to the intercalated cells of this renal segment. In addition, this study shows a PRR gene expression upregulation in the CK of the Goldblatt hypertensive rats, indicating a parallel upregulation of renin and PRR in distal nephron segments of the CK.

Receptor-bound renin exhibits 5-fold increased catalytic activity, and receptor-bound prorenin exhibits full enzymatic activity comparable to that of active renin.⁷ Initially, Nguyen and associates⁷ reported the presence of this receptor predominantly in glomerular mesangial cells and in vascular smooth muscle cells of renal arteries and coronary arteries visualized by immunofluorescence on frozen tissues. However, immunohistochemistry and in situ hybridization studies have reported that the main site of synthesis of PRR in the kidneys of normal rats and humans is at the distal tubules. ^8,9

The present study, using immunofluorescence and immunoperoxidase techniques, confirms the predominant immunoexpression of the PRR in AQP2-negative cells specifically expressed on the apical aspects of the intercalated cells of cortical and medullary collecting ducts. Advani et al⁸ and Gonzalez et al⁹ previously reported this renal localization. The presence of the PRR on the cell surface of the intercalated cells and not on the principal cells supports previous suggestions that renin in the principal cells of the collecting duct is locally synthesized and not caused by uptake via its receptor.^3,6,9 The immunolocalization of the PRR at the apical side of the cells in the distal nephron segments might be of great relevance in light of new findings of renin synthesis and secretion from the principal cells of the collecting ducts.^2,3,6

We propose that the PRR located on the surface of the intercalated cells may play a pivotal role in increasing the efficiency of intratubular angiotensin peptide generation by the distal nephron segments via anchoring of the renin or prorenin synthesized and released by principal cells. Nguyen et al⁷ also described the immunoexpression of this receptor on the basolateral side of distal tubular cells as well as on the macula densa cells, which may be particularly significant in regulating interstitial AngII levels. Increases in renal interstitial fluid AngII levels have been reported for 2 models of hypertension. Siragy and Carey¹³ found that renal interstitial AngII is increased in the wrapped kidney of rats with 2-kidney, 1-wrap Grollman hypertension. Nishiyama et al^14,15 reported that renal interstitial fluid AngII concentrations are increased in rats infused with AngII for 2 weeks. Because the renal interstitial values are greater than what can be explained on the basis of equilibration with the plasma concentrations, it has been suggested that local regulation of AngII formation in the renal interstitial compartment and enhanced production of interstitial AngII might be secondary to specialized AngII-forming pathways or accumulation mechanisms.¹⁵ The presence of the PRR at the basolateral side of distal tubular segments cells may contribute to the pool of AngII in this renal compartment.

To investigate further the mechanisms underlying the maintained capability to form intrarenal AngII in both kidneys of the 2K1C Goldblatt rat model regardless of the suppression of renin produced by the juxtaglomerular cells, we also analyzed the gene expression of the PRR in both kidneys of 2K1C rats. During 2K1C Goldblatt hypertension, a cascade of events initiated by the increases of renin secretion from the CK is followed by early increases in circulating AngII that ultimately inhibit juxtaglomerular renin synthesis and release in the NCK. High-circulating AngII levels return to normal after 2-3 weeks of unilateral renal clipping.¹⁶ We have suggested that increased renin immunoreactivity and protein levels in the collecting duct cells with augmented mRNA renin levels in the renal medullary tissues of both CKs and NCKs from the 2K1C rats indicate enhanced local synthesis and stimulation of renin in the distal nephron segments independent of blood pressure. Importantly, in this model of hypertension we also found that renin in the medullary tissues of both kidneys of 2K1C hypertensive rats is mostly active.⁵

In the present study, we show that PRR gene expression is augmented in the CK but not in the NCK of 2K1C hypertensive rats. Krebs et al¹⁷ also reported an upregulation of PRR in the CKs of Goldblatt rats associated with a redistribution and upregulation of renin upstream from the glomerulus in preglomerular vessels and in distal tubules.¹⁷ The researchers did not study the NCKs. More recently, however, Muller et al¹⁸ did not find differences in PRR gene expression in either the CKs or the NCKs compared with sham controls.

We have also demonstrated that the PRR is augmented in the collecting ducts of rats infused with AngII during 14 days⁹ and in Cyp1a1Ren2-transgenic rats.¹⁹ In addition, a soluble form of the PRR [s(P)RR] has been described²⁰ and found augmented in the urine of chronic AngII hypertensive rats.⁹ Urine collected from AngII-infused rats incubated with recombinant prorenin showed higher AngI formation correlated with the augmentation of s(P)RR and the augmentation of the PRR mRNA levels.⁹ Augmented renin synthesis and secretion by the principal cells of the collecting ducts can combine with the membrane-bound PRR and/or bind to the s(P)RR in the tubular fluid, leading to AngI formation. Thus, regardless of whether the PRR is upregulated or not in the nonclipped kidney of the 2K1C hypertensive rat model, its presence in the distal nephron segments cannot be neglected. We suggest that in the 2K1C Goldblatt rat hypertension model—particularly in the NCK—and in the chronic AngII rat⁹ and the Cyp1a1-Ren2 transgenic rat¹⁹ models, the presence of PRR in this segment of the nephron may contribute substantially to the continued capability to form AngI by binding renin or prorenin locally synthesized and augmented in the collecting duct cells of 2K1C hypertensive rats.^5,6,17

CONCLUSIONS

Recent investigations of the intrarenal renin-angiotensin system have raised our awareness about its multiple components that are regulated independently from the systemic renin-angiotensin system. We propose that in AngII-dependent hypertension, augmented proximal tubular secretion of angiotensinogen into the lumen spills over to the distal nephron and that the coordinated actions of enhancement of renin and the PRR in distal nephron segments further enhance intratubular AngII formation in distal nephron segments, thus allowing for greater distal tubular sodium reabsorption.⁶ Reduced sodium excretion contributes to the development and progression of hypertension. The luminal localization of the PRR suggests the intriguing possibility of serving to anchor renin and/or prorenin, thus preventing or minimizing its washout into the urine.