Expression of Components of the Renin-angiotensin System in Autosomal Recessive Polycystic Kidney Disease
Department of Pediatrics and Pediatric Research Institute, Saint Louis University, St. Louis, Missouri
Correspondence to: Mahmoud Loghman-Adham, MD, Basking Ridge, NJ 07920. E-mail: mloghman{at}att.net
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Summary |
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Key Words: angiotensinogen hypertension polycystic kidney pressure-natriuresis renin
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Introduction |
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Extracellular volume expansion has been reported in both ADPKD and ARPKD patients before the onset of renal failure (Kaplan et al. 1989; Nash 1977
). A few studies have examined the involvement of the RAS in hypertension in ADPKD patients, but no consistent relationship has been found between blood pressure and plasma renin activity (Chapman et al. 1990
; Seeman et al. 1997
). It is now well accepted that increased activity of the intrarenal rather than the systemic RAS is involved in many forms of hypertension (Davisson et al. 1999
; Navar et al. 2002
). It is postulated that persistent elevations of intrarenal angiotensin II (Ang II) production coupled with the inability to reduce Ang II formation in response to a high sodium intake, will lead to a resetting of the pressure-natriuresis relationship toward higher blood pressures, thus leading to hypertension (Gross et al. 1994
; Hall et al. 1996
,1999
).
We recently found that renin and other components of the RAS are expressed by cysts and dilated tubules in ADPKD kidneys (Loghman-Adham et al. 2004). Our results confirmed and extended earlier reports by Torres et al. (1992)
of renin immunostaining in cystic tubules and cysts of ADPKD kidneys. Despite different genetic basis and different gene products, ARPKD and ADPKD share many phenotypic features such as renal cysts and a high prevalence of hypertension. Because of similarities between ARPKD and ADPKD, we hypothesized that ectopic expression of components of the RAS may also be a feature of ARPKD kidneys. We undertook the present immunohistochemical studies on kidney tissues obtained from two infants with ARPKD who had died in the neonatal period and two infants who had died from causes other than kidney disease. We show the ectopic presence of immunoreactive renin and angiotensinogen (AGT) and other components of the RAS in tubules and cysts of ARPKD kidneys. The results provide further support for the hypothesis that, in polycystic kidney disease, ectopic overexpression of components of the RAS could result in increased intrarenal and intratubular Ang II production. Sustained increases in Ang II concentrations might result in hypertension via resetting of the pressure-natriuresis mechanism.
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Materials and Methods |
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Immunohistochemistry of Tissue Sections
Four-micron tissue sections were used for immunohistochemistry. The sections were deparaffinized in HemoDe (Fisher Scientific, Pittsburgh, PA), then rehydrated in graded alcohols. Antigen retrieval was performed with 0.01 M citrate buffer, pH 6.0, at 60C for 30 min. The slides were rinsed twice with phosphate-buffered saline (PBS), followed by the addition of 0.6% H2O2 in 20% methanol for 20 min at room temperature to block endogenous peroxidase. They were washed three times with PBS, then blocked with normal horse serum for 20 min at room temperature. The primary antibodies were added at dilutions indicated in Table 1. The slides were incubated either 1 hr at room temperature or overnight at 4C, then washed three times with PBS/0.1% Tween-20, followed by the addition of the second biotinylated antibody and incubated for 30 min at room temperature. For lectin-binding studies, biotinylated lotus tetragonolobus (LTA) and arachis hypogaea (PNA) lectins were used directly at this stage. LTA is a marker of proximal tubules and PNA is a marker of distal and collecting tubules. The slides were washed three times with PBS, followed by the addition of 1 drop of the ABC reagent (Vectastain Elite kit, Vector Laboratories, Burlingame, CA) and incubated at room temperature for 30 min. The slides were washed three times with PBS, followed by the addition of peroxidase substrate for 610 min. They were rinsed in distilled water, counterstained with hematoxylin (Gill No 3, Sigma Diagnostics, St Louis, MO) for 6090 sec, washed extensively in running water, and mounted. They were viewed with a Zeiss Axioplan microscope or with an Olympus CX41 microscope, equipped with a DP12 digital camera, and photographed.
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Results |
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Previous studies have shown that in ARPKD kidneys, cysts are derived primarily from the collecting ducts (Verani et al. 1989). Proximal tubule-derived cysts have been observed in human ARPKD fetal kidney specimens, ranging in gestational age from 14 to 26 weeks (Nakanishi et al. 2000
). To ascertain the tubule origin of the cysts in our infantile ARPKD kidney specimens, we used staining with lectins specific for proximal and distal tubules (Faraggiana et al. 1982b
). LTA lectin was used as a marker of proximal tubules and PNA was used as a marker of distal/collecting tubules (Faraggiana et al. 1982b
). In normal control kidneys, using the LTA lectin, we observed distinct staining of the brush borders of proximal tubules (Figure 1A)
. In ARPKD kidneys, we observed LTA staining of proximal tubules, which were larger and with less distinct brush borders than those of the normal kidney (Figure 1B). The distal tubules and the surrounding cysts did not bind LTA. Using PNA lectin, in normal control kidney sections, we observed extensive staining of distal tubules (Figure 1C). In ARPKD kidneys, we further showed PNA staining of many tubules and a large majority of the cysts, identifying their origin as distal tubule/collecting duct (Figure 1D). Occasional cysts did not stain with either LTA or PNA, suggesting they either originated from other nephron segments or had lost their ability to bind lectins.
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Discussion |
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Recently, renin mRNA and protein expression were described in developing tubules and ureteric bud branches in avascular metanephric organ cultures (Norwood et al. 2000). Ureteric bud is the precursor of collecting tubules, which are dilated in ARPKD, suggesting that distal nephron renin expression may be derepressed in ARPKD. Accordingly, in ARPKD kidneys, renin was expressed almost exclusively by the distal tubules and cysts, identified by PNA lectin staining. Only occasional proximal tubules showed renin staining (Figure 2 and Figure 3). Because in ARPKD, cysts are derived from the collecting ducts (Verani et al. 1989
; Silva et al. 1993
), renin expression by cysts expands on previous reports that renin can be produced by the distal nephron (Rohrwasser et al. 1999
; Prieto-Carrasquero et al. 2004
). AGT was expressed in proximal tubules and in
15% of the cysts. To our knowledge, this is the first observation of AGT expression by cells originating from distal tubules or collecting ducts.
It could be argued that renin staining of tubules is a nonspecific finding and merely represents filtered renin endocytosed from the tubule lumen. This would be unlikely because, in ARPKD, the cysts originate from the collecting ducts. Filtered renin is degraded in the proximal tubule and 2% may reach the distal tubules and excreted in the urine (Kim et al. 1987
). Because of its large molecular weight (
57,000 kDa) (Tewksbury et al. 1978
), AGT cannot be filtered by the glomerulus. However, AGT produced within the proximal tubules could reach the collecting ducts and the final urine (Rohrwasser et al. 1999
). Therefore, we cannot exclude the possibility that AGT expression in cysts is a result of nonspecific uptake of AGT from the tubule lumen.
The use of archival kidney tissues to study the presence and distribution of components of the RAS provides limited information. Fresh tissue from nephrectomy specimens is difficult to obtain because of the rare occurrence of ARPKD. If available, additional studies such as mRNA analysis and more quantitative protein analysis by Western blotting could be performed. An immortalized cell line derived from ARPKD kidneys was recently described (Rohatgi et al. 2003). Attempts at demonstrating renin staining in these cells were unsuccessful. Additional studies, perhaps using a different antibody are warranted.
In a recent study of ADPKD kidneys, we showed ectopic renin and AGT expression by cyst epithelium (Loghman-Adham et al. 2004). Contrary to the findings reported here, in ADPKD kidneys, renin staining was not observed in afferent arterioles. We speculate that JGA renin may be downregulated in ADPKD. An alternative hypothesis is that many glomeruli were destroyed in these end-stage kidneys, leading to involution of their arterioles (Loghman-Adham et al. 2004
). In contrast, ARPKD kidneys studied here were from young infants and had relatively well-preserved glomeruli. The expression of RAS components in cysts and tubules in two models of polycystic kidney disease suggests a common mechanism of hypertension in different forms of polycystic kidney disease.
This study provides strong evidence in support of the existence of a paracrine/autocrine intrarenal RAS in ARPKD kidneys (Figure 6) . Renin and AGT production by tubules and cysts along with the presence of ACE at the same locations could result in the formation of Ang I and Ang II. Immunoreactive Ang I and Ang II is seen in tubules and cysts and is likely secreted into the lumen. Ang II could bind to specific receptors along the functioning tubules (Harrison-Bernard et al. 1997) and lead to increased salt and water retention via increased tubular sodium reabsorption both by the proximal tubules (Geibel et al. 1990
) and by the collecting ducts (Wang and Giebisch 1996
; Peti-Peterdi et al. 2002
). Continued increased tubular sodium reabsorption may lead to a shift of the pressure-natriuresis curve to the right such that higher blood pressures would be needed to maintain sodium excretion (Hall et al. 1996
,1999
). The end result would be chronic hypertension, unless sodium intake is reduced. In support of this hypothesis is a recent study in immortalized cyst-lining epithelial cells from ARPKD kidneys that shows that these cells absorb sodium by an amiloride-sensitive pathway (Rohatgi et al. 2003
). Another study, using collecting duct principal cells from a mouse model of ARPKD, suggests decreased amiloride-sensitive sodium absorption (Veizis et al. 2004
). Despite these discrepancies observed in vitro, the net in vivo effect of Ang II excess appears to be increased sodium reabsorption perhaps due to a predominance of the absorptive compared with the secretory sodium fluxes in the functioning tubules.
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Reduced intracellular Ca2+ concentration is known to stimulate renin production by the JGA cells (Schnermann 1998). Renin release from JGA cells is also controlled by changes in early distal flow rate. Increasing the flow rate depresses renin release, whereas reducing flow rate increases renin release (Leyssac 1986
). Bending of cilia by mechanical flow initiates Ca2+ transients resulting in increased intracellular Ca2+ concentrations. Polycystins act both as chemosensors and as flow sensors in renal tubules and transduce mechanical fluid flow signals into Ca2+ signals (Nauli et al. 2003
; Zhang et al. 2004
). Nauli et al. (2003)
have recently shown that the cilia in epithelial cells with a homozygous polycystin mutation fail to sense fluid flow. One could hypothesize that increased renin expression by cyst-lining epithelia in ARPKD is related to inability of the primary cilia to detect and transduce changes in flow or in the composition of the tubular fluid.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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