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Due to its pivotal role in blood pressure control and renal pathologies there is renewed interest in renin and its precursor prorenin. Also, the newly discovered (pro)renin receptor is a new element of the ever broadening renin-angiotensin system (RAS). The complexity of RAS including the recently recognized collecting duct site of (pro)renin (a term denoting both renin and prorenin) synthesis requires the use of advanced research techniques such as multiphoton fluorescence microscopy. With the help of this technology we have pioneered an imaging approach to directly visualize (pro)renin content, release and tissue activity in the living kidney. The use of this technology is reviewed here and exemplified by the direct visualization of (pro)renin activity in the collecting duct. New pharmacological tools, the renin inhibitor aliskiren and the handle region peptide (decoy peptide) was used to further characterize the intra-renal, collecting duct RAS.
Multiphoton excitation laser-scanning fluorescence microscopy is a state-of-the-art imaging approach to study basic organ functions in a real-time, quantitative manner with deep optical sectioning capability and with high spatial and temporal resolution. In contrast to conventional confocal imaging that uses UV or visible laser beams (193–694 nm), multiphoton excitation applies infrared lasers (680–1080 nm) that are advantageous for deep penetration into living tissues with complex, light scattering anatomical structure such as the renal cortex. The technology is based on the concept that the simultaneous absorption of two photons of equal energy can cause excitation of a fluorophore equivalent to the absorption of a single photon with double energy (1–3). With multiphoton imaging, these longer (double) wavelength photons (half of the energy) allow deeper penetration into tissues with much less scattering and phototoxic effects.
We applied multiphoton microscopy to visualize living renal tissues in vitro as well as in vivo including direct quantitative imaging of basic functions in renal (patho)physiology in the intact, whole kidney such as single nephron filtration rate (SNGFR) (4), changes in blood flow and tubular flow (4–5), renal concentration, dilution, and permeability of the glomerular filtration barrier (4, 6–7). Very recently we established in vivo imaging of cytosolic variables like intracellular pH and [Ca2+] in the intact kidney (3, 8) as well as more integrated and complex functions such as tubuloglomerular feedback (TGF) (4, 9–11). In addition to the above we have successfully visualized renin granule content, release, and tissue renin activity in the juxtaglomerular apparatus (JGA), the main structural component of the renin-angiotensin system (RAS) (12–16) which is described in detail below. In combination with molecular, cell culture and transgenic animal techniques, the multiphoton imaging approach is a novel, complex tool to study the RAS and (pro)renin in health and disease.
The systemic RAS plays a key role in the regulation of blood pressure and in the maintainance of body fluid and electrolyte homeostasis. In addition, in many organs a local tissue RAS is involved in tissue growth, remodeling and development (17). The release of renin from the juxtaglomerular granular cells in the kidney is considered as the rate limiting step of RAS activation and it is controlled by several factors such as the sympathetic nervous system, renal perfusion pressure and the distal tubular salt content at the macula densa. According to the existing paradigm, renin and its biosynthetic precursor prorenin are mainly produced in the kidney by the granular cells of the JGA in the terminal afferent arteriole (18). However, recent work has revealed the expanded presence of the RAS, revealing its complexity compared to the traditional model, in particular in pathological states such as diabetes mellitus. Activation of the intra-renal RAS in diabetes has been well established and it involves dissimilar cell types including mesangial cells, podocytes, immune cells and the tubular epithelium with special regard to the connecting tubule and the cortical collecting duct (CD) as detailed below (19–20).
Renin is known as a hormone, enzyme and more recently a signaling molecule as well (21–23). Considering its pivotal role in RAS activation the renal renin content and release have been studied by using many methods including electron microscopy (24), radioimmunoassays (25), and patch-clamp techniques (26). However, none of these techniques allowed direct visualization of the renin granules in the living tissue and in real-time. Fluorescence imaging of renin granules using the dye quinacrine was first performed in hemorrhagic and ischemic models of rats (27), but the dynamics of renin exocytosis was visualized only recently. Our laboratory established a multiphoton microscopy approach to directly visualize both renin content, release and activity in freshly dissected JGA preparations in vitro as well as in the intact kidney in vivo with high spatial and temporal resolution down to the individual granule level (3–4, 12–16).
As general rules for any contrast agent, non toxic, water soluble fluorophores must be used with fluorescence imaging applications as well. One good example is quinacrine which is freely permeant to cell membranes and accumulates in the cellular organelles with low pH. This dye clearly and intensely labels renin granules as illustrated in Figure 1A and in previous publications (12–16). However, weak staining can be observed in all cell types most probably due to its accumulation in the lysosomes and cell nuclei as well. Co-localization of intravital quinacrine fluorescence with renin immunofluorescence validated the use of this dye to label renin granules in vivo (3). The application of this imaging approach allowed the investigation of JGA renin secretion in more detail (12–16). Low salt diet for 1 week caused an approximately 5-fold increase in both the number of individual granules and renin-positive JG cells (12). Following treatment with isoproterenol, a beta-agonist, the classic signs of exocytosis were observed by real-time imaging: the association of the emptying granule content with an extracellular cloud of quinacrine fluorescence. The dimming and disappearance of large fluorescence renin granules (1–2 micrometer in diameter) were fast and took place within 300 ms, while no signs of trafficking were observed (12).
In addition to imaging renin content and release, the enzymatic activity of renin or that of activated prorenin can be visualized. A new fluorogenic peptide substrate has been recently developed for renin (AnaSpec, San Jose, CA) which contains a pair of donor-acceptor fluorophores connected by a sequence (decapeptide) of rodent angiotensinogen, including the renin cleavage site at the Leu-Leu bond (a substrate for human renin is available as well). In the absence of renin activity, the donor (5-FAM) fluorescence is quenched by the acceptor molecule (QXL™ 520) due to their close proximity and fluorescence resonance energy transfer (FRET) between them. However, following cleavage into two separate fragments by rat or mouse renin, the intense green fluorescence of 5-FAM is recovered, and can be detected. This fluorescence-based technique permits real-time measurement of renin activity, does not utilize radioactivity, and is conveniently performed within minutes, as opposed to conventional renin assays using radioimmuno-methods. We recently reported that by using this technique the most intense renin activity was observed in the renin-containing terminal part of the afferent arteriole, in the JGA and inside the glomerulus (3, 12). In addition to active renin, this fluorogenic renin substrate can be used to measure the enzymatic activity of activated prorenin (32). The linear relationship between the amount of (pro)renin and the fluorescence intensity of the renin substrate was demonstrated in an earlier study from our laboratory which found 3–35-fold elevations in (pro)renin activity in plasma, renal tissue and cells in response to diabetes and angiotensin II (ANGII) (32). Additional details and examples of imaging renin content, release and activity in the intact kidney tissue can be found in recent reviews on this topic (3, 13–14).
While the JGA is considered to be the classic anatomical site of the RAS within the kidney (18), a body of evidence has revealed that the principal cells of the connecting tubule and collecting duct (CD) can synthesize significant amounts of (pro)renin de novo (28–32). Tubular (pro)renin expression is especially high in pathological states associated with high ANGII such as renovascular hypertension and diabetes mellitus (30–32).
Diabetes mellitus is strongly associated with high RAS activity, high plasma prorenin levels but paradoxically low plasma renin activity (33–34). Using multiphoton imaging we found a significant supply of (pro)renin in the CD under diabetic conditions which finding was confirmed using specific molecular techniques (32). Conversion of prorenin to renin by trypsinization revealed that the vast majority of CD renin is actually prorenin leading to the conclusion that the CD is the major source of prorenin in diabetes (32). Previous studies by our laboratory and others showed that ANGII differentially regulates the synthesis of JGA renin (inhibition) and cortical CD renin (stimulation) (30–32). These findings offered an explanation for the paradoxically high prorenin levels and the persistent RAS activity despite the suppressed JGA in diabetes (32).
What is the functional significance of this local, distal nephron RAS? Interestingly, (pro)renin seems to be secreted toward both the tubular lumen and the interstitium suggested by the high density of (pro)renin granules visualized at both the apical and basal regions of principal cells (32). Cells of the proximal tubule secrete angiotensinogen into the tubular fluid under the same high ANGII conditions, which spills over the entire nephron and collecting ducts (17, 35). Angiotensinogen in the tubular fluid of the collecting duct could be cleaved by the locally secreted (pro)renin (32) with further conversion to ANGII by luminal ACE (36). It is well known that the distal nephron-CD segments contains ANGII type 1 receptors (17, 37). This local, intra-tubular RAS can facilitate salt reabsorption by acting on the epithelial Na+ channel (38). On the other hand, (pro)renin release into the interstitium can exert both systemic endocrine effects via reabsorption into peritubular capillaries and paracrine effects via the recently described (pro)renin receptor [(P)RR] (39). Taken together, the exact role of the distal nephron-CD RAS is likely complex and it should be further studied. Multiphoton microscopy offers great advantages for future work in this area of research using cortical and medullary tissues in vitro or the intact kidney in vivo (Figure 1A, B).
The recently identified (pro)renin receptor [(P)RR] functions to amplify renin enzymatic activity (ANGI generation) and to trigger intracellular signaling (39–40). In addition to renin, the (P)RR binds prorenin equally well and its binding to (P)RR causes a conformational change in prorenin leading to non-proteolytic activation and full enzymatic activity (39–40). (P)RR activation by (pro)renin also results in intracellular signaling via the ERK1/2 MAP kinases, p38 and COX-2 which are essential players in various pathological processes including cell proliferation and tissue fibrosis (39–46).
Previous immunohistochemical studies localized the (P)RR to glomerular mesangial cells, vascular smooth muscle cells of renal and coronary arteries on frozen sections, while it was observed in glomerular mesangial cells and in distal and collecting tubular cells, along their basolateral cell membrane, using paraffin embedded tissue (45).
To gain further insights in the function of the (P)RR in the kidney we now report for the first time the visualization of CCD (pro)renin activity (either active renin or (P)RR-bound, activated prorenin) using multiphoton fluorescence microscopy. This approach was similar to our previous studies using the FRET-based fluorogenic renin substrate to visualize renin and its activity in the JGA (3, 12). The imaging of tissue (pro)renin activity in situ proved to be a great tool to indirectly visualize the localization and activity of the (P)RR in real-time. The isolated CCDs were freshly dissected from C57BL6 mouse kidneys and prepared as described before by our laboratory (47), but were not perfused in the present study. Similarly, the methods of imaging and the use of the fluorogenic renin substrate have been reported earlier in detail (3, 12). The addition of 10 microM renin FRET substrate (5-FAM/QXL™ 520, AnaSpec, San Jose, CA) to the bathing solution resulted in the labeling of most cells of the CCD within seconds (Figure 2A). In addition to significant intracellular labeling, the most intense (pro)renin activity was found at the base of the tubular cells. The pattern of labeling was reminiscent of the basolateral cell membrane and its infoldings (Figure 2A). There was no detectable fluorescence in the bathing solution surrounding the tubule preparation suggesting that the (pro)renin activity (ANGI generation) was membrane-bound. These findings are consistent with the immunohistochemical localization of the (P)RR by Nguyen who found intense labeling at the basolateral membrane of the distal tubule and CCD (45).
Next we applied newly developed pharmacological tools to support (pro)renin and (P)RR-specificity of these data. It is known that the (P)RR interacts with (pro)renin via its handle region which is essential to maintain the prosegment over the active site. Due to its binding to the (P)RR, prorenin undergoes a conformational change and as a result the active site becomes accessible for angiotensinogen (or the renin substrate in our case) as well as for a renin inhibitor. The so-called handle region peptide (HRP) or decoy peptide (NH2-IPLKKMPS-COOH) has been recently developed that mimics a specific amino acid sequence in the prorenin handle region within the prosegment and acts as a peptide antagonist of (P)RR in a competitive manner (46, 48–49). Therefore, in the present experiments we used HRP and aliskiren, a newly developed and marketed renin inhibitor. Figure 2B shows that the renin substrate’s fluorescence intensity ((pro)renin activity) was almost completely prevented by pre-incubating the preparation for 10 min with 100 microM aliskiren. (Pro)renin activity in cells of the CCD was 174±13 (average±SEM) fluorescence units (U) without (n=19), and 67±8 U with aliskiren pretreatment (n=10, p<0.05 using ANOVA followed by post-hoc tests (Bonferroni correction) for multiple comparisons). Importantly, a significant inhibitory effect was observed also when the preparation was pre-incubated with 100 microM HRP (129±13 U, n=11, p<0.05)(Figure 2C). Similar pretreatment with a control peptide of unrelated amino acid sequence (NH2-MTRLSAE-COOH) had no effect on baseline (pro)renin activity (158±45 U, n=9). These data indicate that the renin substrate’s fluorescence intensity was specifically due to (pro)renin activity and suggests that (pro)renin binding to the (P)RR was an important determinant of tissue (pro)renin activity in terms of both intensity and localization. The level of endogenous tissue fluorescence before the application of the renin substrate (Figure 2D) was below the limits of detection which further supports (pro)renin specificity of our technique. Although both aliskiren and HRP, administered in equimolar concentrations (100 microM) significantly inhibited (pro)renin activity in the CCD, aliskiren was more effective (Figure 2E). Our data are in agreement with the basolateral membrane localization of the (P)RR (45), where the HRP can act by inhibiting either the (P)RR-dependent activation of prorenin or the amplification of the enzymatic effect of mature renin.
The combination of multiphoton fluorescence imaging of the living kidney tissue with new fluorescent tools (like the FRET-based fluorogenic renin substrate) and pharmacological inhibitors (aliskiren, HRP) provide novel experimental approaches to study the localization and activity of the (P)RR in the intact kidney. The present imaging studies provided new clues regarding the function of (P)RR in the renal collecting duct which is a newly recognized site of intra-renal (pro)renin synthesis, particularly in high ANGII states. Consistent with the results of previous (P)RR immunolocalization studies showing intense CCD labeling, and with the important role of (P)RR to amplify ANGI generation, high (pro)renin enzymatic activity was found in the basolateral cell membrane of living CCDs. Importantly, the (P)RR decoy peptide HRP effectively reduced (pro)renin activity in this in vitro preparation suggesting that HRP does inhibit at least one function of (P)RR, namely the amplification of ANGI generation by receptor-bound (pro)renin. Since the ability of HRP to block (P)RR activation and subsequent intracellular signaling is currently debated (50–51), the present studies provide an alternative mechanism of HRP’s action. Inhibition of ANGI generation by HRP likely resulted in reduced intra-renal RAS activation in the disease models in which HRP was found to be protective. However, HRP was reported to prevent the development of pathology also in mice deficient of the AT1 receptor (22). Clearly, more studies are needed to carefully test the effects of this interesting decoy peptide and the binding characteristics of the (P)RR.
This work was supported by grants from NIH DK064324, and AHA EIA 0640056N to J. P-P. Á. P. was supported by the Council on International Educational Exchange (CIEE) during these studies. The authors thank Drs. Ildikó Toma and Arnold Sipos for their help with some of the imaging projects.