The kidney proximal tubule is a major target tissue of the renin-angiotensin system (RAS). Megalin is an endocytic multiligand receptor abundantly expressed in the proximal tubule where it drives reabsorption of peptides and proteins from the glomerular ultrafiltrate. All major RAS components are present in the kidney proximal tubules. Here, megalin drives endocytosis of angiotensinogen (AGT), prorenin, and renin, while angiotensin-converting enzyme is localised at the brush border of the proximal tubule cells. Intrarenal formation of the key RAS effector angiotensin II (ANG II) occurs, and liver-derived AGT appears to be the primary source. New studies further suggest that megalin-mediated reabsorption of liver-derived AGT contributes to renal ANG II levels and thereby may influence renal RAS activity. This mini-review presents the recent advances on RAS in the proximal tubule and the involvement of megalin in the uptake and regulation of local RAS and discusses the possibility that megalin is involved in blood pressure regulation.

© 2022 S. Karger AG, Basel

Introduction

Hypertension affects more than 1.5 billion people worldwide and is one of the most important risk factors for development, progression, and mortality of cardiovascular disease. The renin-angiotensin system (RAS) is a molecular cascade causing elevation of systemic blood pressure [1]. Angiotensin II (ANG II) is the key effector of the RAS [2]. It is produced from angiotensinogen (AGT) by the enzymes renin and angiotensin-converting enzyme (ACE) [1]. ANG II induces blood pressure elevation by stimulating the ANG II type 1 receptor (AT1R) [2]. The AT1R is expressed in various body compartments [2], including the renal proximal tubule where it plays an important role in blood pressure regulation [3-5]. ANG II, either derived from the circulation or locally produced within the kidney [6, 7], stimulates the AT1R in the proximal tubules, thereby activating downstream signalling pathways involved in sodium reabsorption and blood pressure elevation [3-5]. Hence, increased renal ANG II levels contribute to progressive hypertension and kidney injury [8], making it an important topic to study.

Sodium Reabsorption in the Proximal Tubule

Regulation of renal sodium excretion is a key determinant of systemic blood pressure [9]. Proximal tubule cells are responsible for ∼70% of sodium reabsorption from the glomerular ultrafiltrate [8]. The sodium hydrogen exchanger 3 (NHE3), which resides in the apical membrane of the proximal tubule cells [10], is the most important proximal tubule sodium transporter [8]. Accordingly, proximal tubule specific deletion of NHE3 lowers sodium reabsorption and systemic blood pressure [8, 10, 11]. It is further established that ANG II-induced blood pressure elevation involves proximal tubular NHE3, and ANG II infusion has been shown to cause lower blood pressure elevation in kidney NHE3 knockout mice than in control mice [8, 12]. Collectively, it is widely accepted that ANG II, via the AT1R, stimulates sodium retention in the proximal tubules, thereby increasing blood pressure [5].

The Megalin Receptor

Megalin is a large (∼600 kDa; 4,655 amino acids) glycosylated endocytic receptor abundantly expressed in the apical membrane of renal proximal tubule epithelial cells [13]. It is a member of the low-density lipoprotein receptor family and comprises a large amino-terminal extracellular domain, a single-spanning transmembrane domain, and a short carboxy-terminal cytoplasmic tail [14]. The extracellular domain constitutes the ligand-binding region and contains four clusters of cysteine-rich complement type/LDLR class A repeats [14]. These repeats are separated by 17 epidermal growth factor-like repeats and eight cysteine-poor spacer regions, which contain YWTD motifs involved in the pH-dependent release of ligands in endosomal compartments [13]. The cytoplasmic tail holds several phosphorylation and protein interaction motifs, including two NPXY motifs and a NPXY-like motif involved in coated pit formation and translocation of megalin to the apical membrane, respectively [15]. Ligand binding to the extracellular domain of megalin leads to coated pit formation and subsequently endocytosis. Endocytic vesicles fuse with the endosomal compartment in which ligands are released from megalin by the acidic environment. Protein ligands are subsequently sent to lysosomes for degradation, whereas megalin recycles to the apical membrane [14].

Megalin and Proximal Tubular RAS

The megalin receptor reabsorbs a wide variety of peptides and proteins from the glomerular ultrafiltrate, including AGT, renin, and its precursor prorenin. Under normal conditions, urine is devoid of prorenin, while only very low levels of AGT and renin can be detected [16]. In patients and mice with impaired megalin function, e.g., patients with Dent’s disease and Lowe syndrome, however, urinary excretion of AGT [7, 17-19], prorenin [19], and renin [7, 17, 19] is significantly increased.

Liver-Derived AGT Contributes to Renal ANG II Levels via Megalin

AGT is predominantly produced in the liver and secreted to the circulation. Liver-derived AGT is a major determinant of renal ANG II levels, as evident by very low renal ANG II levels in liver-specific AGT knockout mice [7, 20]. AGT synthesis has also been detected in segment 3 of the proximal tubule but apparently does not contribute to renal ANG II levels [20].

It is debated to which extent liver-derived AGT is filtered through the glomerulus under normal conditions [21]. However, it has been established that megalin reabsorbs AGT in segment 1 and 2 of the proximal tubule [7, 17, 18, 20]. It is further proposed that megalin-mediated AGT reabsorption contributes to renal ANG II levels. Treatment of wild-type mice with a megalin antisense oligonucleotide, inducing megalin knockdown, had no effect on plasma ANG II levels but resulted in reduced renal ANG II levels measured by mass spectrometry [7]. Moreover, in mice with induced podocyte injury and increased AGT filtration, renal ANG II levels were reduced in megalin knockout mice compared with controls [18]. This study, however, found no difference in renal ANG II levels in megalin knockout mice before podocyte injury induction [18].

Intrarenal Renin and ACE Enable Local ANG II Generation

Renin and ACE, the two enzymes that produce ANG II from AGT, are also both found in the proximal tubules [1]. Renin is the rate-limiting enzyme of ANG II formation. It is derived from its precursor prorenin, which is produced by renal juxtaglomerular cells and secreted into the circulation [2]. Both renin and prorenin are filtered in glomeruli [19] and reabsorbed by megalin [7, 17, 19, 22].

ACE is expressed in various tissues throughout the body, including the brush border of proximal tubule cells [17]. ACE cleaves angiotensin I to ANG II. ACE2, the enzyme that metabolises ANG II to the heptapeptide ANG-(1-7) [17], is also detected in the brush border of proximal tubule cells [17]. ACE2 is found along the entire proximal tubule segment, whereas ACE is expressed with increasing abundance from segment 1 to 3 [17]. Studies in kidney-specific ACE knockout mice found that intrarenal ACE contributes to renal ANG II levels, sodium retention, and development of hypertension. Under chronic ANG II infusion, these mice were unable to produce ANG II in the kidney, which resulted in enhanced sodium excretion and a blunted hypertensive response compared to control mice [23]. In contrast, ACE2 antagonizes ANG II-induced blood pressure elevation. ANG II infusion in ACE2 deficient mice resulted in higher blood pressure levels compared with control mice [24]. Notably, in megalin knockout mice, renal ACE2 protein levels were increased while ACE levels were markedly reduced [17].

Megalin and ANG II

Only few studies have investigated the interplay between megalin and ANG II. ANG II is an 8-amino acid (∼1 kDa) peptide and the main effector molecule of the RAS. Studies in cell cultures expressing megalin have found that ANG II binds to and is internalized by megalin [25, 26]. Surface plasmon resonance analysis confirmed this interaction, suggesting that megalin is a potential ANG II receptor, but no Kd-value was reported [25]. Furthermore, it has been suggested that megalin may be important for ANG II/AT1R-induced downstream signalling events in proximal tubule cells in vitro [26]. Here, ANG II stimulation increased the phosphorylation of MAP kinases ERK1/2 as well as NHE3, whereas treatment with either the AT1R-inhibitor losartan or megalin siRNA both reduced phosphorylated (p-)ERK1/2 and NHE3 levels [26]. Interestingly, megalin-mediated ANG II uptake and effect on downstream signalling in these cells depended on AT1R expression as knockdown of megalin in AT1R knockout cells had no effect on ANG II uptake or p-ERK1/2 and p-NHE3 levels [26]. Further studies are necessary to clarify if and how megalin may affect ANG II/AT1R-endocytosis and signal transduction in vivo. Beyond the cell surface, a role for intracellular ANG II and AT1R in proximal tubule cells has been proposed. The physiologic effect of intracellular ANG II remain poorly understood; however, megalin has been suggested to be involved in mitochondrial ANG II trafficking, and megalin deficiency was associated with impaired mitochondrial respiration and glycolysis in vitro [27, 28].

Pathophysiological Consequences of Megalin-Mediated RAS Reabsorption

Megalin depletion in a number of models leads to alterations in the levels of RAS components in urine and kidney tissue, as summarised in Table 1, suggesting that megalin may play a role in regulating local RAS activity and blood pressure homeostasis. In vivo experiments, in induced podocyte-injured mice with and without megalin knockout, have shown that megalin expression affects renal sodium transporters in mice with podocyte injury. Renal NHE3, but not p-NHE3, protein levels as well as membranous protein levels of epithelial sodium channel subunits were significantly lower in megalin knockout mice with podocyte injury [18]. Furthermore, urinary sodium and chloride excretion as well as urine volume were increased in these mice [18]. These findings suggest that the excessive uptake of filtered AGT via megalin causes increased renal ANG II formation and may aggravate sodium retention and tissue injury in kidney diseases characterized by glomerular filtration barrier defects. Despite changes in sodium excretion and urine volume in these mice, blood pressure measurements, using noninvasive tail-cuff blood pressure measurements, did not find any significant difference in systolic blood pressure [18]. In comparison, proximal tubule-specific AT1R [5] and NHE3 [10] depletion in mice both reduced systemic blood pressure by ∼10 mm Hg. Proportionately, renal megalin-mediated effects on blood pressure would not be expected to exceed a reduction by ∼10 mm Hg and would likely require highly sensitive and continuous blood pressure measurements, such as radiotelemetry.

Table 1.

Summary of RAS component findings upon megalin depletion

In humans, evidence for a role of megalin in blood pressure homeostasis is limited. Dent’s disease and Lowe syndrome patients that have impaired megalin function are reported normotensive [19]. However, a SNP rs133980 polymorphism in the megalin gene, LRP2, has been associated with changes in systolic blood pressure in smokers [29]. In early onset preeclampsia, characterized by new-onset hypertension and proteinuria after 20 weeks of gestation, megalin-mediated AGT uptake has been suggested to play a role in the regulation of placental RAS activity [30]. Placental megalin protein levels were found to be increased in preeclampsia compared with healthy pregnant women, and as placental megalin is suggested to internalize maternal AGT, this increase may contribute to enhanced ANG II generation associated with hypertension in preeclampsia [30].

RAS in Proximal Tubule Cells – Established Findings and Unknowns

The recent findings and some hypotheses on the interplay of proximal tubular RAS and megalin are summarised in Figure 1. Megalin may influence proximal tubular ANG II levels and signalling in several ways. Most clear is the role of filtered liver-AGT, which is taken up by megalin and contributes to renal ANG II levels, particularly in states of increased AGT filtration such as podocyte injury [20]. Megalin deficiency in mice was also associated with reduced ACE, which resides at the luminal brush border membrane [17] and is a determinant of renal ANG II levels [23]. Renin and prorenin are both filtered in the kidney glomeruli and reabsorbed by megalin [7, 17, 19]. Based on these observations, it is speculated that conversion of AGT to ANG II occurs locally, in the tubular lumen [8, 17], intracellularly in endosomes of proximal tubule cells [20], or in the renal interstitium [6]. Locally generated ANG II amplifies ANG II-induced AT1R signalling in the proximal tubule and subsequent downstream events involved in sodium retention and blood pressure regulation [31]. It remains unclear where local ANG II formation occurs and how locally produced ANG II activates intracellular signalling events.

Fig. 1.

Proposed mechanisms of megalin-mediated endocytosis of renin-angiotensin system (RAS) proteins and activity in the proximal tubule. Angiotensinogen (AGT), prorenin, renin, and ANG II are all, at least to some extent, filtered by the kidney glomeruli. In the proximal tubule, megalin binds and internalizes AGT, prorenin, renin and ANG II. Classical megalin-mediated endocytosis directs the megalin:ligand complex to the endosomal compartment. The acidic environment within the endosomal compartment triggers ligand release. Subsequently, megalin is recycled to the brush border membrane, whereas the ligands are delivered to lysosomes for degradation. The angiotensin 1 receptor (AT1R) is both expressed in the brush border and at the basolateral membrane of proximal tubule cells. ANG II stimulation of the AT1R induces downstream signal events involved in sodium retention and blood pressure regulation. ACE is also expressed in the brush border of proximal tubule cells. It is hypothesised (depicted by “?”) that filtered AGT is converted to ANG II in the proximal tubule in the tubular lumen, intracellularly in endosomes, or in the renal interstitium.

Conclusion

Recent advances on RAS in the proximal tubule show that the endocytic receptor megalin plays a role in reabsorbing filtered RAS components. Uptake of RAS proteins by megalin may both be involved in ANG II generation and in ANG II-mediated signalling in the proximal tubule. Future studies are warranted to investigate how and to what extend megalin may influence renal ANG II levels and downstream signalling events involved in blood pressure regulation and hypertension.

Conflict of Interest Statement

The authors declare no conflict of interest.

Funding Sources

This work was supported from the Independent Research Fund Denmark through a Sapere Aude Research Leader Grant to K.W. (DFF 9060-00046B), the Aarhus University Research Foundation Starting Grant to K.W. (AUFF-E-2017-7-25) and by a PhD fellowship to S.H. from Augustinus Fonden (21-1561) and the Danish Cardiovascular Academy (DCA) funded by the Novo Nordisk Foundation (NNF20SA0067242) and The Danish Heart Foundation.

Author Contributions

Sandra Hummelgaard and Kathrin Weyer both wrote and edited this manuscript and approved the final version.

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