Hayo Castrop

Physiology of Kidney Renin

The protease renin is the key enzyme of the renin-angiotensin-aldosterone cascade, which is relevant under both physiological and pathophysiological settings. The kidney is the only organ capable of releasing enzymatically active renin. Although the characteristic juxtaglomerular position is the best known site of renin generation, renin-producing cells in the kidney can vary in number and localization. (Pro)renin gene transcription in these cells is controlled by a number of transcription factors, among which CREB is the best characterized. Pro-renin is stored in vesicles, activated to renin, and then released upon demand. The release of renin is under the control of the cAMP (stimulatory) and Ca(2+) (inhibitory) signaling pathways. Meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control the renin secretion directly at the level of renin-producing cells, by activating either of the signaling pathways mentioned above. The broad spectrum of biological actions of (pro)renin is mediated by receptors for (pro)renin, angiotensin II and angiotensin-(1-7).

Function of the Juxtaglomerular Apparatus: Control of Glomerular Hemodynamics and Renin Secretion

More than a century ago, Golgi observed that “the ascending limb of the loop of Henle returns with invariable constancy to its capsule of origin”. 1 At this point of contact at the glomerular hilum, the afferent and efferent arterioles together with the adherent distal tubule form a wedge-shaped compartment which contains the three defining cell types of the juxtaglomerular apparatus (JGA) ( Figure 23.1 ). The macula densa (MD) cells in the wall of the tubule abut on a cushion of closely packed interstitial cells called Goormaghtigh or lacis cells. These cells are indistinguishable in their fine structure from mesangial cells 2 and are also referred to as extraglomerular mesangial (EGM) cells. The third specialized cell type of the JGA is the juxtaglomerular granular (JG) cell, a modified smooth muscle cell in the media of the arteriolar wall.

Intrarenal Renin Angiotensin System Revisited: ROLE OF MEGALIN-DEPENDENT ENDOCYTOSIS ALONG THE PROXIMAL NEPHRON

The existence of a local renin angiotensin system (RAS) of the kidney has been established. Angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE), angiotensin receptors, and high concentrations of luminal angiotensin II have been found in the proximal tubule. Although functional data have documented the relevance of a local RAS, the dualism between biosynthesis and endocytotic uptake of its components and their cellular processing has been incompletely understood. To resolve this, we have selectively analyzed their distribution, endocytosis, transcytosis, and biosynthesis in the proximal tubule. The presence of immunoreactive AGT, restricted to the early proximal tubule, was due to its retrieval from the ultrafiltrate and storage in endosomal and lysosomal compartments. Cellular uptake was demonstrated by autoradiography of radiolabeled AGT and depended on intact endocytosis. AGT was identified as a ligand of the multiple ligand-binding repeats of megalin. AGT biosynthesis was restricted to the proximal straight tubule, revealing substantial AGT mRNA expression. Transgenic AGT overexpression under the control of an endogenous promoter was also restricted to the late proximal tubule. Proximal handling of renin largely followed the patterns of AGT, whereas its local biosynthesis was not significant. Transcytotic transport of AGT in a proximal cell line revealed a 5% recovery rate after 1 h. ACE was expressed along late proximal brush-border membrane, whereas ACE2 was present along the entire segment. Surface expression of ACE and ACE2 differed as a function of endocytosis. Our data on the localization and cellular processing of RAS components provide new aspects of the functional concept of a “self-contained” renal RAS.

Adenosine Induces Vasoconstriction through Gi-Dependent Activation of Phospholipase C in Isolated Perfused Afferent Arterioles of Mice

Adenosine induces vasoconstriction of renal afferent arterioles through activation of A1 adenosine receptors (A1AR). A1AR are directly coupled to Gi/Go, resulting in inhibition of adenylate cyclase, but the contribution of this signaling pathway to smooth muscle cell activation is unclear. In perfused afferent arterioles from mouse kidney, adenosine and the A1 agonist N(6)-cyclohexyladenosine, when added to the bath, caused constriction in the concentration range of 10(-9) to 10(-6) M (mean diameter: control, 8.8 +/- 0.3 micro m; adenosine at 10(-6) M, 2.8 +/- 0.5 micro m). Adenosine-induced vasoconstriction was stable for up to 30 min and was most pronounced in the most distal part of the afferent arterioles. Adenosine did not cause vasoconstriction in arterioles from A1AR-/- mice. Pretreatment with pertussis toxin (PTX) (400 ng/ml) for 2 h blocked the vasoconstricting action of adenosine or N(6)-cyclohexyladenosine. PTX pretreatment did not affect the constriction response to KCl, whereas the angiotensin II dose-response relationship was shifted rightward. Reverse transcription-PCR revealed expression of Gi but not Go in kidney cortex and preglomerular vessels. The phospholipase C inhibitor U73122 (4 micro M) blocked the constriction responses to both adenosine and angiotensin II. In contrast, the adenylate cyclase inhibitor SQ22536 (10 micro M) and the protein kinase A antagonist KT5720 (0.1 and 1 micro M) did not induce significant vasoconstriction of afferent arterioles. It is concluded that the constriction response to adenosine in afferent arterioles is mediated by A1AR coupled to a PTX-sensitive Gi protein and subsequent activation of phospholipase C, presumably through betagamma subunits released from Galphai.

Dietary Salt Intake Modulates Angiotensin II Type 1 Receptor Gene Expression

This study aimed to characterize the influence of dietary salt intake on the gene expression of angiotensin II type 1 (AT1) receptor subtypes in different organs. Male Sprague-Dawley rats were fed low salt (0.2 mg/g), normal salt (6 mg/g), or high salt (40 mg/g) diets for 5, 10, and 20 days. mRNA levels for the two AT1 receptor subtypes were determined in adrenal gland, kidney, liver, and lung. In all of the organs examined, with the exception of the adrenal glands, low salt diet led to a transient decrease in the abundance of AT1A receptor mRNA but not of AT1B mRNA, which reached their nadirs between days 5 and 10 of feeding. In the adrenal gland, in which the AT1B receptor is predominant, low salt diet led to a transient increase in the expression of this receptor gene, with a maximum around day 10 of feeding. High salt diet exerted no significant influence on AT1 receptor gene expression in these organs. These findings indicate that the rate of salt intake, in particular, a reduction of salt intake, significantly influences AT1 receptor gene expression in an organ-, time-, and subtype-dependent fashion. It appears that AT1 receptor subtypes are differentially influenced by low salt intake, in that AT1B receptor gene expression increases and AT1A receptor gene expression decreases in this situation. This differential response of AT1 receptor gene expression may be relevant for the organism to be able to adapt to a reduction in oral salt intake.

Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A-/- mice were generated by homologous recombination. NKCC2A-/- mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A-/- compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high- and a low-salt diet on plasma renin concentration were similar in NKCC2A+/+ and -/- mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A-/- mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A-/- mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A-/- compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high- and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range.

Mediators of tubuloglomerular feedback regulation of glomerular filtration: ATP and adenosine

In the juxtaglomerular apparatus of the kidney the loop of Henle gets into close contact to its parent glomerulus. This anatomical link between the tubular system and the vasculature of the afferent and efferent arteriole enables specialized tubular cells, the macula densa (MD) cells, to establish an intra‐nephron feedback loop designed to control preglomerular resistance and thereby single nephron glomerular filtration rate. This review focuses on the signalling mechanisms which link salt‐sensing MD cells and the regulation of preglomerular resistance, a feedback loop known as tubuloglomerular feedback (TGF). Two purinergic molecules, ATP and adenosine, have emerged over the years as most likely candidates to serve as mediators of TGF. Data will be reviewed supporting a role of either ATP or adenosine as mediators of TGF. In addition, a concept will be discussed that integrates both ATP and adenosine into one signalling cascade that includes (i) release of ATP from MD cells upon increases in tubular salt concentration, (ii) extracellular degradation of ATP to form adenosine, and (iii) adenosine‐mediated vasoconstriction of the afferent arteriole.

Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene (Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.

Macula Densa Control of Renin Secretion and Preglomerular Resistance in Mice with Selective Deletion of the B Isoform of the Na,K,2Cl Co-Transporter

Na,K,2Cl co-transporter (NKCC2), the primary NaCl uptake pathway in the thick ascending limb of Henle, is expressed in three different full-length splice variants, called NKCC2F, NKCC2A, and NKCC2B. These variants, derived by differential splicing of the variable exon 4, show a distinct distribution pattern along the loop of Henle, but the functional significance of this organization is unclear. By introduction of premature stop codons into exon 4B, specific for the B isoform, mice with an exclusive NKCC2B deficiency were generated. Relative expression levels and distribution patterns of NKCC2A and NKCC2F were not altered in the NKCC2B-deficient mice. NKCC2B-deficient mice did not display a salt-losing phenotype; basal plasma renin and aldosterone levels were not different from those of wild-type mice. Ambient urine osmolarities, however, were slightly but significantly reduced. Distal Cl concentration was significantly elevated and loop of Henle Cl absorption was reduced in microperfused superficial loops of Henle of NKCC2B-deficient mice. Because of the presence of NKCC2A in the macula densa, maximum tubuloglomerular feedback responses were normal, but tubuloglomerular feedback function curves were right-shifted, indicating reduced sensitivity in the subnormal flow range. Plasma renin concentration in NKCC2B-deficient mice was reduced under conditions of salt loading compared with that in wild-type mice. This study shows the feasibility of generating mice with specific deletions of single splice variants. The mild phenotype of mice that are deficient in the B isoform of NKCC2 indicates a limited role for NKCC2B for overall salt retrieval. Nevertheless, the high-affinity NKCC2B contributes to salt absorption and macula densa function in the low NaCl concentration range.

Atrap Deficiency Increases Arterial Blood Pressure and Plasma Volume

The angiotensin receptor-associated protein (Atrap) interacts with angiotensin II (AngII) type 1 (AT1) receptors and facilitates their internalization in vitro, but little is known about the function of Atrap in vivo. Here, we detected Atrap expression in several organs of wild-type mice; the highest expression was in the kidney where it localized to the proximal tubule, particularly the brush border. There was no Atrap expression in the renal vasculature or juxtaglomerular cells. We generated Atrap-deficient (Atrap-/-) mice, which were viable and seemed grossly normal. Mean systolic BP was significantly higher in Atrap-/- mice compared with wild-type mice. Dose-response relationships of arterial BP after acute AngII infusion were similar in both genotypes. Plasma volume was significantly higher and plasma renin concentration was markedly lower in Atrap-/- mice compared with wild-type mice. (125)I-AngII binding showed enhanced surface expression of AT1 receptors in the renal cortex of Atrap-/- mice, accompanied by increased carboanhydrase-sensitive proximal tubular function. In summary, Atrap-/- mice have increased arterial pressure and plasma volume. Atrap seems to modulate volume status by acting as a negative regulator of AT1 receptors in the renal tubules.