Mona Oppermann

Lack of A1 Adenosine Receptors Augments Diabetic Hyperfiltration and Glomerular Injury

Intraglomerular hypertension and glomerular hyperfiltration likely contribute to the pathogenesis of diabetic nephropathy, and tubuloglomerular feedback (TGF) has been suggested to play a role in diabetic hyperfiltration. A1 adenosine receptor (A1AR) null mice lack a TGF response, so this model was used to investigate the contribution of TGF to hyperfiltration in diabetic Ins2(+/-) Akita mice. TGF responses in Ins2(+/-) A1AR(-/-) double mutants were abolished, whereas they were attenuated in Ins2(+/-) mice. GFR, assessed at 14, 24, and 33 wk, was approximately 30% higher in Ins2(+/-) than in wild-type (WT) mice and increased further in Ins2(+/-) A1AR(-/-) mutants (P < 0.01 versus both WT and Ins2(+/-) mice at all ages). Histologic evidence of glomerular injury and urinary albumin excretion were more pronounced in double-mutant than single-mutant or WT mice. In summary, the marked elevation of GFR in diabetic mice that lack a TGF response indicates that TGF is not required to cause hyperfiltration in the Akita model of diabetes. Rather, an A1AR-dependent mechanism, possibly TGF, limits the degree of diabetic hyperfiltration and nephropathy.

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.

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.

Regulation of Renin Secretion and Expression in Mice Deficient in β1- and β2-Adrenergic Receptors

The present experiments were performed in &bgr;1/&bgr;2-adrenergic receptor–deficient mice (&bgr;1/&bgr;2ADR−/−) to assess the role of &bgr;-adrenergic receptors in basal and regulated renin expression and release. On a control diet, plasma renin concentration (in ng angiotensin I per mL per hour), determined in tail vein blood, was significantly lower in &bgr;1/&bgr;2ADR−/− than in wild-type (WT) mice (222±65 versus 1456±335; P<0.01). Renin content and mRNA were 77% and 65±5% of WT. Plasma aldosterone (in picograms per mL) was also significantly reduced (420±36 in &bgr;1/&bgr;2ADR−/− versus 692±59 in WT). A low-salt diet (0.03%) for 1 week increased plasma renin concentration significantly in both &bgr;1/&bgr;2ADR−/− and WT mice (to 733±54 and 2789±555), whereas a high-salt diet (8%) suppressed it in both genotypes (to 85±24 in &bgr;1/&bgr;2ADR−/− and to 676±213 in WT). The absolute magnitude of salt-induced changes of plasma renin concentration was markedly greater in WT mice. Acute stimulation of renin release by furosemide, quinaprilat, captopril, or candesartan caused significant increases of plasma renin concentration in both &bgr;1/&bgr;2ADR−/− and WT mice, but again the absolute changes were greater in WT mice. We conclude that maintenance of normal levels of renin synthesis and release requires tonic &bgr;-adrenergic receptor activation. In the chronic absence of &bgr;-adrenergic receptor input, the size of the releasable renin pool decreases with a concomitant reduction in the magnitude of the plasma renin concentration changes caused by variations of salt intake or acute stimulation with furosemide, angiotensin-converting enzyme, or angiotensin type 1 receptor inhibition, but regulatory responsiveness is nonetheless maintained.

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.

Development of vascular renin expression in the kidney critically depends on the cyclic AMP pathway

During metanephric kidney development, renin expression in the renal vasculature begins in larger vessels, shifting to smaller vessels and finally remaining restricted to the terminal portions of afferent arterioles at the entrance into the glomerular capillary network. The mechanisms determining the successive expression of renin along the vascular axis of the kidney are not well understood. Since the cAMP signaling cascade plays a central role in the regulation of both renin secretion and synthesis in the adult kidney, it seemed feasible that this pathway might also be critical for renin expression during kidney development. In the present study we determined the spatiotemporal development of renin expression and the development of the preglomerular arterial tree in mouse kidneys with renin cell-specific deletion of G(s)alpha, a core element for receptor activation of adenylyl cyclases. We found that in the absence of the G(s)alpha protein, renin expression was largely absent in the kidneys at any developmental stage, accompanied by alterations in the development of the preglomerular arterial tree. These data indicate that the maintenance of renin expression following a specific spatiotemporal pattern along the preglomerular vasculature critically depends on the availability of G(s)alpha. We infer from our data that the cAMP signaling pathway is not only critical for the regulation of renin synthesis and secretion in the mature kidney but that it also is critical for establishing the juxtaglomerular expression site of renin during development.

Loss of WNK3 is compensated for by the WNK1/SPAK axis in the kidney of the mouse

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport.

Dietary salt intake modulates differential splicing of the Na-K-2Cl cotransporter NKCC2

Both sodium reabsorption in the thick ascending limb of the loop of Henle (TAL) and macula densa salt sensing crucially depend on the function of the Na/K/2Cl cotransporter NKCC2. The NKCC2 gene gives rise to at least three different full-length NKCC2 isoforms derived from differential splicing. In the present study, we addressed the influence of dietary salt intake on the differential splicing of NKCC2. Mice were subjected to diets with low-salt, standard salt, and high-salt content for 7 days, and NKCC2 isoform mRNA abundance was determined. With decreasing salt intake, we found a reduced abundance of the low-affinity isoform NKCC2A and an increase in the high-affinity isoform NKCC2B in the renal cortex and the outer stripe of the outer medulla. This shift from NKCC2A to NKCC2B during a low-salt diet could be mimicked by furosemide in vivo and in cultured kidney slices. Furthermore, the changes in NKCC2 isoform abundance during a salt-restricted diet were partly mediated by the actions of angiotensin II on AT1 receptors, as determined using chronic angiotensin II infusion. In contrast to changes in oral salt intake, water restriction (48 h) and water loading (8% sucrose solution) increased and suppressed the expression of all NKCC2 isoforms, without changing the distribution pattern of the single isoforms. In summary, the differential splicing of NKCC2 pre-mRNA is modulated by dietary salt intake, which may be mediated by changes in intracellular ion composition. Differential splicing of NKCC2 appears to contribute to the adaptive capacity of the kidney to cope with changes in reabsorptive needs.

Stimulation of Renin Secretion by Angiotensin II Blockade is Gsα-Dependent

Angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) presumably stimulate renin secretion by interrupting angiotensin II feedback inhibition. The increase in cytosolic calcium caused by activation of Gq-coupled AT1 receptors may mediate the renin-inhibitory effect of angiotensin II at the cellular level, implying that ACEI and ARB may work by reducing intracellular calcium. Here, we investigated whether angiotensin II blockade acts predominantly through Gs-mediated stimulation of adenylyl cyclase (AC) by testing the effect of ACEI and ARB in mice with juxtaglomerular cell-specific deficiency of the AC-stimulatory Gsalpha. The ACEI captopril and quinaprilate and the ARB candesartan significantly increased plasma renin concentration (PRC) to 20 to 40 times basal PRC in wild-type mice but did not significantly alter PRC in Gsalpha-deficient mice. Captopril also completely abrogated renin stimulation in wild-type mice after co-administration of propranolol, indomethacin, and L-NAME. Treatment with enalapril and a low-NaCl diet for 7 days led to a 35-fold increase in PRC among wild-type mice but no significant change in PRC among Gsalpha-deficient mice. Three different pharmacologic inhibitors of AC reduced the stimulatory effect of captopril by 70% to 80%. In conclusion, blockade of angiotensin II stimulates renin synthesis and release indirectly through the action of ligands that activate the cAMP/PKA pathway in a Gsalpha-dependent fashion, including catecholamines, prostaglandins, and nitric oxide.

Localization and functional characterization of the human NKCC2 isoforms

Aim:  Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide‐sensitive Na+/K+/2Cl− cotransporter NKCC2. Three full‐length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms.