Publikationen

Hannah Triebel, Hayo Castrop

Abstract

In this review, we will cover (i) the proteolytic cascade of the RAAS, (ii) its regulation by multiple feedback-controlled parameters, and (iii) the major effects of the RAAS. For the effects of the RAAS, we focus on the role of the RAAS in the regulation of volume homeostasis and vascular tone, as major determinants of arterial blood pressure.

https://pubmed.ncbi.nlm.nih.gov/38233636/ (externer Link, öffnet neues Fenster)

Vera Ziegler, Katharina Fremter, Julia Helmchen, Ralph Witzgall, Hayo Castrop 

Abstract

Aim: The intraglomerular mesangial cells are located between the glomerular capillaries. Here we hypothesized that mesangial cells regulate the single nephron glomerular filtration rate (snGFR) and that mesangial cells support the integrity of the glomerular filtration barrier.

https://pubmed.ncbi.nlm.nih.gov/33269519/ (externer Link, öffnet neues Fenster)

Butt L, Unnersjö-Jess D, Höhne M, Edwards A, Binz-Lotter J, Reilly D, Hahnfeldt R, Ziegler V, Fremter K, Rinschen MM, Helmstädter M, Ebert LK, Castrop H, Hackl MJ, Walz G, Brinkkoetter PT, Liebau MC, Tory K, Hoyer PF, Beck BB, Brismar H, Blom H, Schermer B, Benzing T.

Abstract

Mammalian kidneys constantly filter large amounts of liquid, with almost complete retention of albumin and other macromolecules in the plasma. Breakdown of the three-layered renal filtration barrier results in loss of albumin into urine (albuminuria) across the wall of small renal capillaries, and is a leading cause of chronic kidney disease. However, exactly how the renal filter works and why its permeability is altered in kidney diseases is poorly understood. Here we show that the permeability of the renal filter is modulated through compression of the capillary wall. We collect morphometric data prior to and after onset of albuminuria in a mouse model equivalent to a human genetic disease affecting the renal filtration barrier. Combining quantitative analyses with mathematical modelling, we demonstrate that morphological alterations of the glomerular filtration barrier lead to reduced compressive forces that counteract filtration pressure, thereby resulting in capillary dilatation, and ultimately albuminuria. Our results reveal distinct functions of the different layers of the filtration barrier and expand the molecular understanding of defective renal filtration in chronic kidney disease.

https://pubmed.ncbi.nlm.nih.gov/32694662/ (externer Link, öffnet neues Fenster)

Hayo Catsrop

Abstract

The healthy kidney is considered to be a relatively stable organ with little baseline cell turnover. Nevertheless, cells are constantly replaced to conserve the structural and functional integrity of the organ. The mechanisms of the baseline regenerative processes may also be relevant in situations of insults to the kidney, when the need for cellular replacement considerable exceeds the baseline cell turnover. This review will focus on the mechanisms of the regeneration of the tubular system, in particular the proximal tubule. Specifically, we will cover new aspects of (i), the regenerative capacity of the proximal tubule in health and disease, (ii) the relevant cell populations of proximal tubular regeneration, and (iii) the supportive role of renal interstitial cells in regenerative processes of the tubular system. 

https://karger.com/nef/article/141/4/265/227106/The-Role-of-Renal-Interstitial-Cells-in-Proximal (externer Link, öffnet neues Fenster)

Lisa Kelly, Mohammed M Almutairi, Shams Kursan, Romario Pacheco, Eduardo Dias-Junior, Hayo Castrop, Mauricio Di Fulvio 

Abstract

The Na⁺K⁺2Cl^- cotransporter-2 (Nkcc2, Slc12a1) is abundantly expressed in the kidney and its inhibition with the loop-diuretics bumetanide and furosemide has been linked to transient or permanent hyperglycemia in mice and humans. Notably, Slc12a1 is expressed at low levels in hypothalamic neurons and in insulin-secreting β-cells of the endocrine pancreas. The present study was designed to determine if global elimination of one of the Slc12a1 products, i.e., Nkcc2 variant a (Nkcc2a), the main splice version of Nkcc2 found in insulin-secreting β-cells, has an impact on the insulin and glucagon secretory responses and fuel homeostasis in vivo. We have used dynamic tests of glucose homeostasis in wild-type mice and mice lacking both alleles of Nkcc2a (Nkcc2a^KO) and assessed their islet secretory responses in vitro. Under basal conditions, Nkcc2a^KO mice have impaired glucose homeostasis characterized by increased blood glucose, intolerance to the sugar, delayed/blunted in vivo insulin and glucagon responses to glucose, and increased glycemic responses to the gluconeogenic substrate alanine. Further, we provide evidence of conserved quantitative secretory responses of Nkcc2a^KO islets within a context of increased islet size related to hyperplastic/hypertrophic glucagon- and insulin-positive cells (α-cells and β-cells, respectively), normal total islet Cl^- content, and reduced β-cell expression of the Cl^- extruder Kcc2.

https://pubmed.ncbi.nlm.nih.gov/31365295/ (externer Link, öffnet neues Fenster)

Larionov A, Dahlke E, Kunke M, Zanon Rodriguez L, Schiessl IM, Magnin JL, Kern U, Alli AA, Mollet G, Schilling O, Castrop H, Theilig F.

Abstract
The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria-dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod ) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full-length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+ /K+ -ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.

https://pubmed.ncbi.nlm.nih.gov/31368174/ (externer Link, öffnet neues Fenster)

Schiessl IM, Grill A, Fremter K, Steppan D, Hellmuth MK, Castrop H.

Abstract
Background: The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-β (PDGFRβ) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined.

Methods: In mice with fluorescent protein expression in renal tubular cells and PDGFRβ-positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration.

Results: Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRβ-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFRβ-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFRβ-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFRβ impeded PDGFRβ-positive cell migration to the tubular injury site and compromised the recovery of tubular function.

Conclusions: Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFRβ-mediated communication between the renal interstitium and the tubular system.

https://pubmed.ncbi.nlm.nih.gov/29444905/ (externer Link, öffnet neues Fenster)

Grill A, Schießl IM, Gess B, Fremter K, Hammer A, Castrop H.

Abstract
Aim: The basolateral chloride channel ClC-Kb facilitates Cl reabsorption in the distal nephron of the human kidney. Functional mutations in CLCNKB are associated with Bartter's syndrome type 3, a hereditary salt-losing nephropathy. To address the function of ClC-K2 in vivo, we generated ClC-K2-deficient mice.

Methods: ClC-K2-deficient mice were generated using TALEN technology.

Results: ClC-K2-deficient mice were viable and born in a Mendelian ratio. ClC-K2-/- mice showed no gross anatomical abnormalities, but they were growth retarded. The 24-h urine volume was increased in ClC-K2-/- mice (4.4 ± 0.6 compared with 0.9 ± 0.2 mL per 24 h in wild-type littermates; P = 0.001). Accordingly, ambient urine osmolarity was markedly reduced (590 ± 39 vs. 2216 ± 132 mosmol L-1 in wild types; P < 0.0001). During water restriction (24 h), urinary osmolarity increased to 1633 ± 153 and 3769 ± 129 mosmol L-1 in ClC-K2-/- and wild-type mice (n = 12; P < 0.0001), accompanied by a loss of body weight of 12 ± 0.4 and 8 ± 0.2% respectively (P < 0.0001). ClC-K2-/- mice showed an increased renal sodium excretion and compromised salt conservation during a salt-restricted diet. The salt-losing phenotype of ClC-K2-/- mice was associated with a reduced plasma volume, hypotension, a slightly reduced glomerular filtration rate, an increased renal prostaglandin E2 generation and a massively stimulated renin-angiotensin system. Clckb-/- mice showed a reduced sensitivity to furosemide and were completely resistant to thiazides.

Conclusion: Loss of ClC-K2 compromises TAL function and abolishes salt reabsorption in the distal convoluted tubule. Our data suggest that ClC-K2 is crucial for renal salt reabsorption and concentrating ability. ClC-K2-deficient mice in most aspects mimic patients with Bartter's syndrome type 3.

https://pubmed.ncbi.nlm.nih.gov/27421685/ (externer Link, öffnet neues Fenster)

Mederle K, Gess B, Pluteanu F, Plackic J, Tiefenbach KJ, Grill A, Kockskämper J, Castrop H

Abstract

Aims: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor.

Methods and results: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca(2+)-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca(2+) uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca(2+)]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap-/- (725 ± 48 µL/s) compared with WT mice (1065 ± 122 µL/s; P = 0.01; n = 15).

Conclusion: We identified Atrap as a novel regulatory protein of the cardiac Ca(2+)-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation.

https://pubmed.ncbi.nlm.nih.gov/27015675/ (externer Link, öffnet neues Fenster)

Limmer F, Schinner E, Castrop H, Vitzthum H, Hofmann F, Schlossmann J.

Abstract

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2.

https://pubmed.ncbi.nlm.nih.gov/26183401/ (externer Link, öffnet neues Fenster)

Schießl IM, Castrop H.

Abstract

Purpose of review: Transepithelial salt transport in the thick ascending limb of Henle's loop (TAL) crucially depends on the activity of the Na/K/2Cl cotransporter NKCC2. The pharmacologic blockade of NKCC2 leads to pronounced natriuresis and diuresis, which indicate key roles for NKCC2 in renal salt retrieval. The inadequate regulation of NKCC2 and the loss of NKCC2 function are associated with the disruption of salt and water homoeostasis. This review provides a specific overview of our current knowledge with respect to the regulation of NKCC2 by differential splicing and phosphorylation.

Recent findings: Several mechanisms have evolved to adapt NKCC2 transport to reabsorptive needs. These mechanisms include the regulation of NKCC2 gene expression, the differential splicing of the NKCC2 pre-mRNA, the membrane trafficking, and the modulation of the specific transport activity. Substantial progress has been made over the past few years in deciphering the function of kinases in the regulatory network controlling NKCC2 activity and in elucidating the underlying mechanism and the functional consequences of the regulated differential splicing of the NKCC2 pre-mRNA.

Summary: NKCC2 differential splicing and phosphorylation are critically involved in the modulation of the thick ascending limb of Henle's loop reabsorptive capacity and, consequently, in salt homoeostasis, volume regulation, and blood pressure control.

https://pubmed.ncbi.nlm.nih.gov/26125642/ (externer Link, öffnet neues Fenster)

Schießl IM, Hammer A, Kattler V, Gess B, Theilig F, Witzgall R, Castrop H.

Abstract

Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (P<0.001), and 239.4±34.6 µm(3) (P<0.001) albumin-containing vesicles per glomerulus, respectively, compared with none at baseline or 10 ng/kg per minute Ang II. Immunostaining of Ang II-infused kidneys confirmed the presence of albumin-containing vesicles, which colocalized with megalin, in podocin-positive cells. Furthermore, podocyte endocytosis of albumin was markedly reduced in the presence of gentamicin, a competitive inhibitor of megalin-dependent endocytosis. Ang II infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and gentamicin further increased this concentration. Some endocytic vesicles were acidified and colocalized with LysoTracker. Most vesicles migrated from the capillary to the apical aspect of the podocyte and were eventually released into the urinary space. This transcytosis accounted for approximately 10% of total albumin filtration. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. Ang II enhances the endocytosis and transcytosis of plasma albumin by podocytes, which may eventually impair podocyte function.

https://pubmed.ncbi.nlm.nih.gov/26116357/ (externer Link, öffnet neues Fenster)

Mederle K, Meurer M, Castrop H, Höcherl K.

Abstract

Thromboxane (Tx) A2 has been suggested to be involved in the development of sepsis-induced acute kidney injury (AKI). Therefore, we investigated the impact of cyclooxygenase (COX)-1 and COX-2 activity on lipopolysaccharide (LPS)-induced renal TxA2 formation, and on endotoxemia-induced AKI in mice. Injection of LPS (3 mg/kg ip) decreased glomerular filtration rate (GFR) and the amount of thrombocytes to ∼50% of basal values after 4 h. Plasma and renocortical tissue levels of TxB2 were increased ∼10- and 1.7-fold in response to LPS, respectively. The COX-1 inhibitor SC-560 attenuated the LPS-induced fall in GFR and in platelet count to ∼75% of basal levels. Furthermore, SC-560 abolished the increase in plasma and renocortical tissue levels of TxB2 in response to LPS. The COX-2 inhibitor SC-236 further enhanced the LPS-induced decrease in GFR to ∼40% of basal values. SC-236 did not alter thrombocyte levels nor the LPS-induced increase in plasma and renocortical tissue levels of TxB2. Pretreatment with clopidogrel inhibited the LPS-induced drop in thrombocyte count, but did not attenuate the LPS-induced decrease in GFR and the increase in plasma TxB2 levels. This study demonstrates that endotoxemia-induced TxA2 formation depends on the activity of COX-1. Our study further indicates that the COX-1 inhibitor SC-560 has a protective effect on the decrease in renal function in response to endotoxin. Therefore, our data support a role for TxA2 in the development of AKI in response to LPS.

https://pubmed.ncbi.nlm.nih.gov/26017977/ (externer Link, öffnet neues Fenster)

Schießl IM, Kattler V, Castrop H.

Abstract

Angiotensin-converting enzyme (ACE) inhibitors are commonly used antiproteinuric drugs. Here we assessed the effect of the ACE inhibitor enalapril on the glomerular sieving coefficient of albumin (GSCA) using intravital multiphoton microscopy. Munich Wistar Frömter (MWF) rats were used as a model of hypertension-related glomerular lesions. Young (9-week-old) MWF rats were nonproteinuric, similar to what was observed in control Wistar rats. However, urinary albumin excretion in the MWF rats gradually increased during aging, averaging 0.00062 ± 0.0001 at age 9 weeks and 0.0054 ± 0.0003 (mg/mOsmol per liter) at age 52 weeks (P < 0.0001). Albuminuria in aged MWF rats was accompanied by structural changes, which were indicative of glomerular lesions. The GSCA was low in young MWF rats but increased markedly during aging, averaging 0.00057 ± 4.7 × 10(-5) (n = 25) in young MWF rats and 0.0027 ± 0.00036 in 52-week-old MWF rats (n = 36; P < 0.0001). Treatment of proteinuric 12-month-old MWF rats with enalapril over a 4-week period reduced the GSCA from 0.0027 ± 0.00036 to 0.00139 ± 0.00013 (P = 0.0005). Similarly, urinary albumin excretion was reduced, averaging 0.0051 ± 0.0003 and 0.0036 ± 0.0005 mg/mOsmol per liter before and after enalapril administration, respectively (P = 0.0089). In parallel, enalapril treatment reduced the mean arterial blood pressure (144.6 ± 6.5 mm Hg in untreated versus 110.9 ± 0.6 mm Hg in enalapril-treated MWF rats) and increased the glomerular filtration rate from 1.64 ± 0.3 ml/min to 3.58 ± 0.3 ml/min (P = 0.0025 versus baseline). In summary, enalapril reduced the GSCA in proteinuric MWF rats, which was paralleled by a similar reduction in urinary albumin excretion. These data suggest that glomerular rather than tubular mechanisms account for the beneficial antiproteinuric effects of the ACE inhibitor.

https://pubmed.ncbi.nlm.nih.gov/25680709/ (externer Link, öffnet neues Fenster)

Castrop H.

Abstract

The renin angiotensin-system is one of the most important humoral regulators of blood pressure. The recently discovered angiotensin receptor-associated proteins serve as local modulators of the renin angiotensin-system. These proteins interact with the AT1 receptor in a tissue-specific manner and regulate the sensitivity of the target cell for angiotensin II. The predominant effect of the AT1 receptor-associated proteins on angiotensin II-induced signaling is the modulation of the surface expression of the AT1 receptor. This review provides an overview of our current knowledge with respect to the relevance of AT1 receptor-associated proteins for blood pressure regulation. Two aspects of blood pressure regulation will be discussed in detail: angiotensin II-dependent volume homoeostasis and vascular resistance.

https://pubmed.ncbi.nlm.nih.gov/25562590/ (externer Link, öffnet neues Fenster)

Castrop H.

Abstract

The activity of the renin-angiotensin system crucially depends on the rate of renal renin secretion. Changes in renin secretion result in fluctuations of angiotensin II concentrations in the circulation and subsequently in the activation of angiotensin receptors in all accessible target organs. Consequently, various mechanisms have evolved to regulate the local sensitivity to angiotensin II. In this review, an overview of angiotensin II receptor-associated proteins is addressed. These proteins regulate the local sensitivity of receptor-expressing cells by modulating the receptor surface expression and the receptor sensitivity. A hypothesis will be discussed that integrates the existence of various angiotensin receptor-associated proteins into an overall functional model.

https://pubmed.ncbi.nlm.nih.gov/22588461/ (externer Link, öffnet neues Fenster)

Schießl IM, Bardehle S, Castrop H.

Abstract

Multiphoton microscopy (MPM) offers a unique approach for addressing both the function and structure of an organ in near-real time in the live animal. The method however is limited by the tissue-specific penetration depth of the excitation laser. In the kidney, structures in the range of 100 µm from the surface are accessible for MPM. This limitation of MPM aggravates the investigation of the function of structures located deeper in the renal cortex, like the glomerulus and the juxtaglomerular apparatus. In view of the relevance of gene-targeted mice for investigating the function of these structures, we aimed to identify a mouse strain with a high percentage of superficially located glomeruli. The mean distance of the 30 most superficial glomeruli from the kidney surface was determined in 10 commonly used mouse strains. The mean depth of glomeruli was 118.4±3.4, 123.0±2.7, 133.7±3.0, 132.3±2.6, 141.0±4.0, 145.3±4.3, 148.9±4.2, 151.6±2.7, 167.7±3.9, and 207.8±3.2 µm in kidney sections from 4-week-old C3H/HeN, BALB/cAnN, SJL/J, C57BL/6N, DBA/2N, CD1 (CRI), 129S2/SvPas, CB6F1, FVB/N and NMRI (Han) mice, respectively (n = 5 animals from each strain). The mean distance from the kidney surface of the most superficial glomeruli was significantly lower in the strains C3H/HeN Crl, BALB/cAnN, DBA/2NCrl, and C57BL/6N when compared to a peer group consisting of all the other strains (p<.0001). In 10-week-old mice, the most superficial glomeruli were located deeper in the cortex when compared to 4-week-old animals, with BALB/cAnN and C57BL/6N being the strains with the highest percentage of superficial glomeruli (25% percentile 116.7 and 121.9 µm, respectively). In summary, due to significantly more superficial glomeruli compared to other commonly used strains, BALB/cAnN and C57BL/6N mice appear to be particularly suitable for the investigation of glomerular function using MPM.

https://pubmed.ncbi.nlm.nih.gov/23349687/ (externer Link, öffnet neues Fenster)

Mederle K, Mutig K, Paliege A, Carota I, Bachmann S, Castrop H, Oppermann M.

Abstract

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.

https://pubmed.ncbi.nlm.nih.gov/23427142/ (externer Link, öffnet neues Fenster)

Mederle K, Schweda F, Kattler V, Doblinger E, Miyata K, Höcherl K, Oike Y, Castrop H.

Abstract

Introduction: Hypotension in septic patients results from hypovolemia, vasodilatation and hyporeactivity to vasoconstrictors, such as angiotensin II. The AT1 receptor-associated protein 1 (Arap1) is expressed in vascular smooth muscle cells and increases the surface expression of the AT1-receptor in vitro. We hypothesized that dysregulation of Arap1 may contribute to vascular hyporeactivity to angiotensin II during endotoxemia.

Methods: Arap1-deficient mice were used to assess the role of Arap1 in sepsis-induced hypotension. The isolated perfused kidney was used as an in vitro model to determine the relevance of Arap1 for vascular resistance and sensitivity to angiotensin II.

Results: During endotoxemia, mean arterial blood pressure (MAP) decreased in both genotypes, with the time course of sepsis-induced hypotension being markedly accelerated in Arap1-/- compared to +/+ mice. However, baseline MAP was similar in Arap1-/- and wildtype mice (102 ± 2 vs.103 ± 2 mmHg; telemetry measurements; n = 10; P = 0.66). Following lipopolysaccharide (LPS) injections (3 mg/kg), Arap1 expression was successively down-regulated in the wildtype mice, reaching levels below 10% of baseline expression. The endotoxemia-related decline in Arap1 expression could be recapitulated in cultured mesangial cells by incubation with pro-inflammatory cytokines, such as tumor necrosis factor α and interferon γ. Plasma renin concentration was increased in Arap1-/- mice compared to wildtype mice (66 ± 6 vs. 41 ± 4 ng AngI/ml/h; n = 23; P = 0.001), presumably contributing to preserved MAP under baseline conditions. The sensitivity of the vasculature to angiotensin II was reduced in Arap1-/- compared to +/+ mice, as determined in the isolated perfused kidney.

Conclusions: Our data suggest that down-regulation of Arap1 expression during sepsis contributes to the development of hypotension by causing reduced vascular sensitivity to angiotensin II.

https://pubmed.ncbi.nlm.nih.gov/23844607/ (externer Link, öffnet neues Fenster)

Doblinger E, Höcherl K, Mederle K, Kattler V, Walter S, Hansen PB, Jensen B, Castrop H.

Abstract
Arap1 is a protein that interacts with angiotensin II type 1 (AT(1)) receptors and facilitates increased AT(1) receptor surface expression in vitro. In the present study, we assessed the tissue localization and regulation of Arap1 in vivo. Arap1 was found in various mouse organs, with the highest expression in the heart, kidney, aorta, and adrenal gland. Renal Arap1 protein was restricted to the vasculature and to glomerular mesangial cells and was absent from tubular epithelia. A similar localization was found in human kidneys. To test the hypothesis that angiotensin II may control renal Arap1 expression, mice were subjected to various conditions to alter the activity of the renin-angiotensin system. A high-salt diet (4% NaCl, 7 days) upregulated Arap1 expression in mice by 47% compared with controls (0.6% NaCl, P = 0.03). Renal artery stenosis (7 days) or water restriction (48 h) suppressed Arap1 levels compared with controls (-64 and -62% in the clipped and contralateral kidney, respectively; and -50% after water restriction, P < 0.01). Angiotensin II infusion (2 μg·kg(-1)·min(-1), 7 days) reduced Arap1 mRNA levels compared with vehicle by 29% (P < 0.01), whereas AT(1) antagonism (losartan, 30 mg·kg(-1)·day(-1), 7 days) enhanced Arap1 mRNA expression by 52% (P < 0.01); changes in mRNA were paralleled by Arap1 protein abundance. Experiments with hydralazine and epithelial nitric oxide synthase-/- mice further suggested that Arap1 expression changed in parallel with angiotensin II, rather than with blood pressure per se. Similar to in vivo, Arap1 mRNA and protein were suppressed by angiotensin II in a time- and dose-dependent manner in cultured mesangial cells. In summary, Arap1 is highly expressed in the renal vasculature, and its expression is suppressed by angiotensin II. Thus Arap1 may serve as a local modulator of vascular AT(1) receptor function in vivo.

https://pubmed.ncbi.nlm.nih.gov/22357923/ (externer Link, öffnet neues Fenster)

Barro-Soria R, Stindl J, Müller C, Foeckler R, Todorov V, Castrop H, Strauß O.

Abstract
Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca(2+)inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+)response. RPE cells from Atrap(-/-) mice showed smaller AngII-evoked Ca(2+)peak (by 22%) and loss of sustained Ca(2+)elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca(2+)-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca(2+)-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+)transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+)transients in the RPE by releasing Ca(2+)from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+)elevation.

https://pubmed.ncbi.nlm.nih.gov/23185387/ (externer Link, öffnet neues Fenster)

Castrop H, Höcherl K, Kurtz A, Schweda F, Todorov V, Wagner C.

Abstract
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).

https://pubmed.ncbi.nlm.nih.gov/20393195/ (externer Link, öffnet neues Fenster)

Castrop H

Abstract
Genetically modified mice, created by random integration of a transgene into the genome or by targeted mutation of a specific gene, have proven to be extremely powerful tools for studying gene function in vivo. In this article, we give (1) a short overview of the traditional methods in mouse transgenesis and (2) a discussion of the problems with these methods, (3) more recent methods that were developed to overcome these problems, and (4) an outlook on future directions in gene targeting.

https://pubmed.ncbi.nlm.nih.gov/20140450/ (externer Link, öffnet neues Fenster)

Castrop H, Kurtz A.

Abstract
Erythropoietin (EPO) is the central hormone in the regulation of erythropoiesis. Synthesis of EPO and its constitutive release into the circulation are controlled by the transcriptional activity of the EPO gene. A key regulator of EPO is the dimeric hypoxia-inducible transcription factor (HIF), which has three isoforms. Paliege and co-workers provide convincing morphological evidence that it is the HIF-2 isoform that essentially triggers EPO in the kidney, which is the primary source of EPO in the body.

https://pubmed.ncbi.nlm.nih.gov/20118899/ (externer Link, öffnet neues Fenster)

Oppermann M, Gess B, Schweda F, Castrop H.

Abstract
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.

https://pubmed.ncbi.nlm.nih.gov/20093357/ (externer Link, öffnet neues Fenster)

Oppermann M, Qin Y, Lai EY, Eisner C, Li L, Huang YG, Mizel D, Fryc J, Wilcox CS, Briggs JP, Schnermann JB, Castrop H.

Abstract
Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.38-kb fragment of the rat smooth muscle alpha-actin promoter and first intron (12). Two founder lines with highest expression in the kidney [353 +/- 42 and 575 +/- 43% compared with the wild type (WT)] were used in the experiments. Enhanced expression of A1AR at the expected site in these lines was confirmed by augmented constrictor responses of isolated afferent arterioles to administration of the A1AR agonist N6-cyclohexyladenosine. Maximum TGF responses (0-30 nl/min flow step) were increased from 8.4 +/- 0.9 mmHg in WT (n = 21) to 14.2 +/- 0.7 mmHg in A1AR-transgene (tg) 4 (n = 22; P < 0.0001), and to 12.6 +/- 1.2 mmHg in A1AR-tg7 (n = 12; P < 0.02). Stepwise changes in perfusion flow caused greater numerical TGF responses in A1AR-tg than WT in all flow ranges with differences reaching levels of significance in the intermediate flow ranges of 7.5-10 and 10-15 nl/min. Proximal-distal single-nephron glomerular filtration rate (SNGFR) differences (free-flow micropuncture) were also increased in A1AR-tg, averaging 6.25 +/- 1.5 nl/min compared with 2.6 +/- 0.51 nl/min in WT (P = 0.034). Basal plasma renin concentrations as well as the suppression of renin secretion after volume expansion were similar in A1AR-tg and WT mice, suggesting lack of transgene expression in juxtaglomerular cells. These data indicate that A1AR expression in vascular smooth muscle cells is a critical component for TGF signaling and that changes in renal vascular A1AR expression may determine the magnitude of TGF responses.

https://pubmed.ncbi.nlm.nih.gov/19741017/ (externer Link, öffnet neues Fenster)

Gadau J, Peters H, Kastner C, Kühn H, Nieminen-Kelhä M, Khadzhynov D, Krämer S, Castrop H, Bachmann S, Theilig F.

Abstract
Glomerulonephritis is characterized by hematuria, proteinuria, hypertension, and edema, but the mechanisms contributing to volume disorders are controversial. Here we used the rat anti-Thy1 model of mesangioproliferative glomerulonephritis to test the hypothesis that disturbed salt and water homeostasis is based on tubular epithelial changes that cause salt retention. In this model there was an early onset of pronounced proteinuria and lipiduria associated with reduced fractional sodium excretion and a lowering of the renin-angiotensin-aldosterone system. The glomerular filtration rate and creatinine clearance were decreased on day 6. There was a reduced abundance of the major salt and water transport proteins on the proximal tubular brush border membrane and which paralleled cellular protein overload, enhanced membrane cholesterol uptake and cytoskeletal changes. Alterations in thick ascending limb were moderate. Changes in the collecting ducts were characterized by an enhanced abundance and increased subunit cleavage of the epithelial sodium channel, both events consistent with increased sodium reabsorption. We suggest that irrespective of the proximal tubular changes, altered collecting duct sodium reabsorption may be crucial for volume retention in acute glomerulonephritis. We suggest that enhanced proteolytic cleavage of ion transporter subunits might be a novel mechanism of channel activation in glomerular diseases. Whether these proteases are filtered or locally secreted awaits determination.

https://pubmed.ncbi.nlm.nih.gov/19190681/ (externer Link, öffnet neues Fenster)

Blattmann A, Denk L, Strehl R, Castrop H, Minuth WW.

Abstract
Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1 x 10(-7)m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133+/-26 nm followed by a medium-sized pore type of 317+/-35 nm. Another category of pores with a diameter of 605+/-101 nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina.

https://pubmed.ncbi.nlm.nih.gov/18400296/ (externer Link, öffnet neues Fenster)

Schreiber R, Castrop H, Kunzelmann K.

Abstract
Adenosine is formed from extracellular purines by ecto-5'-nucleotidase (CD73) and is an essential player in allergic airway inflammation. The contribution of adenosine and other purines to electrolyte transport and mucociliary clearance was studied in airways of allergen challenged mice. No signs for allergen-induced inflammation were found in CD73-/- mice, and adenosine monophosphate (AMP) was unable to elicit airway Cl(-) secretion in these animals. Tracheas of ovalbumin (OVA)-treated BALB/c and CD73+/+ mice were hyperresponsive towards methacholine when assessed by Penh and direct optical measurement of contraction. In addition Cl(-) secretion activated by ATP and ADP was enhanced. These changes were not observed in CD73-/- mice. Expression of CFTR or CLCA was unchanged upon OVA treatment of CD73 mice, suggesting enhanced Cl(-) secretion due to upregulated purinergic pathways. Mucociliary clearance was determined by measuring particle transport in excised mouse tracheas and was strongly enhanced in OVA-challenged CD73+/+ mice, but remained unchanged in CD73-/- mice. While mucociliary clearance is activated by allergen exposure independent of functional ecto-5'-nucleotidase, airway inflammation is largely dependent on CD73. Thus, ecto-5'-nucleotidase may provide a novel target for therapeutic intervention, probably by local application of ecto-5'-nucleotidase inhibitors through inhalation.

https://pubmed.ncbi.nlm.nih.gov/18607626/ (externer Link, öffnet neues Fenster)