Eberhard Schlatter

Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.

Background/Aims: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood. Methods: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing. Results: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified. Conclusions: This au-tosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.

New clues for nephrotoxicity induced by ifosfamide: preferential renal uptake via the human organic cation transporter 2.

Anticancer treatment with ifosfamide but not with its structural isomer cyclophosphamide is associated with development of renal Fanconi syndrome leading to diminished growth in children and bone problems in adults. Since both cytotoxics share the same principal metabolites, we investigated whether a specific renal uptake of ifosfamide is the basis for this differential effect. First we studied the interaction of these cytotoxics using cells transfected with organic anion or cation transporters and freshly isolated murine and human proximal tubules with appropriate tracers. Next we determined changes in membrane voltage in proximal tubular cells to understand their differentiated nephrotoxicity. Ifosfamide but not cyclophosphamide was significantly transported into cells expressing human organic cation transporter 2 (hOCT2) while both did not interact with organic anion transporters. This points toward a specific interaction of ifosfamide with hOCT2, which is the main OCT isoform in human kidney. In isolated human proximal tubules ifosfamide also interacted with organic cation transport. This interaction was also seen in isolated mouse proximal tubules; however, it was absent in tubules from OCT-deficient mice, illustrating the biological importance of this selective transport. Ifosfamide decreased the viability of cells expressing hOCT2, but not that of control cells. Coadministration of cimetidine, a known competitive substrate of hOCT2, completely prevented this ifosfamide-induced toxicity. Finally, ifosfamide but not cyclophosphamide depolarized proximal tubular cells. We propose that the nephrotoxicity of ifosfamide is due to its selective uptake by hOCT2 into renal proximal tubular cells, and that coadministration of cimetidine may be used to prevent ifosfamide-induced nephrotoxicity.

Natriuretic peptides increase a K+ conductance in rat mesangial cells.

Mesangial cells (MC) are a main target of natriuretic peptides in the kidney and are thought to play a role in regulating glomerular filtration rate. We examined the influence of cGMP-generating (i.e. guanosine 3′,5′-cyclicmonophosphate) peptides on membrane voltages (Vm) of rat MC by using the fast whole-cell patch-clamp technique. The cGMP-generating peptides were tested at maximal concentrations ranging from 140 to 300 nmol/1. Whereas human CNP (C natriuretic peptide), rat guanylin and human uroguanylin had no significant effect on Vm of these cells, human BNP (brain natriuretic peptide), rat CDD/ANP-99-126 (cardiodilatin/atrial natriuretic peptide) and rat CDD/ANP-95-126 (urodilatin) hyperpolarized Vm significantly by 1.6 ± 0.4 mV (BNP,n = 8), 3.7 ± 0.3 mV (CDD/ANP-99-126,n = 25) and 2.8 ± 0.4 mV (urodilatin,n = 9), respectively. The half-maximally effective concentration (EC50) for the latter two was around 400 pmol/l each. This hyperpolarization could be mimicked with 0.5 mmol/1 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) and was blocked by 5 mmol/1 Ba2+. The K+ channel blocker 293 B (1O)) μmol/l) depolarized basal Vm by 4.3 ± 0.4 mV (n = 8), but failed to inhibit the hyperpolarization induced by CDD/ANP-99-126 (160 nmol/1) (n = 8). The K+ channel opener cromakalim (10 μmol/1) neither influenced basal Vm nor altered the hyperpolarization induced by 160 nmol/1 CDD/ANP-99-126 (n = 8). Adenosine (100 μmol/1) hyperpolarized Vm by 13.4 ± 1.3 mV (n = 16). At 100 μmol/1, 293 B did not inhibit the adenosine-induced hyperpolarization (n = 6). At 160 nmol/l, CDD/ANP-99-126 enhanced the adenosine-induced hyperpolarization significantly by 1.5 ± 0.6 mV (n = 10). CDD/ANP-99-126 (160 nmol/1) failed to modulate the value to which Vm depolarized in the presence of 1 nmol/l angiotensin II (n = 10), but accelerated the repolarization to basal Vm, by 49 ± 20% (n = 8). These results indicate that the natriuretic peptides CDD/ANP-99-126, CDD/ANP-95-126 and BNP hyperpolarize rat MC probably due to an increase of a K+ conductance. This effect modulates the voltage response induced by angiotensin II. The natriuretic-peptide-activated conductance can be blocked by Ba2+, but not by 293 B and cannot be activated by cromakalim. This increase in the K+ conductance seems to be additive to that inducable by adenosine, indicating that different K+ channels are activated by these hormones.

Effects of diadenosine polyphosphates, ATP and angiotensin II on cytosolic Ca2+ activity and contraction of rat mesangial cells

Diadenosine polyphosphates (ApnA) are known to influence cellular Ca2+ activity ([Ca2+]i) in several cells. Their vasoactive potency has been described in various systems including the kidney. We examined the effects of diadenosine polyphosphates, adenosine 5′-triphosphate (ATP) and angiotensin II (Ang II) on cytosolic Ca2+ activity of mesangial cells (MC) in culture obtained from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. [Ca2+]i was measured as a fluorescence ratio F340/F380 with the fura-2 technique using three excitation wavelengths (340 nm, 360 nm and 380 nm) and a photon counting tube. Resting [Ca2+]i was not significantly different in MC from WKY and SHR rats and was measured as 132±9 nmol/l (n=65) and 114±12 nmol/l (n=36), respectively. Diadenosine polyphosphates (Ap3A–Ap6A) increased [Ca2+]i transiently with an initial peak and a secondary plateau phase comparable to the effects of ATP or Ang II. Increases in [Ca2+]i induced by all these agonists were not significantly different between MC of WKY and SHR rats. ATP, Ap3A, Ap4A, Ap5A, Ap6A (each 5 μmol/l) increased the fura-2 fluorescence ratio initially by 0.66±0.09 (n=33), 0.52±0.08 (n=18), 0.25±0.05 (n=16), 0.09±0.06 (n=7), 0.09±0.04 (n=11), respectively. A half-maximal initial increase in the fura-2 fluorescence ratio was reached at 22 nmol/l, 0.9 μmol/l, 2.0 μmol/l and 4.0 μmol/l with Ang II, Ap3A, ATP and Ap4A, respectively. Ap4A (100 μmol/l, n=18) led to a reversible contraction of MC. Diadenosine polyphosphates increase [Ca2+]i in rat MC, in a similar manner to ATP or Ang II and lead to a contraction of MC, suggesting that these nucleotides are also involved in the control of glomerular haemodynamics.

Natriuretic Peptides and Diadenosine Polyphosphates Modulate pH Regulation of Rat Mesangial Cells

Modulation of cell proliferation has often been thought to be connected to changes in the activity of pH-regulatory transporters and consequently intracellular pH (pH_i). The influence of natriuretic peptides, diadenosine polyphosphates, adenosine and ATP as well as platelet-derived growth factor (PDGF) on pH_i regulation of cultured rat mesangial cells was examined with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The inhibitors of Na⁺/H⁺ exchange, amiloride and HOE694, blocked pH_i recovery completely in the absence of and by approximately 50% in the presence of HCO₃^–/CO₂. Natriuretic peptides (ANP, BNP, CNP, urodilatin) completely inhibited pH_i recovery in the absence of and by approximately 40% in the presence of HCO₃^–/CO₂. These effects were abolished by the cGMP-dependent protein kinase inhibitor KT5823. Diadenosine polyphosphates (Ap3A-Ap6A), ATP and adenosine also inhibited pH_i recovery completely in the absence of and partially (30–40%) in the presence of HCO₃^–/ CO₂. The effect of adenosine was abolished in the presence of the cAMP-dependent protein kinase inhibitor KT5720, and that of Ap5A by the protein kinase C inhibitor calphostin C. PDGF activated acid extrusion in these cells by approximately 40%. From the four cloned isoforms of the Na⁺/H⁺ exchanger in the rat, only transcripts of NHE-1 were found in these mesangial cell cultures using RT-PCR analysis. These data suggest that in these rat mesangial cells the Na⁺/H⁺ exchanger, specifically the NHE-1 isoform, accounts for around 50% of pH_i recovery from an acid load under physiological conditions, and that Na⁺/H⁺ exchange stimulated by acidification can be inhibited by activation of PKG, PKA, and PKC and stimulated by PDGF after acute exposition to these agonists.

Effects of Diadenosine Polyphosphates on Systemic and Regional Hemodynamics in Anesthetized Rats

Diadenosine polyphoshates (Ap4A, Ap5A, Ap6A) induce vasodilatation or vasoconstriction in various isolated vessels and influence central and peripheral hemodynamics. The influence of diadenosine polyphoshates on hemodynamics was studied in anesthetized rats in vivo. Mean arterial blood pressure (MABP) and heart rate (HR) measured in the carotid artery decreased with Ap4A, Ap5A and Ap6A. Renal blood flow (RBF), femoral blood flow (FBF) and cardiac output (CO) were evaluated by an ultrasonic transit-time method. Renal superficial blood flow (RSBF) was measured by laser Doppler flowmetry. CO, RBF and RSBF were decreased initially by all three diadenosine polyphosphates. FBF was also slightly decreased. Total peripheral (TPR), renal (RVR) and femoral (FVR) vascular resistances were calculated. TPR was transiently increased by the dinucleotides following by a decrease. RVR and, to a lesser extent, FVR were also increased. These data show that diadenosine polyphosphates have effects on both the heart and the peripheral blood vessels. The effects on the heart and MABP were dominated by bradycardia and hypotension. In the kidney, diadenosine polyphosphates induced a predominant vascoconstriction. The effects on skeletal muscle blood flow were much smaller. Thus, the three diadenosine polyphosphates studied differ in the effects on heart and peripheral vessels.

A novel assay for determination of diadenosine polyphosphates in human platelets: studies in normotensive subjects and in patients with essential hypertension

Objective Diadenosine polyphosphates (APnAs, n = 3–6) are a family of endogenous vasoactive purine dinucleotides which have been isolated from thrombocytes. Diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) are more potent than diadenosine tetraphosphate (AP4A) and diadenosine triphosphate (AP3A) and cause skeletal muscle vasoconstriction in rats. Little is known about their physiological and pathophysiological significance in humans. The aims of the present study were to compare thrombocyte APnA concentrations in patients with essential hypertension (HYP) and in healthy normotensive humans (CON) using a novel quantitative assay and to assess a possible relationship between thrombocyte APnA concentrations and skeletal muscle vascular resistance. Design and methods We describe a novel assay for quantification of APnAs in human platelets, involving platelet isolation from human blood, a solid-phase extracting procedure with a derivatized resin, desalting and quantitative determination of the substances with an ion-pair reversed-phase high-performance liquid chromatography (HPLC) system. The structural integrity of the isolated APnAs was confirmed by mixed assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) measurements and co-elution with added standards. The detection threshold for all four APnAs was 1 pmol/l and the inter-assay coefficients of variation were < 11% (n = 12). After venous blood sampling, mean arterial blood pressure (MAP) and forearm blood flow (FBF, using venous occlusion plethysmography) were measured in HYP and CON. Forearm vascular resistance (FVR) was calculated as MAP/FBF. Results HYP (n = 15) and CON (n = 15) did not significantly differ in platelet AP3A and AP4A content, but HYP had significantly higher thrombocyte concentrations of AP5A (56 ± 7 versus 32 ± 3 ng/μg β-thromboglobulin, P = 0.003) and AP6A (10 ± 1 versus 6 ± 1 ng/μg β-thromboglobulin, P = 0.015) than CON. HYP had significantly elevated FVR (50 ± 6 versus 33 ± 2 arbitrary units, P = 0.01) compared to CON. Significant correlations were found between AP5A and FVR (ρ = 0.38, P = 0.04) as well as between AP6A and FVR (ρ = 0.42, P = 0.02). In contrast, there were no significant correlations between APnAs and MAP. Conclusions The study shows that thrombocyte concentrations of AP5A and AP6A are elevated in patients with essential hypertension. Vasoconstriction caused by release of AP5A and AP6A from thrombocytes may contribute to the increase of vascular resistance in hypertensive patients.

Hydroxyfasudil-Mediated Inhibition of ROCK1 and ROCK2 Improves Kidney Function in Rat Renal Acute Ischemia-Reperfusion Injury

Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by 18F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, 18F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.

Effects of diadenosine polyphosphates, ATP and angiotensin II on membrane voltage and membrane conductances of rat mesangial cells.

Diadenosine polyphosphates have been shown to influence renal perfusion pressure. As mesangial cells may contribute to these effects we investigated the effects of diadenosine triphosphate (Ap3A), diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A) and diadenosine hexaphosphate (Ap6A) on membrane voltage (Vm) and membrane conductance (gm) in mesangial cells (MC) of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats in primary and long-term culture. We applied the patch-clamp technique in the fast-whole-cell configuration to measure Vm and gm. To compare the effects of diadenosine polyphosphates with hitherto known agonists we also tested adenosine 5′-triphosphate (ATP) and angiotensin II (Ang II). As there was no significant difference in the Vm values in MC of WKY (−42±1 mV, n=70) and SHR rats (−45±2 mV, n=99) as well as in the agonist-induced changes of Vm, all data were pooled. The Vm of all the cells was −44±1 mV (n=169) and gm was 15.9±1.8 nS (n=141). Ion-exchange experiments showed the presence of a K+ and a non-selective cation conductance in resting MC whereas a Cl− conductance or a Na+selective conductance could not be observed. Ap3A, Ap4A, Ap5A, AP6A and ATP each at a concentration of 5 μmol/l, led to a significant depolarization of Vm by 5±2 mV (n=14), 7±1 mV (n=25), 3±1 mV (n=23), 2±1 mV (n=16), and 14±2 mV (n=23), respectively. For Ap4A, the most potent diadenosine polyphosphate, we determined the half-maximally effective concentration (EC50) as 6 μmol/l (n=5–25), for ATP as 2 μmol/l (n=9–37), and for Ang II as 8 nmol/l (n=6–18). Ap4A 100 μmol/l increased gm significantly by 55±20% (n=16), 100 μmol/l ATP by 135±60% (n=18). The diadenosine polyphosphates examined were able to depolarize Vm (Ang II >ATP> Ap4A>Ap3A>Ap5A>Ap6A) by activation of a Cl− conductance and a non-selective cation conductance, as do ATP or Ang II.

Effects of Diadenosine Polyphosphates on the Intracellular Ca2+ Concentration in Endothelial Cells

Diadenosine polyphosphates have differential hemodynamic effects. The role of the endothelium in the vascular effects of these agonists is still unclear. Primary cultures of rat aortal endothelial cells and Ea.hy 926 cells (a continuous endothelial cell line) were used to investigate the effects of Ap3A–Ap6A, adenosine triphosphate (ATP), and for comparison, arginine vasopressin (AVP) and angiotensin II (A II) on the intracellular Ca2+ concentration, [Ca2+]i. Fura-2 was used as Ca2+ indicator. In rat aortal endothelial cells, ATP and Ap4A concentration dependently increased [Ca2+]i with an initial peak followed by an elevated plateau. The half-maximal effects were reached at approximately 7 µmol/l for ATP and at approximately 10 µmol/l for Ap4A. The maximal peak effects at 100 µmol/l were 1,035 ± 413 nmol/l (n = 3) and 437 ± 271 nmol/l (n = 8) for ATP and Ap4A, respectively. At 100 µmol/l Ap3A and Ap6A slightly increased [Ca2+]i, while Ap5A had no significant effect. The known endothelial agonists AVP (100 nmol/l) and A II (10 nmol/l) increased [Ca2+]i initially by 1,549 ± 913 nmol/l (n = 7) and 209 ± 45 nmol/l (n = 9), respectively. In Ea.hy 926 cells an increase in [Ca2+]i was obtained only with ATP (10 µmol/l) and with Ap4A (100 µmol/l). Ap3A, Ap5A, and Ap6A (each 100 µmol/l) and also AVP (100 nmol/l) and A II (10 nmol/l) had no significant effects in these cells. These results show that a considerable increase in [Ca2+]i in endothelial cells can only be induced by Ap4A among the diadenosine polyphosphates, indicating that the vasoactive effects of only this polyphosphate could at least partly be mediated via Ca2+-dependent mechanisms in endothelial cells, comparable to the known effects of AVP, A II, and ATP. The fact that A II and AVP did not influence [Ca2+]i in Ea.hy 926 cells is probably due to the loss of the respective receptors in this cell line.