Gert Gabriëls

Non-Invasive Imaging of Acute Renal Allograft Rejection in Rats Using Small Animal 18F-FDG-PET

Background At present, renal grafts are the most common solid organ transplants world-wide. Given the importance of renal transplantation and the limitation of available donor kidneys, detailed analysis of factors that affect transplant survival are important. Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection (AR) is still a major risk for graft survival. Nowadays, AR can only be definitively by renal biopsy. However, biopsies carry a risk of renal transplant injury and loss. Most important, they can not be performed in patients taking anticoagulant drugs. Methodology/Principal Findings We present a non-invasive, entirely image-based method to assess AR in an allogeneic rat renal transplantation model using small animal positron emission tomography (PET) and 18F-fluorodeoxyglucose (FDG). 3 h after i.v. injection of 30 MBq FDG into adult uni-nephrectomized, allogeneically transplanted rats, tissue radioactivity of renal parenchyma was assessed in vivo by a small animal PET-scanner (post operative day (POD) 1,2,4, and 7) and post mortem dissection. The mean radioactivity (cps/mm3 tissue) as well as the percent injected dose (%ID) was compared between graft and native reference kidney. Results were confirmed by histological and autoradiographic analysis. Healthy rats, rats with acute CSA nephrotoxicity, with acute tubular necrosis, and syngeneically transplanted rats served as controls. FDG-uptake was significantly elevated only in allogeneic grafts from POD 1 on when compared to the native kidney (%ID graft POD 1: 0.54±0.06; POD 2: 0.58±0.12; POD 4: 0.81±0.06; POD 7: 0.77±0.1; CTR: 0.22±0.01, n = 3–28). Renal FDG-uptake in vivo correlated with the results obtained by micro-autoradiography and the degree of inflammatory infiltrates observed in histology. Conclusions/Significance We propose that graft FDG-PET imaging is a new option to non-invasively, specifically, early detect, and follow-up acute renal rejection. This method is potentially useful to improve post-transplant rejection monitoring.

Impact of renal transplantation on small vessel reactivity.

Background. The function of large arteries is altered after renal transplantation. Whether transplantation also induces agonist-dependent functional changes in small arterial renal and extrarenal vessels has not yet been studied. Methods. Chronic rejection was induced by grafting Lewis rats with kidneys from Fischer rats (FL). Rats that underwent transplantation were bilaterally nephrectomized. Rats that underwent syngeneic transplantation, uninephrectomized rats, uninephrectomized rats with denervated kidneys or with kidneys made ischemic, and native rats served as controls. All animals were treated with cyclosporine for 10 days. Eighteen weeks after surgery, the reactivity of small arteries (220–270 &mgr;m) was tested by myography. Results. Weight gain, glomerular filtration rate, and arterial pressure were similar in all groups, whereas proteinuria was elevated in FL. Only kidneys from FL showed glomerular lesions, tubular atrophy, and vasculopathy. Responsiveness of coronary, mesenteric, and femoral resistance vessels to both constrictor and dilator agonists was similar in transplanted and nontransplanted animals. Resistance vessels obtained from both allogeneically and syngeneically transplanted kidneys were more sensitive to norepinephrine, phenylephrine, angiotensin II, and vasopressin than renal vessels from weight-matched controls. Vasodilation in response to acetylcholine and sodium nitroprusside was mitigated in transplanted versus nontransplanted kidneys. Conclusions. In rat renal transplantation, renal resistance vessel responsiveness to constrictor or dilator stimuli is altered. Extrarenal small vessel function is not affected. The changes in function of renal resistance vessels are not explained by reduction of nephron mass, denervation, ischemia, or chronic rejection.

Acute Rejection After Rat Renal Transplantation Leads to Downregulation of Na + and Water Channels in the Collecting Duct

Renal transplantation is associated with alterations of tubular functions and of the renin–angiotensin–aldosterone system. The underlying cellular and molecular mechanisms are unclear. We used an allogeneic rat renal transplantation model of acute rejection with and without immunosuppression by cyclosporine A (CsA) and a syngeneic model as control. Uninephrectomized Lewis or Lewis–Brown‐Norway (LBN) rats received a kidney from LBN‐rats. Renal transporters and receptors were analyzed by immunohistochemistry, semiquantitative RT‐PCR and Western‐blot analysis. Intracellular Na+ was analyzed microfluorimetrically in isolated cortical collecting ducts. mRNA expression and function of the epithelial Na+‐channel (ENaC) and mRNA and protein expression of the water‐channel AQP2 were downregulated in transplanted kidneys undergoing rejection. Expression of the serum‐ and glucocorticoid‐kinase (Sgk1) was decreased and that of the ubiquitin–protein ligase Nedd4‐2 was increased. These changes were absent under CsA‐therapy and in syngeneic model. Expression and function of the Na+–K+‐ATPase, expression of the secretory K+‐channel and of the mineralocorticoid receptor remained unchanged. Reduced ENaC function is likely due to decreased Sgk1‐ and increased Nedd4‐2 mRNA expression leading to reduced ENaC expression in the membrane. These acute downregulations of ENaC and AQP2 may be triggered to reduce energy consumption in the distal nephron to protect the kidney immediately after transplantation.

Renal Transplantation Modulates Expression and Function of Receptors and Transporters of Rat Proximal Tubules

Kidney transplantation often leads to disturbances of solute and volume maintenance in humans. To investigate underlying mechanisms, expression and function of renal transporters and receptors of the proximal tubule (PT) were analyzed in an acute rejection model of rat kidney transplantation. Semiquantitative RT-PCR and Western blot, histology, immunohistochemistry, and microfluorometry were performed on whole kidneys and isolated PT. With acute rejection, Na+/H+-exchanger type-3 (NHE-3) was markedly downregulated. Na+-HCO(3)(-)-cotransporter (NBC-1) and Na+-glucose transporter type-2 (SGLT2) were upregulated after transplantation. Expressions of Na+/H+-exchanger type-1 (NHE-1), Na+/K+-ATPase (NKA), angiotensin II (AngII) receptor (AT-1), or natriuretic peptide receptor (GC-A) were unaltered. Microfluorometric analyses of intracellular pH, Na+, and Ca2+ demonstrated a decrease in NHE-3 function and AngII-mediated stimulation of NHE-3. AngII-mediated inhibition of NHE-1 and function of all other transporters tested remained unaltered. Function of AT-1 and GC-A were unaffected. Reduced expression of NHE-3 was also confirmed by semiquantitative immunohistochemistry. These findings suggest that expression and function of transmembrane proteins involved in Na+-transport after transplantation and rejection is specifically modulated. The local renin-angiotensin-system is apparently not altered. Downregulation of NHE-3 may be a protective mechanism occurring in the graft.

Acute Rejection Modulates Gene Expression in the Collecting Duct

Kidney transplantation, especially when associated with acute rejection, leads to changes in the expression of many genes, including those encoding solute transporters and water channels. In a rat model of acute rejection after allogeneic renal transplantation, impaired renal function, increased urine volume, and increased fractional excretion of sodium were observed. Gene array analysis revealed that these findings were associated with significant downregulation of water channels (aquaporin-1, -2, -3, and -4) and transporters of sodium, glucose, urea, and other solutes. In addition, changes in expression of various receptors, kinases, and phosphatases that modulate the expression or activity of renal transport systems were observed. Syngeneic transplantation or treatment with cyclosporine A following allogeneic transplantation did not impair graft function but did lead to the downregulation of aquaporin-1, -3, and -4 and several solute transporters. However, expression of aquaporin-2 and the epithelial sodium channel did not change, suggesting that the downregulation of these transporters following allogeneic transplantation is rejection-dependent. In conclusion, changes in gene expression may explain the impaired handling of solute and water after allogeneic transplantation, especially during acute rejection. Treatment with cyclosporine A improves the regulation of solute and water by preventing the downregulation of aquaporin-2 and epithelial sodium channel, even though many other transporter genes remain downregulated.

Role of the calcium/calmodulin-dependent protein kinase II in the regulation of the renal basolateral PAH and dicarboxylate transporters.

Abstract— The aim of the present study was to investigate whether the activities of the renal basolateral organic anion transporter (PAH transporter) and the sodium‐dependent dicarboxylate transporter are modulated by the calcium/calmodulin‐dependent multifunctional protein kinase II (CaM kinase II). The studies were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H‐PAH was used as marker substance of the anion transporter, and 14C‐glutarate as a marker of the sodium/dicarboxylate cotransporter. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of the marker substances reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s tubular uptake measurements were performed. The results show that a selective inhibitor of CaM kinase II, KN93, inhibited tubular PAH uptake. The smallest effective dose was 10−7 M. An inactive analogue of KN93, KN92, was without effect, even at the high concentration of 105 M. In contrast to PAH transport, tubular 14C‐glutarate uptake was not affected by KN93 (10−5 M). PAH transport was also inhibited after elevation of intracellular Ca2+ by the Ca2+‐ionophore A 23187 and by the polycationic antibiotic neomycin, but not by the intracellular Ca2+ modulators thapsigargin and ryanodine. The effect of the Ca2+‐ionophore could be abolished by KN93, but not by Rp‐cAMPs, an inhibitor of protein kinase A, indicating that this event was mediated by CaM kinase II, but not by PKA. The results provide the first evidence that, in addition to the protein kinases A and C (previous studies from this lab), CaM kinase II has a role in the regulation of the renal basolateral PAH transporter, whereas the renal basolateral dicarboxylate transporter does not depend on CaM kinase II activity.

Kidney transplantation down-regulates expression of organic cation transporters, which translocate β-blockers and fluoroquinolones.

Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), β-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, β-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.

Potassium-Sparing Renal Effects of Trimethoprim and Structural Analogues

Background/Aims: The antibiotic antagonists of folic acid trimethoprim, tetroxoprim, pyrimethamine and their antineoplastic analogue methotrexate have structural characteristics in common with the potassium-sparing diuretic triamterene. They may, therefore, share with triamterne potassium-sparing renal effects. Methods: Clearance studies were performed on anesthetized male Sprague-Dawley rats, and the drug effects on glomerular filtration rate and on urinary excretion of sodium, chloride, and potassium were studied. Results: Trimethoprim and tetroxoprim, injected intravenously at doses ranging from 0.3 to 30 mg/kg, induced dose-dependent natriuretic and antikaliuretic renal effects, whereas pyrimethamine at doses ranging from 1 to 10 mg/kg and methotrexate at doses ranging from 10 to 50 mg/kg did not affect urinary sodium and potassium excretion. An antikaliuretic effect was also observed after application of the structurally related antiprotozoal compound pentamidine at doses ranging from 3 to 10 mg/kg. In contrast to trimethoprim and tetroxoprim, the antikaliuresis was accompanied by a marked decrease of urinary sodium and chloride excretion at all of the doses tested. At 10 mg/kg, pentamidine induced a pronounced fall of the glomerular filtration rate (by 43.5%). Conclusions: Trimethoprim and tetroxoprim share with potassium-sparing diuretics natriuretic and antikaliuretic renal effects which may be caused by similar mechanisms in the distal nephron, whereas pyrimthamine and methotrexate do not. A depression of renal hemodynamics is an important factor involved in the antikaliuretic effect of pentamidine.

Breast pain in a patient on dialysis: a rare manifestation of calcific uraemic arteriolopathy

A 63-year-old woman presented with progredient bilateral breast pain. Her medical history included rheumatoid arthritis, AA amyloidosis and end-stage renal disease treated by peritoneal dialysis. Inflamed skin alterations of the breast and laboratory values suggested mastitis non-puerpuralis but antibiotics did not resolve the symptoms. Sonography and mammography showed severe vessel calcification suggesting calcific uraemic arteriolopathy (calciphylaxis) as a rare complication of chronic kidney disease. Treatment included intensified haemodialysis, thiosulfate application, analgaesia and wound management leading to significant improvement, however, without complete remission.

Involvement of protein phosphatases in differential regulation of renal proximal tubular PAH and sodium-dependent dicarboxylate transport*

Abstract— It has been demonstrated that the basolateral organic anion (PAH) transporter and the sodium‐dependent dicarboxylate transporter of rabbit renal proximal tubules are regulated differentially. A variety of protein kinases has been shown to be involved in the regulation of organic anion transport while dicarboxylate uptake, to which the first is coupled functionally, is not inlluenced by these kinases. This study was undertaken to elucidate whether respective transporter activities are modulated differentially by protein phosphatases as well. The experiments were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H‐PAH was used as marker substance of the PAH transporter. 14C‐glularate as a marker of the sodium/dicarboxylate cotransporter. 30 s tubular uptake measurements were performed. Vanadate (10−3M). a selective inhibitor of tyrosine phosphatase, did not reduce PAH uptake significantly, while inhibitors of the serine/threonine phosphatases I and 2A. okadaic acid and calyculin A (10 6M. each) induced a significant decrease of 30 s PAH uptake (by 32.3% ± 7.9% and 25.6% ± 6.4%) but not a change in dicarboxylate transport. These findings indicate that, in addition to a variety of protein kinases, serine/threonine phosphatases have a role in the regulation of renal basolateral PAH transport. There is no effect of these phosphatases on basolateral 30s glularate transport. Thus, additional evidence for differential regulation of short‐time activity of the transporters for PAH and dicarboxylales is provided.