Renal PET-imaging with (11)C-metformin in a transgenic mouse model for chronic kidney disease.

BACKGROUND: Organic cation transporters (OCTs) in the renal proximal tubule are important for the excretion of both exo- and endogenous compounds, and chronic kidney disease (CKD) alter the expression of OCT. Metformin is a well-known substrate for OCT, and recently, we demonstrated that positron emission tomography (PET) with 11C-labelled metformin ((11)C-metformin) is a promising approach to evaluate the function of OCT. The aim of this study is therefore to examine renal pharmacokinetics of (11)C-metformin and expression of OCTs in a transgenic (RenTGF-ß1) mouse model of CKD. METHODS: Age- and sex-matched RenTGF-ß1 (Tg) and wildtype (WT) mice were used (5-8/group). Animals received an iv bolus of (11)C-metformin followed by 90-min dynamic PET and MRI scan. PET data were analysed using a one-tissue compartment model. Renal protein abundance of OCT2 (by Western blot) as well as OCT1, OCT2, and MATE1 messenger RNA (mRNA) (by RT-PCR) was examined. RESULTS: Protein expression of the basolateral uptake transporter OCT2 was 1.5-fold lower in Tg mice compared to WT mice while OCT1 and MATE1 mRNA expression did not differ between the two groups. The influx rate constant of (11)C-metformin in renal cortex (K 1) was 2.2-fold lower in transgenic mice whereas the backflux rate constant (k 2) was similar in the two groups, consistent with protein expression. Total body clearance (TBC) correlated within each group linearly with K 1. CONCLUSIONS: In conclusion, this study demonstrates that both renal OCT2 expression and (11)C-metformin uptake are reduced in CKD mice. This potentially makes (11)C-metformin valuable as a PET probe to evaluate kidney function.

Restoration of Haemoglobin Level Using Hydrodynamic Gene Therapy with Erythropoietin Does Not Alleviate the Disease Progression in an Anaemic Mouse Model for TGFß1-Induced Chronic Kidney Disease.

Erythropoietin, Epo, is a 30.4 kDa glycoprotein hormone produced primarily by the fetal liver and the adult kidney. Epo exerts its haematopoietic effects by stimulating the proliferation and differentiation of erythrocytes with subsequent improved tissue oxygenation. Epo receptors are furthermore expressed in non-haematopoietic tissue and today, Epo is recognised as a cytokine with many pleiotropic effects. We hypothesize that hydrodynamic gene therapy with Epo can restore haemoglobin levels in anaemic transgenic mice and that this will attenuate the extracellular matrix accumulation in the kidneys. The experiment is conducted by hydrodynamic gene transfer of a plasmid encoding murine Epo in a transgenic mouse model that overexpresses TGF-ß1 locally in the kidneys. This model develops anaemia due to chronic kidney disease characterised by thickening of the glomerular basement membrane, deposition of mesangial matrix and mild interstitial fibrosis. A group of age matched wildtype littermates are treated accordingly. After a single hydrodynamic administration of plasmid DNA containing murine EPO gene, sustained high haemoglobin levels are observed in both transgenic and wildtype mice from 7.5 ± 0.6 mmol/L to 9.4 ± 1.2 mmol/L and 10.7 ± 0.3 mmol/L to 15.5 ± 0.5 mmol/L, respectively. We did not observe any effects in the thickness of glomerular or tubular basement membrane, on the expression of different collagen types in the kidneys or in kidney function after prolonged treatment with Epo. Thus, Epo treatment in this model of chronic kidney disease normalises haemoglobin levels but has no effect on kidney fibrosis or function.

Differential effects of Smad3 targeting in a murine model of chronic kidney disease.

Transforming growth factor (TGF)-ß1 has a pivotal role in the pathogenesis of progressive kidney diseases that are characterized by fibrosis. The main intracellular signaling pathway of TGF-ß1 is the Smad system, where Smad2 and Smad3 play a central role in transcriptional regulation of target genes involved in extracellular matrix (ECM) metabolism. This study analyzes the hypothesis that blockade of Smad3 attenuates the development of TGF-ß1-driven renal fibrosis. This was examined in vivo in a transgenic model of TGF-ß1-induced chronic kidney disease with Smad3 or without Smad3 expression and in vitro in mesangial cells and glomerular endothelial cells with Smad2/3 inhibitors or Smad3-knockdown. Electron microscopy was used for evaluation of morphological changes, real-time polymerase chain reaction for detection of RNA expression, and immunohistochemistry for localization of ECM components. Matrix metalloproteinase (MMP) level was assessed by gelatin zymography electrophoresis and located by in situ zymography. The results show TGF-ß1-induced mesangial matrix expansion, tubulointerstitial fibrosis, and tubular basement membrane thickening that are attenuated by Smad3 deletion, whereas TGF-ß1-induced glomerular basement membrane thickening is not shown. The amount and distribution profile of MMP-2 may suggest a role of the enzyme herein. We conclude that Smad3 targeting is not exclusively beneficial as Smad3 has diverse transcriptional regulatory effects in different cell types in the kidney.

AT1 receptor blockade prevents interstitial and glomerular apoptosis but not fibrosis in pigs with neonatal induced partial unilateral ureteral obstruction.

Obstruction-induced fibrosis is a leading cause of end-stage renal failure in children. The pathophysiological mechanisms may involve apoptosis and the renin-angiotensin system. We studied apoptosis and fibrosis in a well-established neonatal pig model with unilateral partial ureteral obstruction (PUUO) induced during ongoing nephrogenesis in 2-day-old piglets. The role of angiotensin II (ANG II) was studied using the AT(1) receptor blocker CV-11974 (0.12 mg/h candesartan from age 23 to 30 days). At day 30 the kidneys were perfusion fixed and fibrosis, apoptosis, and tubular lengths were quantitated using stereological methods, picro Sirius red staining, and immunohistochemical techniques identifying activated caspase 3, aquaporin-2 (AQP2), and von Willebrand factor. The collagen content was assessed by hydroxyproline density. Neonatal induced PUUO increased interstitial and glomerular cell apoptosis and fibrosis. At this stage, PUUO did not increase tubular cell apoptosis or decrease tubular length and cell number. AT(1) receptor blockade prevented the PUUO-induced interstitial and glomerular cell apoptosis but did not attenuate fibrosis. In conclusion, AT(1) receptor blockade after the end of nephrogenesis may prevent interstitial and glomerular cell apoptosis but not fibrosis, suggesting that pathways not involving AT(1) receptor stimulation contribute to neonatal obstruction-induced fibrosis or that prevention of interstitial cell apoptosis counteracts a potential antifibrotic effect of AT(1) receptor blockade in this pig model of congenital obstructive nephropathy. Our results demonstrate that ANG II plays a role in PUUO-induced glomerular cell apoptosis.