Evaluation of Homobivalent Carbolines as Designed Multiple Ligands for the Treatment of Neurodegenerative Disorders.

Neurodegenerative diseases represent a challenge for biomedical research due to their high prevalence and lack of mechanism-based treatments. Because of the complex pathology of neurodegenerative disorders, multifunctional drugs have been increasingly recognized as potential treatments. We identified homobivalent γ-carbolinium salts as potent inihitors of both cholinesterases, N-methyl-D-aspartate receptors, and monoamine oxidases. Homobivalent γ-carbolines displayed similar structure-activity relationships on all tested targets and may present promising designed multiple ligands for the treatment of neurodegenerative disorders.

Beta and gamma carboline derivatives as potential anti-Alzheimer agents: A comparison.

Nine novel ß- and γ-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recombinantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties were determined for all compounds. Some were additionally tested in vivo for their ability to reverse scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most promising candidates, the interaction with muscarinic M1 receptors was also investigated. With this set of compounds assays the influence of the scaffold itself and the substituents can be investigated separately. 5-Methyl-γ-carboline (6) was the most potent (0.25 µmol/100 g b.w.) compound in the in vivo test and might be a good starting point for the development of novel anti-Alzheimer drugs.

Ultrastructural and ultraimmunohistochemical changes upon partial nephrectomy and uranyl intoxication in the rat kidney.

We studied kidneys of rats intoxicated with uranylnitrate (UN) or subjected to 5/6 nephrectomy (NX) or after a combination of both procedures (NX-UN). Our observations indicate that UN causes impressive changes of ultrastructure (partial loss of brush border, appearance of intercellular clefts in the epithelial barrier) and altered protein expression (α-SMA, collagen I and III) in proximal tubule cells. Renal parameters (creatinine clearance, proteinuria) seemed to be unaffected. Blood pressure recovered to normal values within 12 months. However ultrastructural and functional restoration of modified proximal tubules was not complete. We conclude that changed proximal tubules may induce progression of interstitial fibrosis causing renal failure. NX animals and more pronounced NX-UN animals showed dramatic changes in renal function. We observed increased levels of proteinuria, blood pressure and decreased creatinine clearance. Progressive glomerular reorganization includes loss of filtration gaps and enhanced thickness of glomerular basement membranes (GBM) with increased immunoreactivity for collagen IV. Cells in vicinity of Bowman's capsule contained high amounts of immunoreactive α-smooth muscle actin. The NX-UN group showed more dramatic changes in ultrastructure of proximal tubules including apoptosis. Enhanced expression and secretion of extracellular matrix proteins (ECM e.g. collagens I, III, fibronectin) indicate progressive epithelial-mesenchymal transition (EMT) leading to permanent impairment of renal function.

9-Methyl-ß-carboline-induced cognitive enhancement is associated with elevated hippocampal dopamine levels and dendritic and synaptic proliferation.

ß-Carbolines (BCs) belong to the heterogenous family of carbolines, which have been found exogenously, that is, in various fruits, meats, tobacco smoke, alcohol and coffee, but also endogenously, that is, blood, brain and CSF. These exogenous and endogenous BCs and some of their metabolites can exert neurotoxic effects, however, an unexpected stimulatory effect of 9-methyl-ß-carboline (9-me-BC) on dopaminergic neurons in primary mesencephalic cultures was recently discovered. The aim of the present study was to extend our knowledge on the stimulatory effects of 9-me-BC and to test the hypothesis that 9-me-BC may act as a cognitive enhancer. We found that 10 days (but not 5 days) of pharmacological treatment with 9-me-BC (i) improves spatial learning in the radial maze, (ii) elevates dopamine levels in the hippocampal formation, and (iii) results after 10 days of treatment in elongated, more complex dendritic trees and higher spine numbers on granule neurons in the dentate gyrus of 9-me-BC-treated rats. Our results demonstrate that beyond its neuroprotective/neurorestorative and anti-inflammatory effects, 9-me-BC acts as a cognitive enhancer in a hippocampus-dependent task, and that the behavioral effects may be associated with a stimulatory impact on hippocampal dopamine levels and dendritic and synaptic proliferation.

Influence of age on cognition and scopolamine induced memory impairment in rats measured in the radial maze paradigm.

The influence of age on (1) cognition and (2) scopolamine (CAS 51-34-3) induced memory impairment in female rats was measured in the radial maze paradigm (RAM). (1) First training trials were done with 3 and 12 months old rats. Rats were trained to find all eight food baits in the RAM without errors and within 1 min. Both 3- and 12-month old rats need about 15 trials for the first-time learning of the RAM task. After intervals of 3 6 months, respectively, initially young rats were re-trained with an age of 6 and 12 months. Surprisingly, re-trained rats successfully completed the maze runs already after one re-training trial. Thus the phenomenon of preserved spatial memory was approved for female rats. (2) Memory impairment by scopolamine in the RAM was tested for the time in rats with an age of 3 months. first rats with thesame After a control run,the rats received an i.p. injection of either scopolamine hydrochloride (0.05 mg/100 g b. wt.) or saline vehicle. The effect of scopolamine on working memory was measured 20 min after administration. Training procedure and scopolamine administration were repeated at an age of 6, 12, 18, and 24 months in the same manner. The cognition impairment after scopolamine (number of errors: control: <1; scopolamine: 5-6) remains constant between 3 and 24 months of age. The only significant difference was the increase in run time in rats older than 18 months caused by degenerative changes developing with age.

Investigation into the in vivo effects of five novel tacrine/ferulic acid and beta-carboline derivatives on scopolamine-induced cognitive impairment in rats using radial maze paradigm.

Two tacrine-ferulic acid hybrids (1 A, 1 B) and three beta-carboline derivatives (BCs; 2A, 2B, 2C) were tested in vivo on 3-month-old female rats as multi-potent anti-Alzheimer drug candidates. In vitro, the two tacrine-ferulic acid hybrids show higher acetylcholinesterase (AChE) inhibitory activity and comparable butyrylcholinesterase (BChE) inhibitory activity compared to tacrine (CAS 1684-40-8). However, in vivo both substances have no beneficial effect on scopolamine (CAS 51-34-3) induced cognition impairment. On the contrary, 1B even worsen the scopolamine induced cognition impairment. The beta-carboline derivatives 2A, 2B, and 2 C, the inhibitory potency of which at AChE reaching tacrine activity does not antagonize scopolamine induced impairment of cognition in rats measured in radial maze paradigm. Compounds 2A and 1B might act as positive allosteric modulators of scopolamine action at the muscarinic acetylcholine receptors. On the basis of these results it can be concluded that both ferulic acid- (CAS 537-98-4) and BC-derivatives are not qualified as cognition improving drugs and further studies in this field should be focussed on other pharmaceutical leads to find effective anti-Alzheimer drugs.

Tacrine-NO donor and tacrine-ferulic acid hybrid molecules as new anti-Alzheimer agents: hepatotoxicity and influence on the cytochrome P450 system in comparison to tacrine.

Tacrine (CAS 321-64-2) is a reversible acetylcholine esterase inhibitor that, despite exerting beneficial effects on Alzheimer's disease (AD), displays marked hepatotoxicity. Searching for safer drugs and taking into account the multi-pathogenesis of AD, two tacrine-NO donor hybrid molecules (FL16, FL38) as well as a tacrine-ferulic acid hybrid (FL67) were synthesized. NO donors coupled to the tacrine moiety may exert an additional beneficial effect on AD via an increased blood supply to the brain and by reducing inflammation. Ferulic acid is an antioxidant. To investigate the hepatotoxicity and effects on the cytochrome P450 (CYP) system of the liver, female rats were treated with the highest tolerated dose of tacrine or equimolar doses of the novel compounds 24 or 36 h, respectively, before sacrifice. Tacrine caused pericentral necrosis and fatty degeneration of the hepatocytes, loss of liver glycogen and (indicating oxidative stress) induction of heme oxygenase (HO)-1. No histopathological changes were observed with the hybrids, but a glycogen deficit and an elevation of HO-1 was noticed after FL38 or FL67 treatment. Both tacrine and the hybrids, but mainly FL38 and FL67, caused an induction of CYP1A1, 2B1 and 3A2 expression. CYP activity was noticeably increased after treatment with FL38 and FL67 only. Thus, since it displays much less hepatotoxicity and interaction potential at the CYP system than tacrine and the other hybrids, FL16 is a good candidate for further investigations.

Hybrid molecules from xanomeline and tacrine: enhanced tacrine actions on cholinesterases and muscarinic M1 receptors.

A set of amide- and amine-linked hybrid molecules comprising moieties of the orthosteric M(1) muscarinic receptor agonist xanomeline and the cholinesterase inhibitor and allosteric receptor modulator tacrine were prepared with varying spacer length of 10-17 atoms. The hybrids inhibited acetylcholinesterase with similar or higher potency compared to tacrine. M(1) receptor binding affinity was similar or higher relative to xanomeline and far higher relative to tacrine. Affinities hardly changed when the receptors' orthosteric site was occupied by an inverse agonist ligand. When occupied by the orthosteric activator acetylcholine, affinity for the hybrids declined to unmeasureably low levels. Hybrids did not activate M(1) receptors. In vivo studies assaying cognition impairment in rats induced by scopolamine revealed pronounced enhancement of scopolamine action. Taken together, instead of dualsteric (simultaneous allosteric/orthosteric) binding, the hybrids seem to prefer purely allosteric binding at the inactive M(1) receptor.

NO-donating tacrine hybrid compounds improve scopolamine-induced cognition impairment and show less hepatotoxicity.

A series of tacrine-NO donor hybrid compounds are synthesized and evaluated for cholinesterase inhibitory activity, cognition improving activity, and hepatotoxicity. The pharmacological results indicate that hybrid compounds 1, 2, and 3a potently inhibit cholinesterase in vitro and significantly improve the scopolamine-induced cognition impairment, whereas an analogue (3h) of 2 without the NO donor moiety does not. Compared to tacrine, 1 and 2 show much less hepatotoxicity. Molecular modeling studies suggest that 2 may interact with the catalytic and the peripheral anionic site of acetylcholinesterase.

Synthesis and biological evaluation of NO-donor-tacrine hybrids as hepatoprotective anti-Alzheimer drug candidates.

In search of safer anti-Alzheimer drugs, 14 NO-donor-tacrine hybrids (1- 14) were synthesized and evaluated for their ability to inhibit cholinesterases and for vasorelaxation effects. Compounds 1- 13 showed good cholinesterases inhibitory activities in vitro, while 14, particularly, was highly selective, preferring butyrylcholinesterase rather than acetylcholinesterase. Four selected compounds (1, 9, 11, and 14) moderately relaxed the porcine pulmonary arteries in organ bath. In the hepatotoxicity study, significant hepatotoxicity was caused by tacrine but not by 9.

A new way of data interpretation for cognition tests in rats used to characterise six choline esterase inhibitors with heterocyclic nitrogen bridgehead structure. Application in Alzheimer therapy.

Six new tri- and tetracyclic nitrogen bridgehead compounds known to be moderate to potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro were tested in vivo as experimental therapeutics for treatment of Alzheimer's disease. Cognitive impairment in rats was reversibly induced by scopolamine (CAS 51-34-3). The effect of the new substances was evaluated in an eight-arm radial maze and run times (1), errors (2), correct choices (3), correct choices per second (4), speed (5), and running distance (6) were recorded. For optimisation of data analysis a new strategy was used: A score was created on the basis of the 6 parameters described with score 1 for controls and score 4 for scopolamine rats. Scores above 4 indicate an impairment of cognition function compared to scopolamine. After equimolar dosage compared to the reference drug rivastigmine (CAS 123441-03-2), two of the new substances slightly improved cognition in rats, but only to a significantly lower degree compared to the irreversible inhibitor rivastigmine. Surprisingly, the other four compounds did not improve or even worsened the scopolamine effect on working memory.

Suitability of 5/6 nephrectomy (5/6NX) for the induction of interstitial renal fibrosis in rats--influence of sex, strain, and surgical procedure.

Chronic renal failure (CRF) is a serious clinical problem and currently there are no adequate therapeutic strategies for treatment. Many possible treatment strategies have been tested in rats with CRF induced by subtotal nephrectomy. However, reports in the literature concerning the consequences of this procedure on rat kidney function are contradictory. For instance, such an intervention in male Sprague-Dawley rats apparently initiates the development of interstitial renal fibrosis, while in our similar studies on female Wistar rats (HW) there was minimal renal fibrosis. Therefore, we carried out experiments in adult rats to investigate the long-term consequences of 5/6 nephrectomy (5/6NX) in relation to (1) sex, (2) strain, and (3) two methods of surgical ablation. Ten weeks after 5/6NX, body weight gain, systolic blood pressure, creatinine clearance, and urinary protein were measured, along with renal hydroxyproline concentration determinations to assess the deposition of extracellular matrix. Also, light microscopic investigations were done to characterize renal damage. The functional parameters clearly indicated the development of CRF, while morphologic investigations showed only moderate fibrotic areas containing atrophic tubules and lymphocytic infiltrates. However, 45-60% of glomeruli were sclerotic. In summary, 5/6NX, using either method of partial nephrectomy, induces signs of moderate glomerulonephritis preferentially in female HW rats. Thus 5/6NX in female HW rats can be recommended as a suitable model in the induction of renal fibrosis.

Immunocytochemical characterization of the incubated rat renal cortical slices.

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g., structural and functional integrity of the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25 h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1 h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15 min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the incubated rat renal cortical slices.

Effects of a therapy with losartan and quinaprilat on the progression of chronic renal failure in rats after a single dose of uranyl nitrate or 5/6 nephrectomy.

The renoprotective effect of losartan and quinaprilat was tested in two different animal models of renal failure [female Wistar rats, single administration of 0.5 mg uranyl nitrate (UN)/100 g body wt. or 5/6 nephrectomy (5/6NX)]. Losartan (1 mg/100 g body weight [wt.]) and quinaprilat (1 mg/100 g body wt.) were administered intraperitoneally, once daily, starting 10 days after UN and one week after 5/6NX till the end of 10 weeks experimental period. Parameters characterizing the therapeutic effect were blood pressure, urinary protein excretion 4 and 10 weeks after the injury, and p-aminohippurate accumulation in renal cortical slices in vitro, hydroxy-proline concentration in renal tissue and morphology at the end of the experiment. Summarizing our results we state: (1) the angiotensin 1 receptor blocker losartan is more effective in UN-treated than in 5/6 NX rats, and (2) the angiotensin converting enzyme inhibitor quinaprilat is more effective than losartan because of the amelioration of blood pressure and OH-proline concentration in renal tissue of UN-treated rats.

RT-PCR-based evidence for the in vivo stimulation of renal tubularp-aminohippurate (PAH) transport by triiodothyronine (T3) or dexamethasone (DEXA) in kidney tissue of immature and adult rats.

Our previous studies have shown that a pre-treatment of rats with triiodothyronine (T3) or dexamethasone (DEXA) increases renal PAH excretion significantly. This stimulation was accompanied by an enhanced protein synthesis within the renal cortex. To explore the molecular basis for this sub-chronic induction process, we investigated the stimulation of PAH accumulation in renal cortical slices as well as the expression level of organic anion transporter 1 (OAT1), the recently cloned renal basolateral PAH-transporter, using RT-PCR techniques under the applied conditions. 10- and 55-day-old Han:WIST rats were treated in vivo with T3 (20 microg/100 g b.wt.) or DEXA (60 microg/100 g b.wt.), both for 3 days, once daily. Renal cortical slices were incubated for 2 hours in Cross-Taggart medium and PAH uptake into kidney tissue was measured time dependently (slice to medium ratio, QS/M). The accumulation capacity is comparable between immature and mature rats (control-QS/M: 6.7 +/- 0.1 vs. 6.9 +/- 0.2, respectively). Both age groups showed a significant increase of PAH accumulation capacity after T3 treatment (10-day-old rats: 15.0 +/- 0.2; 55-day-old rats: 11.7 +/- 1.3). After DEXA pre-treatment, PAH accumulation was only slightly changed (10-day-old rats: 5.9 +/- 0.2; 55-day-old rats: 8.2 +/- 1.3). Semi-quantitative measurements of OAT1 mRNA expression level showed a significant increase of OAT1 mRNA after pre-treatment with both T3 and DEXA in the two age groups. Thus, this is the first evidence that T3 and DEXA pre-treatment induces the expression of OAT1.

Determination of renal porphyrin handling in rats suffering from different kinds of chronic renal failure (CRF): uranyl nitrate (UN) induced fibrosis or 5/6-nephrectomy (5/6NX).

The renal handling of porphyrins is reported to be a sensitive marker for chronic renal failure (CRF) for two reasons: heme is synthesised in proximal tubules and porphyrins are reabsorbed in the renal proximal tubule by apical peptide transporter PEPT 2. Two different models of CRF in female Wistar rats have been used for investigation of renal porphyrin handling: (1) single administration of uranyl nitrate (UN; 0.5 mg/100 g b.wt.) and (2) 5/6 nephrectomy (5/6NX). Renal clearance experiments were performed at weeks 2 and 10 after the onset of CRF. The concentrations of porphyrin intermediates (uroporphyrin I and III, coproporphyrin I and II, heptaporphyrin, and pentaporphyrin) were measured by HPLC with fluorescence detection. Both after UN and 5/6NX a significant reduction of body weight occurred. The kidney weight was enhanced 2 weeks after UN compared to controls (+31%). After 5/6NX, the weight of the remnant kidney was 44% (2nd week) and 140% (10th week) higher compared to one control kidney. Urine volumes and GFR were significantly reduced at week 2 and 10 after 5/6NX, but at week 10 after UN values were comparable to controls. Two weeks after UN and 5/6NX the concentrations of heptaporphyrin was moderately decreased in renal tissue whereas after 10 weeks the concentrations of most porphyrins were increased in the kidney. The plasma levels of free porphyrins were only slightly enhanced (week 2). The renal excretion of porphyrins was initially slightly reduced in both models, whereas it increases 10 weeks after UN, but it remained reduced 10 weeks after 5/6NX. UN induces tubulointerstitial fibrosis including atrophic glomeruli, whereas 5/6NX was characterized by distinct proteinuria, dilated tubules containing hyaline casts. A modulation of porphyrin metabolism in the kidney seems first of all to be responsible for UN effect on renal porphyrin handling. Summing up the 5/6NX results, both reduction in intact renal tissue mass and a modification of enzymes involved in heme biosynthesis by uraemic toxins are responsible for accumulation of porphyrins in renal tissue. After 5/6NX reduced excretion of porphyrins into urine and enhanced porphyrin concentrations in the kidney indicate more a damage of renal porphyrin biosynthesis than changes in their reabsorption.

Use of gene chip technology for the characterisation of the regulation of renal transport processes and of nephrotoxicity in rats.

Gene expression profiling using microarrays (rat-specific array RG-U34A, Affymetrix, U.S.A.) was employed for the investigation of: (1) hormonal regulation of renal function and (2) nephrotoxicity. For this purpose about 8,800 genes were analysed in kidney and, additionally, in liver tissue. Ad 1.) Kidney functions develop during postnatal life. Thus, in vivo transport and accumulation of p-aminohippurate (PAH) was investigated on renal cortical slices (RCS) from 10- and 55-day-old rats. The animals were treated with dexamethasone (DEXA; 60 microg/100 g b.wt./day) for 3 days, which caused a significant reduction in the accumulation of PAH in 10-day-old rats (42 +/- 5% whereas it was only slightly reduced in 55-day-old rats (70 +/- 8%). To further clarify the regulation of renal function by DEXA, results were compared with those obtained previously after in vitro stimulation with DEXA. RCS were incubated for 24 hours in DEXA-containing medium (10(-9) M). Under these conditions DEXA significantly increased the PAH uptake capacity in RCS obtained from 10- and 55-day-old rats up to 126 and 136%, respectively. Thus a stimulation of tubular transport capacity is possible in vitro. The effect of DEXA treatment on the gene expression of the kidney (in vivo) was moderate. Focussing especially on transporters, ion channels, ATPases, glucuronyltransferases, glutathione-S-transferase and cytochrome P450, the expression of only few genes were significantly changed (3 to 50-fold up- or down-regulation). Moreover, distinct age differences were found after in vivo administration of DEXA. The investigation of in vitro effects of DEXA is currently been performed. Ad 2.) The kidney is threatened by nephrotoxins because of its ability to accumulate them. We used a single administration of uranyl nitrate (UN; 0.5 mg/100 g b.wt.) as a model for chronic renal failure (CRF). Clearance experiments were performed 10 weeks after UN administration (maximal symptoms of CRF) in adult female rats. As expected, UN induced interstitial cicatrices with reduced GFR and diminished PAH transport capacity. Despite the impressive morphological and functional changes in the kidney after exposure to UN, the gene expression profiles in the kidneys were only minimally affected: we found significantly changed expression levels for only 20 genes (5 genes were up-regulated [e.g. transgelin], 15 down-regulated [among these the Na-K-Cl-symporter, insulin-like growth factor, kallikrein, and ornithine decarboxylase). The lack of agreement between gene expression data and the nephrotoxic effects of UN can probably be explained by the long time interval between dosing and the assessment of the effect. The results confirm that primary genomic responses are likely to be strongest transiently after exposure and then decrease in intensity.

Influence of high-dose methotrexate therapy (HD-MTX) on glomerular and tubular kidney function.

BACKGROUND: The present investigation was intended to further clarify the mechanisms involved in renal dysfunction following high-dose methotrexate (HD-MTX) treatment. PATIENTS AND METHODS: Fifty eight predominately pediatric patients [39 male, 19 female; mean age 12.3 years (range 2.2-34.1)] suffering from acute lymphoblastic leukemia (ALL, n = 28), Non Hodgkins lymphoma (NHL, n = 13), osteosarcoma (n = 8), malignant brain tumor (n = 6), or an ALL relapse (n = 3), were prospectively examined. In the course of 220 infusions of HD-MTX, glomerular and tubular renal function was determined by measuring proteinuria and glomerular filtration rate (GFR), as well as renal excretion of alpha-1-microglobulin (AMG) and N-acetyl-beta-D-glucosaminidase (NAG). It was investigated whether there were differences in MTX toxicity in dependence on the administered dose (1, 5, or 12 g/m(2) BSA), on the combination with other cytostatic agents (ifosfamide or cyclophosphamide), on the metabolism of MTX into 7-OH-MTX, and on pre-treatment with MTX. RESULTS: The administration of HD-MTX has no direct tubulotoxic effect. The disturbance in glomerular function was dose dependently and indicated by an increase in proteinuria as well as by a decrease in GFR; all changes were completely reversible and did not correlate to the metabolism of MTX to 7-OH-MTX. Increasing the number of MTX therapeutic cycles did not increase the nephrotoxicity of MTX. CONCLUSION: MTX is not directly tubulotoxic. Its side effects on glomeruli are usually without clinical relevance.

Fibronectin splice variants--prognostic markers for the stage of renal interstitial fibrosis in the rat.

BACKGROUND/AIMS: Renal interstitial fibrosis (RIF) is the main cause for progressive renal failure, but its pathogenic factors are not well known. In animal models of renal fibrogenesis done thus far an increase of total fibronectin (FN) mRNA has been proved. Recent studies have pointed to a key role of the splice variant EIIIA(+)-FN and EIIIB(+)-FN for the development of organ fibrosis. However, a broader knowledge of the distribution of these different FN mRNA isoforms is still lacking. Our aim was to study the particular expression of the EIIIA(+)-FN and EIIIB(+)-FN during the process of fibrogenesis in two rat models and to evaluate the FN isoforms as diagnostic/prognostic marker for the stage of interstitial damage in rat kidneys. METHODS: Kidneys of unilateral ureteral obstruction (UUO) and control rats were removed in intervals of 5, 14 or 21 days after surgery. For the investigation of kidney damage due to uranyl nitrate (UN), rats obtained a single i.p. dose of 5 mg/kg body weight UN and were killed 2, 10 and 20 weeks thereafter. The quantitative RT-PCR method was used to estimate the total FN, EIIIA(+)-FN and EIIIB(+)-FN transcription rate. RESULTS: In the UUO model, a significant augmentation of both isoforms was obtained in the kidneys in the first 5-day interval, which was more pronounced at the 21-day interval. In the UN-treated kidneys there appeared only a continuous increase of EIIIA(+)-FN and the splice variant EIIIB(+)-FN failed to show a shift in these animals as compared to the controls. CONCLUSION: Both animal models generated fibrogenic damages of the tubulointerstitium, whereas only the UUO resulted in progressive fibrosis. Absence of EIIIB(+)-FN seems to enhance the progression of fibrogenesis.

Renal interstitial fibrosis (RIF): II. Ultrasound follow up study of single uranyl nitrate administration causing renal dysfunction in rats--comparison with histologic and functional renal parameters.

A single administration of uranyl nitrate (UN; 0.5 mg/100 g b. wt. i.p.) to adult female Wistar rats reliably induces renal interstitial fibrosis (RIF) providing an experimental model to investigate therapeutic strategies. It was the aim of this study to further characterise a rat model of UN induced RIF which we have studied previously (Appenroth et al. 2001) by the comparison of functional parameters with ultrasonographic examination over a period of 30 weeks after injury. In the acute phase after UN administration (between days 2 and 17) signs of inflammation (increase in renal blood flow, swelling of renal cortex, enlargement of renal pelvis) could be detected by ultrasound. After four weeks UN led to functional changes (decreased creatinine clearance, increased urinary protein excretion and increased OH-proline concentration in renal tissue). In vitro, the accumulation of p-aminohippurate and the gluconeogenesis were reduced. In accordance with the functional changes, distinct ultrasonographic abnormalities could be seen between weeks 10 and 30 after UN with regard to changes in kidney size and shape, reduced renal perfusion and enlargement of renal pelvis. The sensitivity of ultrasonography in small laboratory animals is limited and most useful for follow-up studies of acute renal changes after administration of nephrotoxins. Ultrasonography can not be recommended for non-invasive screening of the progression of chronic renal failure.