Induction Of Acute Renal Failure Research Articles

Experimental application results of mesenchymal stem cell microvesicles in the mouse model of acute renal failure

An important role in restoration of damaged organs and tissues is played by mesenchymal stem cells (MSCs) and microvesicular particles (MV) produced by them. They can be a source of cytokines, anti- apoptotic and growth stimulating factors. In addition, MVs carry out transport of mRNA, miRNA, and signal proteins into damaged tissues. This increases the ability of cells to regenerate and to inhibit apoptosis, promote to angiogenesis and stimulate cell proliferation. The aim of our research was to study the immunoregulatory and pro-regenerative properties of mesenchymal stem cell microvesicles (MSC-MV) in a model of glycerol- induced acute renal failure (ARF) in mice. The experiments were carried out on CBA mice aged 3-4 months. AKI was induced by a single intramuscular injection of 50% glycerol. MSCs were obtained from the bone marrow of healthy animals and cultivated under standard conditions. Microvesicles were obtained by centrifugation at 12000g of MSC supernatant after induction of their apoptosis by culturing under oxygen deprivation conditions and in serum-free medium. MSC-MV was injected intravenously into the retroorbital sinus one day after induction of ARF. The MV dose was calculated as equivalent to (derived from) 1 million MSCs, which was 100 mL per mouse. Animals were taken out of the experiment on days 4 and 11 after MSC-MV injection. Blood plasma was taken to determine the level of creatinine, urine – for albumin analysis, kidneys – for histological examination. It has been shown that MVs induced by MSCs dose-dependently stimulated splenocyte proliferation in both spontaneous and Con-A induced tests. The addition of MV caused a decrease in doxorubicin-induced apoptosis of splenic lymphocytes in mice. Probably, in this case, MV produced by MSCs had an immunostimulatory and antiapoptotic effect. Also, MVs had a positive impact on the restoration of structure and function kidneys in a model of ARF in mice. The use of MSC-MV in treatment of acute renal failure induced by a single injection of 50% glycerol contributed to decrease albumin level urine and restoration of creatinine level in blood serum of animals. Morphological studies have shown decrease in the height cell and collecting duct diameter in the medulla and a decrease in the largest transverse diameter of superficial glomeruli in the renal cortex of sick mice. Thus, the obtained results indicate significant therapeutic and pro-regenerative properties of MSC-MV, which require further study.

Optimization and Validation of Glycerol-induced Acute Renal Failure Model in Rats in Predicting the Pharmacokinetics in Patients with Acute Renal Failure

The study standardized the glycerol induced (intra-peritoneal and intra-muscular) acute renal failure (ARF) models in Sprague-Dawley rats and validated its potential to predict the change in pharmacokinetics of the selected probe drugs in renal impairment. We assessed the induction of ARF by measuring the serum creatinine and blood-urea-nitrogen (BUN) levels at different time points (baseline, 6 h, 12 h, 24 h, 48 h and 72 h). Glycerol administrations via both intra-peritoneal and intra-muscular routes caused a significant increase in serum creatinine and BUN levels in rats at 6 h; however, the levels decreased within 24 h with intra-peritoneal administration. In contrast, with intra-muscular administration, a sustained increase in serum creatinine and BUN levels was observed up to 72 h post glycerol treatment demonstrating a sustained ARF like condition in rats that was suitable to evaluate the pharmacokinetics of drugs. Therefore, intra-muscular glycerol administration model was used to evaluate the pharmacokinetics of selected probe drugs including metformin, digoxin, atorvastatin and linezolid. The pharmacokinetics of metformin and digoxin were significantly altered in ARF versus control rats with increase in plasma exposure, prolonged elimination half-life and decreased systemic clearance. However, the pharmacokinetics of atorvastatin and linezolid did not show significant differences between ARF and control rats. These results demonstrated the utility of the ARF model in rats in predicting pharmacokinetic changes in patients with renal impairment. Such models may prove useful in early discovery work to screen and prioritize compounds requiring dose adjustment in patients with renal impairment.

Some Physiological Effect of Acacia Gum on Acute Renal Failure Induced by Gentamicin in Male Rabbits

Renal failure a case characterized by the impairment of the kidney to filter waste products from the body.The study aims to evaluate the potential effect of Acacia gum in the prevention of induced Acute renalfailure in the rabbit model. Eighteen healthy male domestic rabbits (Lepus cuniculus) were divided intothree groups. Group 1: normal rabbits as control, Group 2: animals were injected (70mg/kg. B.W) ofintramuscular gentamicin daily for 14 days for induction of acute renal failure, Group 3: animals weretreated with intramuscular injection (70mg/kg B.W) gentamicin daily and offered 15% Acacia gum withdrinking water for 14 days. At the end of the experiment Serum urea, uric acid, creatinine, blood ureanitrogen, calcium, sodium, and malondialdehyde were measured in addition to Urine analysis. The resultsshowed that the treatment of Acacia gum improved significantly serum uric acid, creatinine, urea, bloodurea nitrogen, electrolyte, and proteinuria. Also reduced malonaldehyde, urine Specific gravity, and pHlevels. Our findings conclude that GA can protect the kidney by enhancing antioxidant efficiency, and maybe useful in improving acute renal failure.

The Assay of Quercetin Solid Dispersion as a Potential Nephronprotector in Acute Renal Failure Induced Mice

Quercetin has been used with other nutraceutical components to improve renal function. Its potential to be developed as an active pharmaceutical ingredient, however, is limited by poor aqueous solubility and low rate of dissolution leading to low bioavailability in rats (< 17%) and in human (1%). Solid dispersion of quercetin with PVP K30 has increased its solubility 13.24 times and the amount dissolved (95.12 ± 1.83%) in comparison to pure quercetin. This study aimed to determine the nephron-protection effect of the solid dispersion on Acute Renal Failure (ARF) mice. The animals were divided into 6 groups, normal mice as a negative control group (G1), ARF induced mice as a positive control group (G2), ARF induced mice given pure quercetin 50 mg/kg BW (G3), ARF induced mice given solid dispersion containing 10 mg/ kg BW (G4), 5 mg/kg BW (G5) and 2.5 mg/kg BW (G6) quercetin respectively. The ARF was induced by injection of gentamycin sulphate 100 mg/kg BW for 7 days consecutively. Renal function was monitored by measuring the serum creatinine at day 8th. The protection effect was also observed from the histopathology score of the nephrons. Results showed that ARF induction increased serum creatinine above normal. Solid dispersion doses variations significantly influence the serum creatinine (p < 0.05). The stage of renal impairment based on histopathology score was significantly influenced by the doses of quercetin in solid dispersion (p < 0.05). It was concluded that solid dispersion containing quercetin at doses 2.5 and 5.0 mg/ kg BW respectively did not effective as a nephron-protector. The solid dispersion containing quercetin 10.0 mg/kg BW was effective to reduce the serum creatinine and showed a nephronprotection effect on the ARF induced mice.

In Vivo Study on Mechanism Underlying Increased Pharmacological Effects of Phenobarbital in Rats with Glycerol-Induced Acute Renal Failure.

The mechanism underlying the increased pharmacological effects of phenobarbital in rats with glycerol-induced acute renal failure (ARF) was examined. In the experiments, a surgical cannula was inserted in the lateral ventricle of the rats for phenobarbital infusion, and the ARF induction was performed by intramuscular administration of 50% glycerol. The onset time of anesthesia by phenobarbital was determined with the tail flick method. In addition, cerebral microsomes were prepared from excised cerebral cortices of sham and ARF rats, and the cerebral expression of the γ-aminobutyric acid (GABA)A receptor and two cation-chloride transporters, KCC2 and NKCC1, was evaluated by Western blotting, as their functions are involved in the anesthetic effects of phenobarbital. When phenobarbital was infused in the ventricle, anesthesia was induced 2.2-times faster in ARF rats than in sham rats, and there was no detectable increase in the cerebral expression of the GABAA receptor in ARF rats. It was additionally noted that the cerebral expression of KCC2 decreased, whereas that of NKCC1 was unaltered in ARF rats. These findings indicated that the anesthetic effects of phenobarbital are potentiated in ARF rats, probably due to imbalanced cerebral expression of KCC2 and NKCC1, suggesting that altered cation-chloride handling in nerve cells is associated.

STF-083010, an inhibitor of XBP1 splicing, attenuates acute renal failure in rats by suppressing endoplasmic reticulum stress-induced apoptosis and inflammation.

Endoplasmic reticulum (ER) stress is one of the driving forces of ischemia/reperfusion (IR)-induced acute renal failure (ARF). STF-083010, an inhibitor of the endonuclease activity of inositol-requiring enzyme-1 (IRE1), has the potential to block the initiation of a prolonged unfolded protein response (UPR) that is stimulated by ER stress and alleviates the impairments due to ER stress. In the current study, it was hypothesized that STF-083010 was capable of ameliorating ER stress-related damages in IR-induced ARF. Rats were administrated with STF-083010 and were subjected to induction of ARF using a ligation method. Then the effect of STF-083010 administration on the renal structure and function, oxidative stress, and inflammation in model rats was assessed. Furthermore, the levels of expression of UPR members and downstream effectors regulating apoptosis were detected as well. The results showed that establishment of the ARF model induced ER stress and impaired the renal structure and function. Administration of STF-083010 ameliorated impairments in the structure and function of the kidneys and the effect was associated with the suppressed oxidative stress and inflammation. At the molecular level, STF-083010 inhibited the prolonged UPR by downregulating the expressions of GRP78, p-IRE1, XBP1s, CHOP, and caspase 3, partially explaining the decreased apoptotic rate. The current study evaluated the potential of STF-083010 in treating ER stress-induced symptoms in ARF for the first time, and the findings demonstrated that STF-083010 resulted in effective treatment outcomes of ARF.

Functional Renal Imaging with 2-Deoxy-2-18F-Fluorosorbitol PET in Rat Models of Renal Disorders.

Precise regional quantitative assessment of renal function is limited with conventional 99mTc-labeled renal radiotracers. A recent study reported that the PET radiotracer 2-deoxy-2-18F-fluorosorbitol (18F-FDS) has ideal pharmacokinetics for functional renal imaging. Furthermore, 18F-FDS is available via simple reduction from routinely used 18F-FDG. We aimed to further investigate the potential of 18F-FDS PET as a functional renal imaging agent using rat models of kidney disease. Methods: Two different rat models of renal impairment were investigated: induction of acute renal failure by intramuscular administration of glycerol in the hind legs, and induction of unilateral ureteral obstruction by ligation of the left ureter. At 24 h after these procedures, dynamic 30-min 18F-FDS PET data were acquired using a dedicated small-animal PET system. Urine 18F-FDS radioactivity 30 min after radiotracer injection was measured together with coinjected 99mTc-diethylenetriaminepentaacetic acid urine activity. Results: Dynamic PET imaging demonstrated rapid 18F-FDS accumulation in the renal cortex and rapid radiotracer excretion via the kidneys in healthy control rats. On the other hand, significantly delayed renal radiotracer uptake (continuous slow uptake) was observed in acute renal failure rats and unilateral ureteral obstruction kidneys. Measured urine radiotracer concentrations of 18F-FDS and 99mTc-diethylenetriaminepentaacetic acid correlated well with each other (R = 0.84, P < 0.05). Conclusion:18F-FDS PET demonstrated favorable kinetics for functional renal imaging in rat models of kidney diseases. 18F-FDS PET imaging, with its advantages of high spatiotemporal resolution and simple tracer production, could potentially complement or replace conventional renal scintigraphy in select cases and significantly improve the diagnostic performance of renal functional imaging.

Renal excretion of rhodamine 123, a P-glycoprotein substrate, in rats with glycerol-induced acute renal failure.

To clarify renal handling of rhodamine 123, a substrate for P-glycoprotein, in normal and diseased states, in-vivo clearance studies were performed with normal rats and rats with glycerol-induced acute renal failure. For normal rats the excretion ratio of unbound rhodamine 123-to-inulin was 3.25, indicating the presence of the renal tubular secretion of rhodamine 123. Co-administration of cyclosporin, a P-glycoprotein inhibitor, significantly reduced tubular secretion of rhodamine 123. Administration of glycerol induced both an increase in blood urea nitrogen and a reduction in the glomerular filtration rate, confirming the induction of acute renal failure. Total plasma, renal, and tubular secretory clearances of rhodamine 123 were significantly lower for rats with acute renal failure than for control rats. There was no difference between the ATP content of the renal cortex in control rats and those with acute renal failure. In addition to the decrease in renal clearance, a decrease in the biliary clearance of rhodamine 123 was also observed in rats with acute renal failure. These results imply that rhodamine 123 is secreted via P-glycoprotein in renal tubules and that the renal secretory clearance of rhodamine 123 was reduced after acute renal failure, probably because of impairment of P-glycoprotein.

Histone deacetylase blockade improves renal function and attenuates oxidative stress in rats with cisplatin‐induced acute renal failure

Histone deacetylase (HDAC) inhibition has been shown to prevent cytokine induction and to modulate renal disease in rodent models. We tested the hypothesis that HDAC inhibition would improve various renal and oxidative stress parameters in cisplatin (CDDP)‐induced acute renal failure (ARF). 15‐week old male Sprague‐Dawley rats (n=4 per group) were treated with the HDAC inhibitor, valproic acid (VPA; 300 mg/kg, s.c.), or vehicle, for 1 day prior to and 5 days following induction of ARF with a single 6 mg/kg injection of CDDP (or saline vehicle). At the end of 6 days, anesthetized rats underwent acute renal experiments to determine renal blood flow (RBF) and glomerular filtration rate (GFR) using PAH and inulin clearance techniques, respectively. Rats were sacrificed and kidneys removed; renal cortices were isolated for measurement of tissue and mitochondrial free radical levels using electron paramagnetic resonance spectroscopy. Tissue mRNA and protein levels of HDAC and oxidative stress genes were determined using real‐time RT‐PCR and western blot, respectively. No significant changes in mean arterial pressure (MAP) were noted among groups. VPA treatment improved RBF and GFR (p&lt;0.05) and attenuated the higher levels of total tissue and mitochondrial ROS and tissue superoxide in ARF rats. VPA treatment also significantly decreased tissue mRNA and protein expression levels of HDAC and oxidative stress genes, and normalized ATP production and ADP/ATP ratio in rats with ARF. These findings suggest that inhibition of class I HDACs with VPA improves the renal oxidative stress and tissue and mitochondrial injury that accompany CDDP‐induced ARF. Grant support: NIH grant RO1HL080544‐01 for Dr. Joseph Francis.

Protective role of the inhibitor of apoptosis protein, survivin, in toxin-induced acute renal failure.

Acute renal failure (ARF) is a major worldwide cause of morbidity and mortality, lacking specific targeted, effective therapies. Renal tubular cell apoptosis has been recognized to play a critical role in the pathogenesis of ARF, yet few studies have evaluated whether intervention in apoptotic pathways can ameliorate the deterioration in renal function associated with ARF. Using transgenic mice with diminished levels of the inhibitor of apoptosis protein, survivin, we show that survivin is required to protect the kidney from apoptosis, to suppress renal expression of p53, and to ameliorate renal dysfunction in two models of ARF. Gene delivery of survivin to wild-type mice and mice with 50% levels of survivin, prior to or at the time of induction of ARF, interferes with the deterioration of renal function and preserves the integrity of the kidneys and the renal tubular cells by inhibiting activation of apoptotic pathways in the kidneys and suppressing expression of p53. These results encourage further evaluation of survivin, its active structural domains, and other inhibitors of apoptosis proteins, for preventing and/or treating acute renal failure.

Ciglitazone, a Peroxisome Proliferator-Activated Receptor γ Inducer, Ameliorates Renal Preglomerular Production and Activity of Angiotensin II and Thromboxane A2 in Glycerol-Induced Acute Renal Failure

Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear transcription factor, modulates vascular responses to angiotensin II (AII) or thromboxane A(2) (TxA(2)) via regulation of their gene/receptor. Increased vasoconstriction and deteriorating renal function in glycerol-induced acute renal failure (ARF) may be attributed to down-regulation of PPARgamma. In this study, we investigated the effect of ciglitazone (CG), a PPARgamma inducer, on AII and TxA(2) production and activity in glycerol-induced ARF. Vascular responses to AII or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F(2alpha) (U46619), a TxA(2) mimetic, were determined in preglomerular vessels following induction of ARF with glycerol. Renal damage and function were assessed in CG-treated (9 nmol/kg for 21 days) rats. PPARgamma protein expression and activity, which were significantly lower in ARF rats, were enhanced by CG (26 and 30%). CG also increased PPARgamma mRNA by 67 +/- 6%, which was reduced in ARF. In ARF, there was significant tubular necrosis and apoptosis, a 5-fold increase in proteinuria and a 2-fold enhancement in vasoconstriction to AII and U46619. CG reduced proteinuria (49 +/- 3%), enhanced Na(+) (124 +/- 35%) and creatinine excretion (92 +/- 25%), markedly diminished tubular necrosis, and reduced ARF-induced increase in AII (40 +/- 3%) and TxA(2) (39 +/- 2%) production, the attending increase in vasoconstriction to AII (36 +/- 2%) and U46619 (50 +/- 11%), and the increase in angiotensin receptor-1 (AT(1)) (23 +/- 3%) or thromboxane prostaglandin (TP) receptor (13 +/- 1%). CG reduced free radical generation by 55 +/- 14% while elevating nitrite excretion (65 +/- 13%). Our results suggest that enhanced activity of AII and TxA(2), increased AT(1) or TP receptor expression, and renal injury in glycerol-induced ARF are consequent to down-regulation of PPARgamma gene. CG ameliorated glycerol-induced effects through maintaining PPARgamma gene.

Antioxidant U74389G Improves Glycerol-Induced Acute Renal Failure without Affecting PPARγ Gene

Oxygen metabolites play an important role in the pathogenesis of myoglobinuric acute renal failure (ARF). Previously, we have reported a down regulation of peroxisome proliferator activated receptor γ (PPARγ) in glycerol-induced ARF, and the induction of PPARγ has been shown to provide renal protection. In this study, we determined the protective influence of U74389G, a hydroxyl radical scavenger in myoglobinuric ARF, and its association with PPARγ-mediated renal protection in the rat. Vascular responses to AII were determined in renal pre-glomerular vessels following the induction of ARF with glycerol (50%, v/v, i.m.). The extent of renal damage and function were assessed with or without pre-treatment with U74389G (10 mg/kg × 21 days). In ARF, AII vasoconstriction was enhanced (97%; p < 0.05), and AII production was doubled. U74389G reduced AII vasoconstriction and production by 42% (p < 0.05) and 40% (p < 0.05), respectively. U74389G reduced proteinuria (85%; p < 0.05), which was four times higher in ARF. Similarly, U74389G enhanced Na+ excretion twofold while reducing plasma creatinine (24%; p < 0.05) and BUN (31%; p < 0.05). U74389G attenuated free radical generation in ARF while nitrite excretion was unchanged. In renal pre-glomerular vessel, PPARγ expression, activity, and mRNA were significantly lower in ARF rats; this was unchanged with U74389G treatment. On the other hand, U74389G significantly reduced NFκB protein expression, which was elevated in ARF by 25% (p < 0.05). We suggest that antioxidant U74389G blunted renal injury and improved renal function in glycerol-induced ARF through the reduction of free radical production and/or inhibition of NFκB without affecting PPARγ.

Role of PPAR-γ on the pathogenesis and vascular changes in glycerol-induced acute renal failure

Peroxisome proliferator activated receptor-γ (PPAR-γ), a nuclear transcription factor, modulates angiotensin II (AII) or thromboxane A2 (TxA2) response in the vasculature via transcriptional regulation of their gene or receptor expression. Increased AII or TxA2 vasoconstriction and deteriorating renal function observed in glycerol-induced acute renal failure (ARF) may be attributed to a down-regulated PPAR-γ expression/activity probably via an increased free radical generation. In this study, we investigated the effect of PPAR-γ induction in glycerol-induced ARF by examining renal vascular reactivity to AII and TxA2 and by renal expression/activity of PPAR-γ. Vascular responses to AII or U46619, a TxA2 mimetic were determined in rat isolated perfused kidney following induction of ARF with glycerol (50%, v/v, i.m.). Extent of renal damage and function were assessed with or without pre-treatment with ciglitazone (9nmolkg−1×21 days), a PPAR-γ inducer. In ARF, vasoconstriction was enhanced to AII (three-fold; p<0.05) and U46619 (82%; p<0.05). Ciglitazone reduced AII and U46619 vasoconstriction by 59±1% (p<0.05) and 56±1% (p<0.05), respectively. Ciglitazone reduced proteinuria (38±3%) which was two-fold higher in ARF. Similarly, ciglitazone enhanced Na+ excretion by 1.5 times while reducing BUN by 49±6%. On the contrary, ciglitazone did not change plasma creatinine which was significantly higher in ARF rats. Ciglitazone reduced free radical generation by 30±3% while elevating nitrite excretion ∼2-fold. PPAR-γ expression and activity were significantly lower in ARF rats and ciglitazone enhanced PPAR-γ protein expression and activity by 45±3% and 52±4%, respectively. Data from this study suggest that reduced PPAR-γ expression and activity may be involved in the pathology of glycerol-induced ARF and induction of PPAR-γ by ciglitazone confers protection through reduced AII and TxA2 vasoconstriction and/or enhanced renal function via reducing free radical generation.

Ischemic proximal tubular injury primes mice to endotoxin-induced TNF-α generation and systemic release

Endotoxemia (LPS) can exacerbate ischemic tubular injury and acute renal failure (ARF). The present study tested the following hypothesis: that acute ischemic damage sensitizes the kidney to LPS-mediated TNF-alpha generation, a process that can worsen inflammation and cytotoxicity. CD-1 mice underwent 15 min of unilateral renal ischemia. LPS (10 mg/kg iv), or its vehicle, was injected either 45 min before, or 18 h after, the ischemic event. TNF-alpha responses were gauged 2 h post-LPS injection by measuring plasma/renal cortical TNF-alpha and renal cortical TNF-alpha mRNA. Values were contrasted to those obtained in sham-operated mice or in contralateral, nonischemic kidneys. TNF-alpha generation by isolated mouse proximal tubules (PTs), and by cultured proximal tubule (HK-2) cells, in response to hypoxia-reoxygenation (H/R), oxidant stress, antimycin A (AA), or LPS was also assessed. Ischemia-reperfusion (I/R), by itself, did not raise plasma or renal cortical TNF-alpha or its mRNA. However, this same ischemic insult dramatically sensitized mice to LPS-mediated TNF-alpha increases in both plasma and kidney (approximately 2-fold). During late reperfusion, increased TNF-alpha mRNA levels also resulted. PTs generated TNF-alpha in response to injury. Neither AA nor LPS alone induced an HK-2 cell TNF-alpha response. However, when present together, AA+LPS induced approximately two- to fivefold increases in TNF-alpha/TNF-alpha mRNA. We conclude that modest I/R injury, and in vitro HK-2 cell mitochondrial inhibition (AA), can dramatically sensitize the kidney/PTs to LPS-mediated TNF-alpha generation and increases in TNF-alpha mRNA. That ischemia can "prime" tubules to LPS response(s) could have potentially important implications for sepsis syndrome, concomitant renal ischemia, and for the induction of ARF.

In Vivoandin VitroDegradation of Urea and Uric Acid by Encapsulated Genetically Modified Microorganisms

This study was undertaken to characterize the capacity of a combination of genetically modified bacteria to lower elevated levels of urea and uric acid and thus to serve as a potential adjunct to maintenance dialysis in patients with chronic renal failure. Two strains of genetically modified bacteria expressing enzymes, urease to degrade urea and uricase to degrade uric acid, were identified, combined, and dispersed in 600-microm alginate microcapsules suitable for oral administration. In 24 h in vitro experiments, 5 mL of these capsules completely cleared 95% of the urea and >99% of the uric acid from 100 mL of a challenge solution formulated to the concentration of these solutes in a presenting hemodialysis patient. The process of urea degradation was found to be intracellular and each bacterial strain was specific for its substrate. Solute degradation in vivo was evaluated with a chemically induced model of acute renal failure, using Sprague-Dawley rats. Orally administered capsules were found to remain in the gastrointestinal tract for at least 6 h. The severity of azotemia and hyperuricaemia after chemical induction of acute renal failure was reduced by 64 and 31%, respectively, on administration of the capsules. Reduction of urea concentration (but not uric acid concentration) in vivo required coadministration of an ion-exchange resin to adsorb ammonia. Oral delivery of a combination of genetically modified microorganisms should be further explored in chronic renal failure models as a useful adjunct to dialysis or to immunosorption for the treatment of uremia.

Plasma protein extravasation and vascular endothelial growth factor expression with endothelial nitric oxide synthase induction in gentamicin-induced acute renal failure in rats.

Microvascular hyperpermeability to plasma proteins via vascular endothelial growth factor (VEGF) with endothelial nitric oxide synthase (eNOS) induction may contribute to wound healing through matrix remodeling. However, vascular hyperpermeability is not examined in acute renal failure (ARF), a unique form of wound healing. Subcutaneous injection of gentamicin (400 mg/kg per day for 2 days in divided doses every 8 h) in rats increased serum creatinine levels and induced tubular damage, which peaked at day 6, after the last gentamicin injection. Ki67-positive regenerating proximal tubules (PTs) peaked in number at day 6 and almost covered the bare tubular basement membrane (TBM) by day 10. Staining of fibrinogen and plasma fibronectin began to increase in the peritubular regions as early as day 0, steadily increased in TBM and tubular lumen until day 6 and then decreased. Hyperpermeable peritubular capillaries were identified by extravasation of perfused-fluoresceinated dextran (both 70 kDa and 250 kDa) into peritubular regions as early as day 0 and prominently into TBM and tubular lumen at day 6. Electron microscopy further suggested the intraendothelial pathway of dextran. Immunoreactive VEGF increased in the damaged and regenerating PTs. Immunoreactive VEGF receptors-1 and -2 did not change, but immunoreactive eNOS increased in the peritubular capillaries after induction of ARF. Western blotting for VEGF and eNOS supported the immunostaining findings. In addition, we assessed the effects of NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) on vascular hyperpermeability during the recovery phase of this model. Treatment with L-NAME (s.c. at a dose of 100 mg/kg/day from day 3 to day 6) decreased extravasation of perfused-250-kDa dextran and significantly inhibited the regenerative repair of PTs at day 6 when compared with vehicle-treated rats. In conclusion, plasma protein extravasation occurred, leading to matrix remodeling, such as the process of wound healing during the tubular repair in gentamicin-induced ARF. Since VEGF-induced vascular hyperpermeability may depend on NO production, VEGF/VEGF receptor system with eNOS induction might be responsible for this process.

Role of Thyroid Hormones on the Synthesis and Release of Atrial Natriuretic Peptide in Rats with Acute Renal Failure

Background/Aims: High atrial natriuretic peptide (ANP) serum levels are seen in acute renal failure (ARF) due to impaired ANP clearance, but the response of ANP to blood volume expansion is blunted. ARF-associated hypothyroidism influences ANP clearance but it may also play a role in the synthesis and release of ANP because in primary hypothyroidism both mechanisms are disturbed. The aim of this study is to analyze whether thyroxin (T₄) supplementation improves the synthesis and release of ANP in ARF. Methods: Four groups of rats were studied: group C (control); group ARF; Group ARF-T₄ (ARF supplemented with T₄), and group Tx (thyroidectomized). Serum creatinine (SCr), creatinine clearance (CrCl), total T₄, total triiodothyronin (T₃), plasma ANP, ANP response to acute saline load and atrial content of mRNA-ANP were measured 5 days after ARF induction with potassium dichromate. Results: CrCl was 1.8 ± 0.4 ml/min in group C, and it significantly dropped to 0.6 ± 0.5 in ARF (p < 0.01), T₄ improved it (1.7 ± 0.7 in ARF-T₄, p < 0.01) and Tx did not change it. Plasma ANP was higher in ARF as compared with C (152 ± 23 vs. 52 ± 21 fmol/l, p < 0.01). In ARF-T₄, ANP was higher than in C but lower than in ARF (85 ± 28 fmol/l, p < 0.01). No changes were observed in Tx rats (48 ± 17 fmol/l). The atrial content of mRNA-ANP was 0.06 ± 0.025 mRNA-ANP/mRNA-β-actin ratio units in group C, it was higher in ARF (0.13 ± 0.025, p < 0.01) and lower in Tx (0.04 ± 0.01, p < 0.05) as compared to C. Supplementation with T₄ increased the mRNA-ANF content (0.18 ± 0.2 mRNA-ANP/mRNA-β-actin ratio units, p < 0.05) over ARF. Plasma ANP response to saline load was 45 ± 7% in C, but it was reduced in ARF and Tx (20 ± 10 and 26 ± 10%, p < 0.01). In ARF-T₄ the response was restored (36 ± 9%, p < 0.01 vs. ARF). Conclusion: Thyroid hormones modulate the synthesis and release of ANP in ARF. Although the effect is probably direct, T₄-induced amelioration of renal damage may also play a significant role by improving the uremic milieu.

Important role for fibronectin-EIIIA during renal tubular repair and cellular recovery in uranyl acetate-induced acute renal failure of rats.

The present study was designed to identify the source and kinetics of an alternatively spliced "embryonic" cellular fibronectin EIIIA (cFn-EIIIA) in relation to regenerating renal tubules in uranyl acetate (UA)-induced acute renal failure (ARF) in rats. Damage of the proximal tubules was found as early as day 2 after induction of ARF, peaked at day 5, and was almost substituted by epithelial relining by day 7. Immunohistochemistry showed de novo deposition of cFn-EIIIA in peritubular regions as early as day 2, then on the tubular basement membrane (TBM) after day 4. beta1 Integrin, the receptor for Fn, was mainly found at the basal side of tubules in the normal control and increased in the interstitium after induction of ARF, but the staining pattern gradually returned to the control after day 7. Immunoelectron microscopy revealed that cFn-EIIIA was produced initially by the peritubular endothelium and later by fibroblastic cells and was deposited to the TBM, on which regenerating tubules proliferated, probably with cFn-EIIIA production. beta1 Integrin was expressed in cFn-EIIIA-producing cells, especially in regenerating tubular cells, suggesting that cFn-EIIIA signal transduction affects regenerating tubules. Transforming growth factor (TGF)-beta1 was found in some damaged proximal tubules and interstitial cells after induction of ARF and later in the regenerating tubules. CFn-EIIIA and beta1 integrin mRNA levels were upregulated as early as day 2. TGF-beta1 mRNA level significantly increased after day 3, suggesting a modulatory role for TGF-beta1 on cFn-EIIIA production, but not by day 2. Our data suggest that cFn-EIIIA production by the endothelium during the very early response to tubular injury and by fibroblastic cells and regenerating tubules may play an important role in the cellular recovery of UA-induced ARF in rats.

Decreased Expression of Brain Nitric Oxide Synthase in Macula Densa Cells and Glomerular Epithelial Cells of Rats with Mercury Chloride-Induced Acute Renal Failure

To elucidate the mechanisms responsible for the development of HgCl 2-induced acute renal failure (ARF), we examined the expression of brain type (b) nitric oxide synthase (NOS), which is involved in the generation of the vasodilator nitric oxide (NO), in the renal cortex of rats at 20 h after exposure to 7.5 mg/kg HgCl 2. Both blood urea nitrogen and serum creatinine were significantly increased in rats exposed to HgCl 2 relative to control rats, indicating the induction of ARF resulting from HgCl 2 exposure. Histopathological analysis demonstrated that, in addition to necrosis of proximal tubule epithelial cells, necrosis of macula densa cells and swelling of glomerular epithelial cells were observed in the renal cortex of rats with HgCl 2-induced ARF. Consequently, the number of pars maculata segments was decreased by 42% in rats with HgCl 2-induced ARF compared to control rats. The primary sites of bNOS mRNA and protein expression were macula densa cells and glomerular epithelial cells in the renal cortex of control rats and rats with HgCl 2-induced ARF. The abundance of the bNOS mRNA and protein was significantly decreased in rats with HgCl 2-induced ARF relative to control rats. These observations suggest that the production of the vasodilator NO derived from bNOS is decreased at the glomerulus level in the HgCl 2-induced ARF setting. Thus, the reduction in bNOS expression may in part contribute to the progression of HgCl 2-induced ARF through the deterioration of glomerular hemodynamics. In addition, the decrease in bNOS expression may be primarily the result of cell injury caused by the cytotoxic effect of HgCl 2.

Stimulation of osteoclastic bone resorption in a model of glycerol-induced acute renal failure: evidence for a parathyroid hormone-independent mechanism

This study was undertaken to evaluate the bone changes occurring in rats with acute renal failure (ARF). Acute renal failure was induced in rats 24 hours after dehydration by an intramuscular injection of glycerol. After induction of ARF, the rats were divided into two groups, one of which underwent parathyroidectomy (PTX). Rats with normal renal function, matched for age and weight, were used as controls and divided into two groups, one of them for PTX. At termination of the study blood and urine chemistry and bone histomorphometry were analyzed. Rats with glycerol-induced ARF developed bone changes compatible with mild hyperparathyroid bone disease, characterized mainly by increased osteoclastic bone resorption when compared with control rats having normal renal function. Rats with normal renal function following PTX developed bone disease showing complete suppression of forming and resorptive parameters. Rats with glycerol-induced ARF and PTX showed abolishment of all bone forming parameters, but a dramatic increase in osteoclastic resorption was apparent. Based on these observations we suggest that, in this model of glycerol-induced ARF, osteoclastic bone resorption may develop in the absence of parathyroid hormone, probably stimulated by other potent osteoclastogenic factors.