Rat Renal Tissue Research Articles

The effects of urokinase-type plasminogen activator (uPA) on cell proliferation and phenotypic transformation of rat mesangial cells induced by high glucose

AimsTo investigate the effects of urokinase-type plasminogen activator (uPA) on proliferation and phenotypic transformation of rat mesangial cells (MCs) under high glucose conditions and its possible signal transduction pathway. MethodsRat MC were divided into 4 groups: the control group, the high glucose group, the high glucose and wortmannin group, and the high glucose and uPA group. MC proliferation in all groups was detected by the 3-(4,5-dimethylthiazol-)-2,5-diphenyltetrazolium bromide (MTT) method. MC cell cycle was analyzed by flow cytometry. Expression of cyclin dependent kinase 2 (CDK2), and activity of the signaling protein Akt in MC were detected by Western blot. Expression pattern and quantity of α-smooth muscle actin (α-SMA) in MC were examined using laser confocal microscopy. The expression of plasminogen activator inhibitor-1 (PAI-1), and collagen IV in renal tissues in rats was tested with immunohistochemistry and Western blotting methods. ResultsActivation of Akt induced by high glucose can be reduced significantly by wortmannin and uPA. There was no obvious change in CDK2 protein expression in different groups (P>0.05). Expression of α-SMA in MC cytoplasm increased dramatically (P<0.01). Expression of α-SMA decreased significantly in the high glucose and wortmannin group and the high glucose and uPA group compared with that of the high glucose group (P<0.01). In diabetic rats, uPA down-regulated PAI-1 and collagen IV expression in mesangial matrix (P<0.05). ConclusionuPA antagonizes cell proliferation and phenotypic transformation of MCs induced by high glucose through inhibiting Akt signaling pathway.

Abstract 540: Increased Intrarenal CGRP Release From Afferent Renal Nerves Is Accompanied By Decreased Afferent Electric Activity

Afferent peptidergic renal nerves exhibit a dual function. They influence intrarenal immunological processes by releasing peptides like CGRP and control central sympathetic outflow via afferent electrical activity. The former seems to be important in renal inflammation whereas the modulation of sympathetic outflow by afferent electrical activity is not fully understood. Hence, we wanted to test the hypothesis that augmented effects of CGRP in renal inflammation occur with increased afferent renal nerve activity. As inflammatory model, normotensive renal inflammation (RI) was induced by i.v. injection of 1.75 mg/kg BW OX-7 antibody to rats. Animals were investigated neurophysiologically and pathomorphologically using standard techniques at day 6 after induction of RI. There was no increase in blood pressure (BP) in RI animals, hence confounding effects of BP were unlikely. RI rats exhibited albuminuria (61±6 μg/24h), infiltration of macrophages in the interstitium (26 ± 4 cells/high-power field) and glomeruli (3.7±0.6 cells/glomerular cross-section). Pretreatment with the CGRP antagonist rCGRP 8-37 (Tocris Bioscience 15nmol/kg) significantly reduced proteinuria and macrophage infiltration suggesting increased activity of CGRP in RI. In an in-vitro assay, renal tissue of rats with RI exhibited increased release of CGRP (14 ± 3 pg/ml vs. controls 6.8 ± 2.8 pg/ml; p&lt;0.05, n=8). RI tissue from renally denervated rats showed no detectable levels of CGRP. These results suggested CGRP release from renal nerves at a higher rate in RI. Directly recorded afferent renal nerve activity (ARNA; spikes/sec) was unexpectedly lower in RI compared to controls (8.0 ± 1.8 vs. 27.4 ± 4.1 Hz*,n=6, p&lt;0.05). On the other hand, the burst-frequency of directly recorded renal sympathetic nerve activity (RSNA) in RI was significantly higher than in controls (14.7 ± 0.9 vs. 11.5 ± 0.9 bursts/sec*; n=6, *p&lt;0.05). In contrast to our assumption increased release of neurogenic CGRP aggravating renal inflammation occurred with decreased ARNA. Increased RSNA pointed to a reduced tonic inhibition by ARNA. In hypertension, the later mechanism might worsen sympathetic dysregulation and renal damage.

Immunohistochemical expressions of adropin and ınducible nitric oxide synthase in renal tissues of rats with streptozotocin-ınduced experimental diabetes

Diabetes is characterized by high blood glucose levels; it occurs in 30–35% of the population. Elevated glucose levels can damage a number of organs, including the kidneys. Several peptide hormones participate in maintaining glucose homeostasis including the recently discovered “adropin,” a 42 amino acid peptide hormone. Adropin also alters inducible nitric oxide synthase (iNOS) expression. Therefore, we studied how adropin and iNOS expression is altered in the renal tissues of streptozotocin (STZ) induced diabetic rats. Seven sham, seven control and seven Wistar albino male rats were fed standard rat pellets and water ad libitum for 10 weeks. The rats in the diabetic group were injected i.p. with a single dose of 60 mg/kg STZ dissolved in 0.1 M phosphate-citrate buffer, pH 4.5. After the 10-week experimental period, the rats in both groups were anesthetized and decapitated. Kidney tissues were excised and placed in 10% formaldehyde solution, taken through routine histological procedures, and embedded in paraffin. Sections 5–6 μm thick were stained immunohistochemically using the avidin-biotin complex (ABC) method. Adropin and iNOS immunoreactivity were co-localized in the glomeruli, peritubular interstitial cells and peritubular capillary endothelium of the cortex; the thin limb of the loop of Henle in the medulla; and medullary peritubular interstitial cells and endothelium of the peritubular capillaries in both the control and diabetic groups. The intensities of adropin and iNOS immunoreactivity increased with the severity of the diabetes. Intense adropin immunoreactivity was detected in both the smooth muscle and human small intestine Paneth cells that were used as positive controls. The elevated levels of adropin and iNOS in the kidney indicates that these substances are involved in the pathophysiology of diabetes; this constitutes a compensatory mechanism against the damage inflicted by the disease.

Effect of subchronic inhalation of ethylbenzene on expression of heme oxygenase-1 in rat renal tissues

To investigate the effect of ethylbenzene on the expression of heme oxygenase-1 (HO-1) intrarenal tissues. Forty male Sprague-Dawley rats were randomly and equally allocated to control group, low-dose exposure group, moderate-dose exposure group, and high-dose exposure group to inhale different doses of ethylbenzene (0, 433.5 mg/m(3) (100 ppm), 4335.0 mg/m(3) (1000 ppm), and 6500.0 mg/m(3) (1500 ppm)) for 6 h per day, 5 days per week, for 13 weeks. After the rat model of subchronic ethylbenzene exposure was established, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in renal tissues were measured, and the mRNA and protein expression levels of HO-1 in renal tissues were measured by real-time PCR and Western blot. Compared with the control group, all exposure groups showed significantly decreased activities of GSH-Px and CAT in renal tissues and the moderate- and high-dose exposure groups showed significantly decreased activity of SOD in renal tissues (P < 0.05). All exposure groups showed significantly higher expression of HO-1 than the control group (P < 0.05). The high-dose exposure group showed significantly higher expression of HO-1 than the low- and moderate-dose exposure group (P < 0.05), and the moderate- and high-dose exposure group had significantly higher expression of HO-1 than the control group and low-dose exposure group (P < 0.05). A certain dose of ethylbenzene can induce elevated expression of HO-1 and decreased antioxidant levels in rat renal tissues, thus leading to oxidative stress damage.

Biochemical and histological evaluation of the effect of Sudan IV (a red dye) on renal function

The effect of a red dye, Sudan IV on kidney function was investigated. Some biochemical parameters such as total protein, creatinine, urea, sodium, potassium, alanine aminotransferase and aspartate aminotransferase levels were determined. Histopathological analyses were also carried out. Consequently, thirty-two albino rats (Wistar strain) were divided into 4 groups of eight rats each. The groups were given 90% rat chow supplemented with 10% palm oil. Sudan IV was administered in the diet to provide levels of 0% (PO), 0.005% (PO/0.005), 0.01% (PO/0.01) and 0.015% (PO/0.015). They were given these diets for six weeks along with water ad libitum. Weekly measurements of weights were recorded. Results showed that there were no significant (P&lt;0.05) differences in weight gain although there was slight reduction in feed intake. A significant (P&lt;0.05) increase in the levels of total protein, creatinine, urea, aspartate aminotransferase and alanine aminotransferase levels suggest possible kidney malfunction. Histopathological analyses reveal damage to renal tissues in rats administered with the dye.Keywords: Sudan IV; Kidney; Creatinine; Urea; Histopathological; Biochemical parameters

Protective effects of melatonin against spinal cord injury induced oxidative damage in rat kidney: A morphological and biochemical study

Spinal cord injury (SCI) induced oxidative stress affects multiple organ systems including the kidney. We studied the possible protective effects of melatonin on SCI-induced oxidative damage in renal tissues of rats. Wistar albino rats (n=24) were exposed to SCI and divided into vehicle- or melatonin-treated SCI groups. Melatonin was administred intraperitoneally at a dose of 10mg/kg for seven days. Renal tissues were investigated by light and electron microscopy. Furthermore, tissue malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) and superoxide dismutase (SOD) activities were also determined. In the vehicle-treated SCI group, the renal histology was disturbed compared to controls, whereas the melatonin-treated SCI group showed significantly reduced degeneration of renal tissue as seen by both light and electron microscopy. MDA levels, MPO and SOD activities were increased and GSH levels were decreased in the vehicle-treated SCI group compared to controls. On the other hand, decreased MDA levels and MPO activities and increased GSH levels were observed in the melatonin-treated SCI group compared to vehicle-treated SCI group. These results showed that experimentally induced SCI caused oxidative stress in the rat kidney, whereas melatonin treatment reduced oxidative stress, suggesting that it may be used as a complementary therapy of renal problems occurring following SCI.

Advanced IgA nephropathy with impaired renal function benefits from losartan treatment in rats

Background: Treatment with angiotensin receptor blockers (ARBs) is successful in mitigating IgA nephropathy (IgAN), independent of blood pressure changes, but the therapeutic role of ARB in advanced IgAN with impaired renal function is to be ascertained. The present study was performed to investigate the effect of losartan on advanced IgAN induced by staphylococcal enterotoxin B (SEB) combined with 5/6 nephrectomy in rats. Methods: Fifty-four male SD rats were randomly divided into three group: Rats in the model group were treated with SEB plus 5/6 nephrectomy, and those in the losartan group were gavaged with losartan (33.3 mg kg−1 d−1) besides the treatment with SEB plus 5/6 nephrectomy. The urine and blood biochemical changes of rats were tested. IgA, IgG, IgM and C3 depositions were studied dynamically with immunofluorescence. The renal tissue structures were observed under light microscopy. The expressions of TGF-β1, FN, alpha-SMA and FGF-1 in rat renal tissues were determined with immunohistochemical methods and real-time PCR. Results: At 12 weeks, rats with SEB treatment plus 5/6 nephrectomy showed gradually increased urinary red blood cell (URBC) with a gradual elevation of the 24 h urinary protein, serum BUN and Scr, but losartan treatment lowered the levels of 24 h urinary protein, serum BUN and Scr. A large number of IgA depositions in the mesangial area, glomerulosclerosis and tubulointerstitial fibrosis were found in the model group, and the losartan group showed relieved injury. The expressions of TGF-β1, FN, alpha-SMA and FGF-1 were significantly elevated in the model. Losartan lessened their expressions. Conclusion: Losartan treatment can delay the progression of advanced IgA nephropathy with impaired renal function.

Influence of Aflatoxin B1 on Oxidoreductive Balance in Renal Tissue of Rats

Abstract The toxic effect of various doses of aflatoxin B1 on renal function was studied. Experiments were conducted on randomly chosen Wistar rats. The animals were divided into four groups. Group I received 8% alcohol intragastrically. The other groups received aflatoxin B1 in various doses. The effect of the aflatoxin on renal cells was analysed by means of determination of oxidoreductive balance and development of free radicals. The activity of antioxidative enzymes in renal tissue has decreased with an increase in the dose of aflatoxin B1. Disturbance of oxidation balance in the kidneys confirm a toxic effect of aflatoxin B1 on these organs

The Effects of Melatonin and Endothelin-A Receptor Antagonist BQ-123 in Exposed to Smoke in Rat Renal Tissue

The Effects of Melatonin and Endothelin-A Receptor Antagonist BQ-123 in Exposed to Smoke in Rat Renal Tissue

Association between interleukin-17 expression in renal tissue of rats with diabetic nephropathy and renal tubulointerstitial lesions

Objective To investigate the association between the expression of interleukin-17 （ IL-17 ） in renal tissues of rats with diabetic nephropathy and tubulointerstitial lesions. Methods Ninety rats were randomly divided into three groups： control group, type 1 diabetic mellitus （T1DM） group, and type 2 diabetic mellitus （T2DM） group. The rats in T1DM group were given 50 mg/kg streptozotiein （STZ） , and those in T2DM group fed on a diet enriched with sucrose, triglyceride and cholesterol, and in- jected with 25 mg/kg STZ. At 2nd and 4th week, serum and urine IL-17 levels were determined by using enzyme-linked immunosorbent assay （ELISA）. The expression of IL-17 and transforming growth factor-131 （ TGF-B1 ） in the kidney was detected by using immunohistoehemisty staining and Western blotting. Results Serum and urine IL-17 levels were obviously increase in diabetic rats. The expression of IL-17 was detectable in renal tubulointerstitium of diabetic rats. The expression of IL-17 in T2DM and T1DM groups was 0. 1809 ±0. 0090 and O. 2357 ±0. 0410 respectively （P 〈0. 05）. The expression of TGF-B1 in T2DM and T1DM groups was 0. 2809 ± 0. 0420 and 0. 1412 ± 0. 0610 respectively （ P 〈 0.05 ）. The expression of IL-17 in renal tubulointerstitium of diabetic rats was positively correlated with urine IL-17 （r = 0. 715 ,P 〈 0. 05 ）. Conclusion The increase of IL-17 expression may play an important role in diabetes mellitus-in- dueed tubulointerstitial lesions. IL-17 and TGF-[31 might exert different effects on pathogenesis and pro- gression of diabetic nephropathy from T1DM and T2DM. Key words: Interleukin-17; Diabetes mellitus; Transforming growth faetor-B1

Therapeutic potential of EGCG on acute renal damage in a rat model of obstructive nephropathy

As a major active component in green tea, (-)-epigallocatechin 3-O-gallate (EGCG) has many anti-oxidative activities. This study investigated whether intraperitoneal administration of EGCG was capable of suppressing oxidative stress in rats with unilateral ureteral obstruction (UUO) and probed the potential mechanisms involved. In total, 150 adult male rats were randomly divided into 5 groups (n=30 each): the control group (group N); the unilateral ureteral obstruction (UUO) group (group C), where the unilateral ureter was ligated resulting in an obstructive nephropathy model; and the EGCG group (group T), following unilateral ureteral ligation, rats were intraperitoneally injected with EGCG at a dosage of 2.5 (T1), 5 (T2) and 10 mg/kg/day (T3). Each group of rats was sacrificed 72 h after surgery. We evaluated the effects of EGCG on the reactive oxygen species (ROS), reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione in the renal tissue of rats. Immunohistochemistry and western blot analysis were applied to detect nuclear factor erythoid-derived 2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase (γ-GCS) protein expression. Real-time PCR was performed to detect the mRNA levels of Nrf2 and γ-GCS. Changes in renal ultrastructure were also observed using electron microscopy. There was no significant difference in GSH, and compared with group N, ROS, GSSG and total GSH levels were much higher in the T groups (p<0.01), while much lower than those of group C (p<0.01). Protein levels of Nrf2 and γ-GCS and the mRNA levels of Nrf2 and γ-GCS notably increased in EGCG-treated rats (all p<0.05). Furthermore, electron microscopy showed that renal ultrastructure was improved in the treatment groups. Our findings suggest that, resulting from suppression of oxidative stress influenced by free radicals, EGCG exerts a protective effect on rats with obstructive nephropathy, and this anti-oxidative effect may be partly induced by activating the Nrf2 signaling pathway.

Rituximab regulates the expression of the Raf kinase inhibitor protein via NF-κB in renal tissue of rats with diabetic nephropathy

This study aimed to investigate the expression levels of the Raf kinase inhibitor protein (RKIP) and NF-κB in renal tissues of diabetic nephropathy (DN) rats, and to determine the underlying molecular targets of rituximab (RTX), with the goal of developing new clinical treatment selection for DN. Sprague-Dawley rats were randomly divided into a normal group (N), a DN group (M), and an RTX treatment group (D). Blood glucose and 24-h urine protein levels of rats were determined. The expression levels of RKIP and NF-κB in glomerular tissues were determined by immunohistochemistry staining and Western blotting. Comparisons between the M and N groups revealed that the concentrations of blood glucose and 24-h urine protein were significantly increased by DN (P < 0.01), and the expression levels of RKIP and NF-κB were significantly decreased and increased (P < 0.05), respectively. In the D group, the expression levels of RKIP and NF-κB were, respectively, upregulated and downregulated by RTX, and the concentrations of 24-h urine protein were also decreased by RTX. These results suggest that expression levels of RKIP might be regulated by RTX via NF-κB. This pathway could play an important role in the development and pathogenesis of DN. Therefore, RTX could be selected for clinical treatment of DN.

Regulation of Renal Organic Anion Transporter 3 (SLC22A8) Expression and Function by the Integrity of Lipid Raft Domains and their Associated Cytoskeleton

Background/Aims: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. Methods: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. Results: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MβCD. Conclusion: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.

Observation of extracellular matrix damage in the kidney of rats with multiple organ dysfunction syndrome

Objective To determine the levels of type Ⅳ collagen and matrix metalloproteinase-9 ( MMP-9 ) in the serum and kidney of rats with the multiple organ dysfunction syndrome ( MODS ) and investigate the mechanism of extracellular matrix damage in renal failure of MODS.Methods Forty adult male Sprague- Dawley (SD) rats were randomly (ramdom number) divided into two groups,namely the normal control group ( n =8 ) and the MODS model group ( n =32 ).The rats of model group were further divided into four sub-groups as per different intervals,6 h,12 h,24 h and 48 h,after modeling (n =8 in each).The animal models of MODS were established by two hits,the left eyeball of each model rat was removed to bleed to 2 ml bloocd/100 g body weight and lipopolysaccharide ( LPS,5 mg/kg) was injected into intraperitoneal cavity of model rats four hours later. The same volume of saline instead was injected intraperitoneally into rats of control group.All rats were sacrificed at different intervals.Creatinine (Cr)and blood urea nitrogen (BUN) were determined by using a Hitachi Automatic Biochemical Analyzer.The histological changes in renal tissue were observed under light microscope and transmission electronic microscope.The levels of serum type Ⅳ collagen and MMP - 9 protein were measured by using enzyme linked immunosorbent assay (ELISA).Type Ⅳ collagen and MMP- 9 protein levels in renal tissue were detected by western blot.One - way ANOVA was used for comparison among multiple groups.Results Compared with the control group,Cr and BUN were significantly higher in MODS group ( P ＜0.05 ).There were no histopathological changes in kidney of rats in control group,and the renal injury was serious in rats with MODS.The remarkable edema of basement membrane and defect of collagen fibers in renal tissue were observed in MODS group.Compared with the control group,the levels of MMP-9 in serum increased 6-48 hours after modeling and peaking at interval of 12 hours after modeling ( P ＜ 0.05 ).The protein levels of type Ⅳ collagen increased not significantly in 6 h group and 12 h group in comparison with control group (P ＞ 0.05 ),while those in 24 h group and 48 h group significantly increased ( P ＜ 0.01 ).The levels of MMP -9 in renal tissue of rats with MODS increased in 6 h group and 12 h group (P＜0.05),peaked in 24 h group ( P ＜ 0.05 ),and decreased in 48 hours.However,the level of Ⅳ collagen in serum of rats with MODS decreased significantly 6-48 hours after modeling (P ＜ 0.05 ) Conclusions The injury of extracellular matrix is an important factor to the kidney damage in MODS and it may he used for early diagnose and as a treatment target for kidney injury in MODS. Key words: Multiple organ dysfunction syndrome; Matrixmetalloproteinase-9; Endothelial cell; Extracellular matrix; Collagen Ⅳ; Lipopolysaccharide; Systemic inflammatory response syndrome; Two hits; Rat

Reno-protective effect of NECA in diabetic nephropathy: implication of IL-18 and ICAM-1

Diabetic nephropathy (DN) remains the most common cause of end-stage renal disease. Although, adenosine acts as a local modulator with a cytoprotective function, extracellular adenosine usually disappears quickly due to a rapid uptake into adjacent cells. Therefore, we investigated the effect of 5'-(N-ethylcarboxamido)-adenosine (NECA), a stable, nonselective adenosine receptor agonist, on diabetes-induced increases in inflammatory cytokines and adhesion molecules. The enhancement of adenosine receptor action by NECA was examined in the renal tissues of rats with streptozotocin-induced diabetes. Daily i.p. injections of NECA at 0.3 mg/kg/day were given to rats, over a two-week period, six weeks after the induction of diabetes. Morphological changes were assessed in kidney sections. Oxidative stress was examined by measuring tissue malondialdehyde. Gene expression of interleukin (IL)-18, tumor necrosis factor (TNF)-α and intercellular adhesion molecule (ICAM)-1 was measured by real-time PCR. Activation of cellular, proapoptotic pathways was demonstrated by measuring the activation of c-Jun NH(2)-terminal kinases (JNK)-mitogen-activated-protein kinase (MAPK). We found that diabetes-induced malondialdehyde formation activated the production of IL-18, TNF-α and ICAM-1, which, in turn, activated pro-apoptotic pathways in diabetic rats. Treatment with NECA protected diabetic rats by exerting hypoglycemic and antioxidant effects as well as reducing gene expression of proinflammatory cytokines. These effects were associated with deactivation of JNK-MAPK. In addition, diabetic rats treated with NECA showed mild glomerular effects and vacuolation of tubular epithelium. We can conclude that activation of adenosine receptors is a potential therapeutic target in DN. NECA acts via multiple mechanisms including: reducing diabetes-induced oxidative stress, inhibiting gene expression of IL-18, TNF-α and ICAM-1, and blocking activation of the JNK-MAPK pathway.

The effects of PDE5 inhibitory drugs on renal ischemia/reperfusion injury in rats

The aim of the present study was to evaluate the effects of phosphodiesterase type 5 (PDE5) inhibitory drugs, Tadalafil and Sildenafil, on inducible NOS (iNOS), endothelial NOS (eNOS) and p53 genes expressions and apoptosis in ischemia/reperfusion (I/R) induced oxidative injury in rat renal tissue. Eighty Sprague-Dawley rats (300-350 g) were divided into four groups. In ischemia/reperfusion group, rats were subjected to renal ischemia by clamping the left pedicle for 60 min, and then reperfused for 90 min. On the other hand, in other two groups the rats were individually pretreated with Tadalafil and Sildenafil 1 h before the induction of ischemia. Malondialdehyde (MDA) is determined in renal tissue homogenates by high-performance liquid chromatography, the number of apoptotic cell were calculated by TUNEL method and p53 and eNOS expression were detected with immunohistochemistry. On the other hand, myeloperoxidase (MPO) levels were measured by spectrophotometric method and the mRNA level of iNOS in renal tissue was determined by Real-time PCR (RT-PCR). Our results indicate that MDA and MPO levels were increased in the I/R group than those in the control group. Both Tadalafil and Sildenafil treatment decreased the MDA levels in ischemia/reperfusion group, whereas this effect was more potent with Sildenafil. RT-PCR results showed that, iNOS gen expression increased in the I/R group, but decreased in the PDE5 inhibitory drugs treated group. Apoptotic cells, eNOS levels and p53 positive cells were also decreased in PDE5 inhibitory drugs treated group. We suggest that Tadalafil and Sildenafil have beneficial effects against I/R related renal tissue injury and this protective effect is clearer for Sildenafil than Tadalafil.

Effect of short-term treatment with levosimendan on oxidative stress in renal tissues of rats

The aim of this study is to evaluate the influences of short-term treatment with levosimendan (chemical formula: C14H12N6O) on oxidative stress and some trace element levels in renal tissues of healthy rats. A total of 20 male Wistar-albino rats were randomly divided into two groups, each consisting of 10 rats. Animals in the first group were not treated with levosimendan and served as control. Animals in the second group were injected intraperitoneally with 12µg/kg levosimendan and served as levosimendan group. Animals in both the groups were killed 3days after the treatment, and their kidneys were harvested for the determination of tissue oxidant/antioxidant statues and trace element levels in renal tissues. The tissue malondialdehyde level was significantly (p<0.001) lower in levosimendan group than in controls. The protective enzyme activities such as superoxide dismutase, catalase, and glutathione peroxidase and antioxidant glutathione level were significantly (p<0.001) higher in levosimendan group than in controls. It was concluded that levosimendan reduced oxidative stress by avoiding lipid peroxidation and production of reactive oxygen species, and overactivating and/or increasing the protective antioxidant enzyme levels in renal tissues of rats. It is supposed that this experimental study provides beneficial data for clinicians in the management of renal tissue damage related to obstruction and/or ischemia.

Effect of Yishen Huayu Fang on kidney tissue E-cadherin expression in unilateral ureter ligation in rats

ObjectiveTo observe E-calcium sticky protein (E-cadherin) expression in kidney tissues in a rat model of unilateral ureter ligation and the effect of Yishen Huayu Fang (formula of tonifying the kidney and dissolving accumulated blood stasis) on the expression. MethodsA total of 150 clean grade male rats were randomly divided into a control group, model group, low-dose Yishen Huayu Fang group (low-dose group), high-dose Yishen Huayu Fang group (high-dose group), and Lotensin group. A renal fibrosis model was established with unilateral ureteral obstruction (UUO). Pathological changes of rat renal tissue were observed with light microscopy on days 3, 7, 14, 21, and 28 after UUO. Changes in kidney tissue E-cadherin expression were observed with immunohistochemistry. ResultsThree days after modeling, kidney edema appeared followed by gradual inflammatory cell infiltration, and part of the small tubules disappeared while the renal cortex thinned. Meanwhile, the E-cadherin expression level dropped, which was negatively correlated with the obstruction time. After intervention, E-cadherin expression was increased in all treatment groups (P<0.01 or P< 0.05), while there were no significant differences between the high-dose and Lotensin groups. ConclusionYishen Huayu Fang delays the renal fibrosis process by promoting E-cadherin expression in renal tissues and reducing extracellular matrix deposition.

Sublytic C5b-9 Complexes Induce Apoptosis of Glomerular Mesangial Cells in Rats with Thy-1 Nephritis through Role of Interferon Regulatory Factor-1-dependent Caspase 8 Activation

The apoptosis of glomerular mesangial cells (GMC) in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis, is accompanied by sublytic C5b-9 deposition, but the mechanism of sublytic C5b-9-mediated GMC apoptosis has not been elucidated. In the present study, the gene expression profiles both in the GMC stimulated by sublytic C5b-9 and the rat renal tissue of Thy-1N were detected using microarrays. Among the co-up-regulated genes, the up-regulation of interferon regulatory factor-1 (IRF-1) was further confirmed. Increased caspase 8 and caspase 3 expression and caspase 8 promoter activity in the GMC were also identified. Meanwhile, overexpression or knockdown of IRF-1 not only enhanced or inhibited GMC apoptosis and caspase 8 and 3 induction but also increased or decreased caspase 8 promoter activity, respectively. The element of IRF-1 binding to the caspase 8 promoter was first revealed. Furthermore, silencing IRF-1 or repressing the activation of caspases 8 and 3 significantly reduced GMC apoptosis, including other pathologic changes of Thy-1N. These novel findings indicate that GMC apoptosis of Thy-1N is associated with the IRF-1-activated caspase 8 pathway.

Isolation and characterisation of lipid rafts containing the kidney‐specific Na(+)K(+)2Cl(−) cotransporter

Transport activity of the thick ascending limb (TAL) Na(+)K(+)2Cl(−) cotransporter (NKCC2) critically depends on its association with lipid raft membrane microdomains (LR). Aim of the present study was to characterize the protein composition of NKCC2‐containing LR. To this end we isolated detergent‐resistant LR from rat renal outer medullary tissue samples using a discontinuous sucrose gradient and ultracentrifugation (floating assay). NKCC2‐containing LR were purified from total LR by immunoprecipitation. Protein content of the precipitates was analyzed by western blot and mass spectrometry (MS). Floating assay confirmed accumulation of NKCC2 in LR‐enriched fractions. Analysis of the immunoprecipitate demonstrated the presence of NKCC2 and, among others, the lipid raft proteins flotillin‐1 and annexin A2. In conclusion, we have successfully established a protocol for the isolation of NKCC2‐containing LR from kidney samples. Further analysis of the protein content of these LR may provide insight into the regulation of NKCC2 function.Support: DFG FOR667