Ischemic Rat Kidneys Research Articles

Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

IntroductionAcute kidney injury (AKI) was a disease with a high mortality mainly caused by renal ischemia/reperfusion injury (I/R). Although the current non-targeted administration of vascular endothelial growth factor (VEGF) for AKI had been revealed to facilitate the recovery of renal I/R, how to targeted deliver VEGF and to retain it efficiently in the ischemic kidney was critical for its clinical application. MethodsIn present study, bi-functional KIT-PR1P peptides were constructed which bond VEGF through PR1P domain, and targeted ischemic kidney through KIT domain to interact with biomarker of AKI-kidney injury molecule-1 (Kim-1). Then the targeted and therapeutic effects of KIT-PR1P/VEGF in AKI was explored in vitro and in vivo. ResultsThe results showed KIT-PR1P exhibited better angiogenic capacity and targeting ability to hypoxia HK-2 cells with up-regulated Kim-1 in vitro. When KIT-PR1P/VEGF was used for the treatment of renal I/R through intravenous administration in vivo, KIT-PR1P could guide VEGF and retain its effective concentration in ischemic kidney. In addition, KIT-PR1P/VEGF promoted angiogenesis, alleviated renal tubular injury and fibrosis, and finally promoted functional recovery of renal I/R. ConclusionThese results indicated that the bi-functional KIT-PR1P peptides combined with VEGF would be a promising strategy for the treatment of AKI by targeting to Kim-1.

Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

Activin A, a member of the TGF-beta superfamily, is a negative regulator of tubular regeneration after renal ischemia. Activin action is controlled by an endogenous antagonist, follistatin. However, the role of follistatin in the kidney is not fully understood. In the present study, we examined the expression and localization of follistatin in normal and ischemic rat kidneys and measured urinary follistatin in rats with renal ischemia to assess whether urinary follistatin could serve as a biomarker for acute kidney injury. Using vascular clamps, renal ischemia was induced for 45 min in 8-week-old male Wistar rats. In normal kidneys, follistatin was localized in distal tubules of the cortex. In contrast, in ischemic kidneys, follistatin was localized in distal tubules of both the cortex and outer medulla. Follistatin mRNA was mainly present in the descending limb of Henle of the outer medulla in normal kidneys but was upregulated in the descending limb of Henle of both the outer and inner medulla after renal ischemia. Urinary follistatin, which was undetectable in normal rats, was significantly increased in ischemic rats and peaked 24 h after reperfusion. There was no correlation between urinary follistatin and serum follistatin. Urinary follistatin levels were increased according to ischemic duration and were significantly correlated with the follistatin-positive area as well as the acute tubular damage area. These results suggest that follistatin normally produced by renal tubules increases and becomes detectable in urine after renal ischemia. Urinary follistatin might be useful to assess the severity of acute tubular damage.

Melatonin attenuates renal sympathetic overactivity and reactive oxygen species in the brain in neurogenic hypertension.

Sympathetic overactivation contributes to the pathogenesis of both experimental and human hypertension. We have previously reported that oxidative stress in sympathetic premotor neurons leads to arterial baroreflex dysfunction and increased sympathetic drive to the kidneys in an experimental model of neurogenic hypertension. In this study, we hypothesized that melatonin, a potent antioxidant, may be protective in the brainstem regions involved in the tonic and reflex control of blood pressure (BP) in renovascular hypertensive rats. Neurogenic hypertension was induced by placing a silver clip (gap of 0.2 mm) around the left renal artery, and after 5 weeks of renal clip placement, the rats were treated orally with melatonin (30 mg/kg/day) by gavage for 15 days. At the end of melatonin treatment, we evaluated baseline mean arterial pressure (MAP), renal sympathetic nerve activity (rSNA), and the baroreflex control of heart rate (HR) and rSNA. Reactive oxygen species (ROS) were detected within the brainstem regions by dihydroethidium staining. Melatonin treatment effectively reduced baseline MAP and sympathoexcitation to the ischemic kidney in renovascular hypertensive rats. The baroreflex control of HR and rSNA were improved after melatonin treatment in the hypertensive group. Moreover, there was a preferential decrease in ROS within the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS). Therefore, our study indicates that melatonin is effective in reducing renal sympathetic overactivity associated with decreased ROS in brainstem regions that regulate BP in an experimental model of neurogenic hypertension.

Rat mRNA expression profiles associated with inhibition of ischemic acute kidney injury by losartan.

Objective: Losartan was reported to inhibit the progression of acute kidney injury (AKI), but little is known about the underlying pharmacological mechanisms. In the present study, the mRNA expression profiles in ischemic AKI rat kidney altered by losartan treatment were analyzed by next-generation deep sequencing technology.Methods: Ischemia and reperfusion treatment was applied to induce AKI in Sprague–Dawley (SD) rats. The urea and creatinine contents in rat blood were measured. H&E staining was performed to evaluate the histological alteration of rat kidney tissues under a microscope. The TUNEL method was applied to analyze apoptosis in rat kidney tissues. The mRNA profiles in rat kidney were analyzed using next-generation deep sequencing. Differential gene expression was confirmed by quantitative qRT-PCR.Results: The rat model of AKI induced by ischemia and reperfusion showed significant increases in urea and creatinine levels, accompanied by a disrupted kidney tubular structure and renal cell apoptosis. Losartan treatment effectively inhibited the changes in urea and creatinine, tubular structure, and apoptosis in AKI rat kidney. A large number of mRNAs were found to be differentially expressed in the kidneys of AKI rats treated with losartan, which are involved in multiple processes and signaling pathways. The expression of nine differentially expressed genes such as monocyte chemoattractant protein-1 (CCL2) and suppressor of cytokine signaling 3 (SOCS3) was confirmed by qRT-PCR and Western blot.Conclusion: Losartan caused significant alterations in the gene expression profile in AKI rat kidney, which mediated its anti-AKI effects.

TNF-\u03b1-induced Inflammation Stimulates Apolipoprotein-A4 via Activation of TNFR2 and NF-\u03baB Signaling in Kidney Tubular Cells

Apo-A4 expression was increased in tissues from chronic kidney disease (CKD) patients compared to that in normal kidney tissue. We determined the association of apo-A4 and its regulatory signals following acute kidney injury and elucidated the effects of apo-A4 on cell signaling pathways related to kidney injury in vitro and in vivo. Tumor necrosis factor (TNF)-α, which causes inflammatory cell injury, induced significantly increased expression of apo-A4 protein levels, and these levels were related to pro-inflammatory acute kidney injury in human kidney cells. Apo-A4 expression was also increased in experimented rat kidney tissues after ischemic reperfusion injury. The expression of tumor necrosis factor receptor (TNFR) 2 was increased in both kidney cell lines and experimented rat kidney tissues following acute kidney injury. The expression of apo-A4 and TNFR2 was increased upon treatment with TNF-α. Immunohistochemistry revealed positive apo-A4 and TNFR2 staining in ischemic reperfusion injury rat kidneys compared with levels in the sham operation kidneys. After neutralization of TNF-α, NF-κB expression was only observed in the cytoplasm by immunofluorescence. Therefore, the apo-A4 expression is increased by stimulation of injured kidney cells with TNF-α and that these effects occur via a TNFR2-NFκB complex.

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia

BackgroundIt was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met80 to Met80 sulfoxide (Met-O) in isolated hearts after ischemia–reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met80-Fe ligation of cyt c, forming pentacoordinated heme Fe2+, which inhibits electron transport (ET) and promotes oxygenase activity. MethodsSS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met80-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. ResultsBiochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met80-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. ConclusionOur results show that ischemia disrupts the Met80-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe2+ that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. General significanceCyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met80-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

Effect of Remote Ischemic Perconditioning on Renal Water and Salt Handling in Rats Subjected to Renal Ischemia/Reperfusion

Renal ischemia/reperfusion (I/R) can lead to acute kidney injury, characterized by increased urine output and fractional excretion of sodium (FE(Na)). Previous studies have shown an improvement of these parameters when applying local ischemic preconditioning (IPC) to ischemic kidneys. Here we investigate the effect of remote ischemic perconditioning (rIPeC) on the renal water and salt handling in ischemic rat kidneys.Male Wistar rats were subjected to 37 min of unilateral renal ischemia and three days reperfusion. rIPeC was applied to the abdominal aorta. Blood and urine was collected on day 2 and 3 post‐operatively for clearance studies. The expression and localization of renal aquaporins (AQP1, AQP2, phosphorylated S256‐AQP2 and AQP3) and the sodium transports Na‐K‐ATPase and NKCC2 was analyzed using immunoblotting and immunohistochemistry (IHC). Finally, the renal injury was evaluated by morphological examination and detection of renal injury markers.As previously shown, renal I/R resulted in an increase in urine output and FE(Na) and a decrease in urine osmolality compared to Sham group. rIPeC attenuated all these parameters. The protein level of AQPs, Na‐K‐ATPase and NKCC2 was down regulated in the I/R group compared to Sham, and this reduction was attenuated in the I/R+rIPeC group. IHC showed weak labeling of AQP2 and pAQP2 in collecting ducts in I/R kidneys compared to sham kidneys. rIPeC increased the labeling and showed more apical localization of AQP2 and pAQP2.In conclusion, data suggests that rIPeC prevents dysregulation of renal water and salt handling by attenuating down regulation of aquaporins and sodium transporters in ischemic rat kidneys.

Krüppel -Like Factor 8 is a Stress-Responsive Transcription Factor that Regulates Expression of HuR

Background/Aims: HuR is an RNA-binding protein that regulates the post-transcriptional life of thousands of cellular mRNAs and promotes cell survival. HuR is expressed as two mRNA transcripts that are differentially regulated by cell stress. The goal of this study is to define factors that promote transcription of the longer alternate form. Methods: Effects of transcription factors on HuR expression were determined by inhibition or overexpression of these factors followed by competitive RT-PCR, gel mobility shift, and chromatin immunoprecipitation. Transcription factor expression patterns were identified through competitive RT-PCR and Western analysis. Stress responses were assayed in thapsigargin-treated proximal tubule cells and in ischemic rat kidney. Results: A previously described NF-κB site and a newly identified Sp/KLF factor binding site were shown to be important for transcription of the long HuR mRNA. KLF8, but not Sp1, was shown to bind this site and increase HuR mRNA levels. Cellular stress in cultured or native proximal tubule cells resulted in a rapid decrease of KLF8 levels that paralleled those of the long HuR mRNA variant. Conclusions: These results demonstrate that KLF8 can participate in regulating expression of alternate forms of HuR mRNA along with NF-κB and other factors, depending on cellular contexts.

N-cadherin is depleted from proximal tubules in experimental and human acute kidney injury

Ischemia remains the most common cause of acute kidney injury (AKI). Decreased intercellular adhesion and alterations in adhesion molecules may contribute to the loss of renal function observed in AKI. In the present study, we evaluated the distribution of adhesion molecules in the human kidney and analyzed their expression in human and experimental AKI. Specimens of human kidneys obtained from patients with and without AKI were stained for the cell adhesion molecules E-cadherin, N-cadherin and beta-catenin. Experimental AKI in rats was induced by renal artery clamping. Immunostaining and immunoblotting were carried out for E-cadherin, N-cadherin and beta-catenin. Proximal tubule cells from opossum kidneys (OKs) were used to analyze the effect of chemical hypoxia (ATP depletion) in vitro. In the adult human kidney, N-cadherin was expressed in proximal tubules, while E-cadherin was expressed in other nephron segments. beta-Catenin was expressed in both proximal and distal tubules. In human AKI and in ischemic rat kidneys, N-cadherin immunostaining was depleted from proximal tubules. There was no change in E-cadherin or beta-catenin. In vitro, OK cells expressed N-cadherin only in the presence of collagen, and ATP depletion led to a depletion of N-cadherin. Collagen IV staining was reduced in ischemic rat kidneys compared to controls. The results of the study suggest that N-cadherin may play a significant role in human and experimental AKI.

The carboxyproxyl-derived spin trap (CP-H) is an appropriate detector-compound for oxidative stress

Reperfusion of ischemic tissue disturbs the balance between reactive oxygen species (ROS) and the cellular antioxidative defense. This imbalance is known as oxidative stress. In this study the spin trap 3-carboxy-2,2,5,5-tetramethylpyrrolin-1-hydroxide (CP-H) with its ESR-detectable paramagnetic analogue 3-carboxy-2,2,5,5-tetramethylpyrrolin-1-oxyl (*CP) was analyzed in vitro and in vivo. In preliminary in vitro experiments we studied the interaction of CP-H with reactive compounds like hydroxyl radicals (*OH) and alkylperoxyl radicals (ROO*) which are formed during organ reperfusion or tissue reoxygenation. The increase in the peak intensity of the ESR signal of the *CP-radical was used as a measure for CP-H oxidation by the above-mentioned oxidizing radicals. It could be clearly shown that *OH as well as ROO* induce CP-H oxidation. The intensity of the ESR signal (*CP) depends on the concentration of the applied oxidant. In a further set of in vitro experiments we analyzed some factors influencing the stability of the generated *CP. Cellular reductants are able to interact with many radicals whereby their paramagnetic signal intensity decreases. We could show that glutathione (GSH) up to 5 mM does not influence *CP concentration. On the other hand, ascorbate at a concentration of 0.6 mM significantly reduces 55% of *CP within 60 min to the ESR-silent CP-H. At 1 mM ascorbate the *CP derived ESR signal is reduced within 60 min by 90%. Lower concentrations of ascorbate (0.1-0.3 mM) do not significantly decrease signal intensity within 1 h. Homogenization of ischemic rat kidney in the presence of an air-equilibrated buffer obviously induces the formation of oxidizing radicals which in turn are able to convert diamagnetic CP-H into paramagnetic *CP. The intensity of the formed *CP was analyzed in a 600 g supernatant with ESR spectroscopy at 25 degrees C. It could be demonstrated that at least 3.0 +/- 0.5 microM *CP is formed 15 min after starting tissue homogenization and reoxygenation. Subsequent measurements of the *CP concentration indicated that its signal intensity continuously decreases. After 75 min a residual *CP concentration of 0.7 +/- 0.3 microM was monitored. Removal of mitochondria from the homogenate by centrifugation at 6,000g decelerates the disappearance of *CP but does not block it completely. In summary it could be shown that the marker (CP-H) is able to indicate the formation of oxidizing radicals during reoxygenation of ischemic tissue. This method underestimates the amount of produced oxidizing radicals. One reason for this is the reduction of *CP by some cellular reductants. Other reasons will be discussed. We assume that the used method allows a nearly real-time determination of radical production during organ reoxygenation.

Attenuation of Folic Acid-Induced Renal Inflammatory Injury in Platelet-Activating Factor Receptor-Deficient Mice

Platelet-activating factor (PAF), a potent lipid mediator with various biological activities, plays an important role in inflammation by recruiting leukocytes. In this study we used platelet-activating factor receptor (PAFR)-deficient mice to elucidate the role of PAF in inflammatory renal injury induced by folic acid administration. PAFR-deficient mice showed significant amelioration of renal dysfunction and pathological findings such as acute tubular damage with neutrophil infiltration, lipid peroxidation observed with antibody to 4-hydroxy-2-hexenal (day 2), and interstitial fibrosis with macrophage infiltration associated with expression of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in the kidney (day 14). Acute tubular damage was attenuated by neutrophil depletion using a monoclonal antibody (RB6-8C5), demonstrating the contribution of neutrophils to acute phase injury. Macrophage infiltration was also decreased when treatment with a PAF antagonist (WEB2086) was started after acute phase. In vitro chemotaxis assay using a Boyden chamber demonstrated that PAF exhibits a strong chemotactic activity for macrophages. These results indicate that PAF is involved in pathogenesis of folic acid-induced renal injury by activating neutrophils in acute phase and macrophages in chronic interstitial fibrosis. Inhibiting the PAF pathway might be therapeutic to kidney injury from inflammatory cells.

The effect of gradually increased blood flow on ischemia-reperfusion injury in rat kidney

Background Gradually increased blood flow to the ischemic rat kidney was studied to assess the ability to diminish ischemia-reperfusion injury. Methods The left renal artery and vein were isolated in 25 rats. Microclamps were applied for 45 minutes and were released at once (group II) or gradually (group III). Renal arterial blood flow and K+ activity were measured. Bilateral kidneys were harvested for histopathology and for malonyldealdehyde and myeloperoxidase levels. Results Increased K+ activity returned to preischemic values faster in group III than in group II. No statistically significant difference existed in malonyldealdehyde and myeloperoxidase levels; histopathologic scoring showed less tissue damage in group III ( P < .05). Contralateral kidney samples showed signs of ischemia in group II. Conclusions Gradually increased blood flow to the ischemic kidney decreases ischemic changes. Ischemic insult to 1 kidney causes histopathologically detectable changes to the contralateral kidney, which can be diminished by gradual reperfusion.

STAT3 is a potential modulator of HIF‐1‐mediated VEGF expression in human renal carcinoma cells

Aberrantly enhanced vascular endothelial growth factor (VEGF) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies, including human renal carcinomas. Persistent activation of STAT3 is linked to tumor-associated angiogenesis, but underlying mechanisms remain unclear. Therefore, we examined whether STAT3 modulates the stability and activity of hypoxia-inducible factor-1alpha (HIF-1alpha), and in turn enhances VEGF expression. We found that STAT3 was activated in ischemic rat kidneys and hypoxic human renal carcinoma cells. We also found that hypoxia-induced activation of STAT3 transactivated the VEGF promoter and increased the expression of VEGF transcripts. Consistent with these findings, STAT3 inhibition attenuated the hypoxic induction of VEGF. Interestingly, activated STAT3 increased HIF-1alpha protein levels due to the HIF-1alpha stability by blocking HIF-1alpha degradation and accelerated its de novo synthesis. The novel interaction of STAT3 with HIF-1alpha was identified in hypoxic renal carcinoma cells. Furthermore, hypoxia recruited STAT3, HIF-1alpha, and p300 to the VEGF promoter and induced histone H3 acetylation. Therefore, these findings provide compelling evidence that a causal relationship exists between STAT3 activation and HIF-1-dependent angiogenesis and suggest that therapeutic modalities designed to disrupt STAT3 signaling hold considerable promise for the blocking tumor growth and enhancing apoptosis of cancer cells and tissues.

Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis.

To investigate regional aspects of hypoxic regulation of adrenomedullin (AM) in kidneys, we mapped the distribution of AM in the rat kidney after hypoxia (normobaric hypoxic hypoxia, carbon monoxide, and CoCl(2) for 6 h), anemia (hematocrit lowered by bleeding) and after global transient ischemia for 1 h (unilateral renal artery occlusion and reperfusion for 6 and 24 h) and segmental infarct (6 and 24 h). AM expression and localization was determined in normal human kidneys and in kidneys with arterial stenosis. Hypoxia stimulated AM mRNA expression significantly in rat inner medulla (CO 13 times, 8% O(2) 6 times, and CoCl(2) 8 times), followed by the outer medulla and cortex. AM mRNA level was significantly elevated in response to anemia and occlusion-reperfusion. Immunoreactive AM was associated with the thin limbs of Henle's loop, distal convoluted tubule, collecting ducts, papilla surface epithelium, and urothelium. AM labeling was prominent in the inner medulla after CO and in the outer medulla after occlusion-reperfusion. The infarct border zone was strongly labeled for AM. In cultured inner medullary collecting duct cells, AM mRNA was significantly increased by hypoxia. AM mRNA was equally distributed in human kidney and AM was localized as in the rat kidney. In human kidneys with artery stenosis, AM mRNA was not significantly enhanced compared with controls, but AM immunoreactivity was observed in tubules, vessels, and glomerular cells. In summary, AM expression was increased in the rat kidney in response to hypoxic and ischemic hypoxia in keeping with oxygen gradients. AM was widely distributed in the human kidney with arterial stenosis. AM may play a significant role to counteract hypoxia in the kidney.

Ischemia Induces Early Expression of a New Transcription Factor (6A3-5) in Kidney Vascular Smooth Muscle Cells: Studies in Rat and Human Renal Pathology

Acute renal failure, characterized by rapid decline in glomerular filtration rate, is a major cause of morbidity and mortality. During the evolution of renal diseases chronic ischemia develops. Indeed, acute or chronic renal failure may occur as a result of renal ischemia, which induces cells to dedifferentiate, proliferate, or become apoptotic. In this study, we have investigated the expression of a newly identified transcription factor, 6A3-5, under in vitro and in vivo conditions. Proliferating vascular smooth muscle were investigated in response to different mitogenic agents. The 6A3-5 expression was then studied in ischemic rat kidney, induced by renal pedicle clamping, followed, or not, by reperfusion. Subsequently human renal biopsies from early kidney grafts and chronic renal diseases were also investigated for 6A3-5 protein expression by immunohistochemistry. In vitro study shows an over-expression of 6A3-5 following 2 to 4 hours stimulation by serum or Angiotensin II, of rat proliferating aortic smooth muscle cell. Moreover, in vivo study shows that this new protein is over expressed in rat kidney submitted to 45 minutes ischemia. An anti-6A3-5 antibody shows the protein to be expressed in smooth muscle cells of the arterioles and intermediate size arteries, in mesangial cells and interstitial myofibroblasts. In human biopsies of early kidney grafts and renal disease, the same up-regulation of 6A3-5, as in acute ischemic situation, is observed. This 6A3-5 expression is intimately associated with alpha-smooth muscle cell actin expression in mesangial cells, arteriolar smooth muscle cells as well as interstitial myofibroblasts. Transcription factor 6A3-5 could potentially be a novel early vascular marker of acute and chronic renal ischemic stress implicated in tissue remodeling.

Preconditioning with cyclosporine A or FK506 differentially regulates mitogen-activated protein kinase expression in rat kidneys with ischemia/reperfusion injury.

The signaling pathways of mitogen-activated protein kinases (MAPKs) are important molecular components responsible for ischemia/reperfusion (I/R) injury in the kidneys. Preconditioning with cyclosporine A (CsA) or FK506 reduces subsequent I/R injury. We studied the effect of preconditioning with CsA or FK506 on MAPK expression in ischemic rat kidneys. Two separate studies were performed using Sprague-Dawley rats. First, MAPK (extracellular signal-regulated kinase [ERK], jun N-terminal kinase [JNK], p38) expressions were observed at 0, 10, 20, 30, 60, 120, and 1,440 min after I/R injury. Second, the effects of preconditioning with CsA or FK506 on MAPK expressions were observed in rat kidneys with I/R injury. I/R injury was induced by clamping both renal arteries for 45 min. Rats were pretreated with intravenous (IV) CsA (3 mg/kg) or IV FK506 (0.3 mg/kg) 6 hr before I/R injury and killed 30 min later. Expression of MAPK was measured using immunoblot and immunohistochemistry. MAPK (ERK, JNK, p38) expressions were significantly increased in kidneys with I/R injury compared with sham-operated controls, and immunohistochemistry revealed increased MAPK immunoreactivity in renal tubules of the outer medulla. Kidneys preconditioned with low-dose CsA or FK506 showed significantly increased ERK expression compared with kidneys with I/R injury alone (CsA, 9.5- vs. 4.5-fold; FK506 10.4- vs. 4.5-fold: P<0.05) but showed decreased JNK (CsA, 3.8- vs. 5.3-fold; FK506, 3.4- vs. 5.3-fold: P<0.05) and p38 expression (CsA, 2.5- vs. 3.7-fold; FK506, 2.1- vs. 3.7-fold: P<0.05). Preconditioning with CsA or FK506 differentially regulates the expression of MAPK in rat kidneys with I/R injury, and this may explain the remarkable protective effects of these agents.

Requirement of Mitogen-activated Protein Kinase Kinase 3 (MKK3) for Activation of p38α and p38δ MAPK Isoforms by TGF-β1 in Murine Mesangial Cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.

Involvement of Pax-2 in the action of activin A on tubular cell regeneration.

It has been recently shown that in ischemic rat kidneys activin A is induced in tubular cells and inhibits their regeneration. The present study was conducted to further investigate the action of activin A in tubular cells during regeneration. Among genes thought to be critical for kidney development, Pax-2 was upregulated in tubular cells during regeneration after renal ischemia. Pax-2 protein was localized in nuclei of tubular and interstitial cells, some of which co-expressed a mesenchymal cell marker, vimentin, suggesting that a population of Pax-2-positive cells have properties of immature progenitor-like tubular cells. The Pax-2-expressing cells co-expressed a cell proliferation marker, BrdU, activin A, and the type II activin receptor. Activin A modulated growth of BrdU/Pax-2 double-positive cells since an administration of follistatin increased; conversely, exogenous activin A decreased the number of BrdU/Pax-2 double-positive cells after renal ischemia. Activin A also reduced the expression of Pax-2 in cultured metanephroi. A proximal tubular cell line, LLC-PK(1) cells, was used to further study the mode of action of activin A. The expression of Pax-2 was not detected in quiescent LLC-PK(1) cells, but it was markedly increased when growth was stimulated. Under this condition, activin A significantly inhibited DNA synthesis and reduced the expression of Pax-2 in LLC-PK(1) cells. In contrast, blockade of the activin signaling by overexpressing dominantly negative mutant receptor enhanced the expression level of Pax-2 in LLC-PK(1) cells and induced an immature phenotype. These results suggest that activin A regulates tubular cell growth and differentiation by modulating the expression of Pax-2 during regeneration.

Cyclosporine or FK506 Decrease Mature Epidermal Growth Factor Protein Expression and Renal Tubular Regeneration in Rat Kidneys with Ischemia/Reperfusion Injury

Background: Epidermal growth factor (EGF) plays an important role in tubular regeneration in kidneys with ischemia/reperfusion (I/R) injury. This study was undertaken to evaluate the influence of cyclosporine A (CsA) or FK506 on mature EGF expression and tubular regeneration in rat kidneys with I/R injury. Methods: Two separate studies were performed. First, the expression of EGF and tubular regeneration was observed in rat kidneys with I/R injury on days 1, 2, 3, 5, and 7. Second, the dose-dependent response of EGF expression and tubular regeneration to CsA (5, 10, and 20 mg/kg) or FK506 (0.25, 0.5, and 1.0 mg/kg) was observed in rat kidneys with I/R injury. I/R injury was induced by clamping both renal arteries for 45 min, and CsA or FK506 was injected just after release of vascular clamps. Rats were sacrificed on day 1 for evaluation of EGF expression, and on day 2 for evaluation of BudU-positive cells. Renal function, tubular injury score, EGF expression assessed by immunoblotting, levels of CsA and FK506 in whole blood, and immunostaining for BrdU was studied. Results: EGF expression was maximal on day 1 (cortex, 29-fold; medulla, 31-fold compared with sham-operated controls), and renal tubular regeneration measured with the number of BrdU-positive cells was maximal on days 2 and 3 in kidney with I/R injury, and thereafter the level of EGF and the number of BrdU-positive cells decreased progressively. CsA or FK506 treatment to ischemic rat kidneys reduced the expression of EGF and the number of BrdU-positive cells in a dose-dependent manner. Conclusions: CsA or FK506 treatment delays recovery from acute tubular necrosis, and this may be associated with decreased EGF expression by CsA or FK506.

Intravital videomicroscopy of peritubular capillaries in renal ischemia.

The recent refinement and computerization of intravital microscopy have permitted us to monitor microcirculation in vivo with minimal invasion. Here, we report on the first findings made with the use of a pencil-lens intravital microscope as applied to the ischemic rat kidney. Peritubular capillary and glomerular blood flow were monitored under basal conditions, during renal artery occlusion, and immediately after release of the clamp. Erythrocyte velocity was calculated as an angle in consecutive spatiotemporal images. Intravital videomicroscopy during the reperfusion period showed intermittent cessation and partial recovery of blood flow in both peritubular and glomerular capillaries. Blood flow was uniformly orthograde under control conditions; however, the retrograde flow occurred on reperfusion. The patency of peritubular capillaries was partially compromised during the early reperfusion period but rapidly recovered. The recovery of glomerular microcirculation occurred faster than that of peritubular capillaries. We suggest that a functional vasculopathy develops very early in the course of ischemia-reperfusion in superficial cortical microvasculature and is more pronounced in peritubular capillaries, thus accounting for the development of patchy injury of tubular epithelia.