Days Of Unilateral Ureteral Obstruction Research Articles

Abstract P376: Collecting Duct Renin And Its Receptor Contributes To Unilateral Ureteral Obstruction-induced Kidney Injury Via Activation Of The Intrarenal Ras

The collecting duct (CD) is well known for its role in fine-tuning urinary excretion of electrolytes and water. Emerging evidence suggests a potential role played by the CD in immunity and renal injury but the underlying mechanism largely remains elusive. Within the CD, (pro)renin receptor (PRR) via site-1 protease (S1P)-derived soluble PRR interacts with renin controls the intrarenal renin-angiotensin system (RAS) to regulate Na+ and water balance and blood pressure. The present study attempted to employ mouse models of CD-specific deletion of PRR and renin (CD PRR KO and CD renin KO, respectively) and a specific S1P inhibitor PF-429242 (PF) to explore the pathogenic role of this local system in renal injury induced by unilateral ureteral obstruction (UUO). Male 3-4-month-old CD PRR KO and CD renin KO, and their respective floxed controls were subjected to UUO surgery or sham operation for 3 days, followed by analysis of renal fibrosis, inflammation, and indices of the intrarenal RAS. Male 3-4-month-old C57BL/6 mice were subjected to the same surgical procedures and infused with vehicle or PF at 20 mg/kg/day via minipump for 3 days, followed by analysis of the same parameters. 3-day UUO in floxed mice induced renal abundance of fibronectin and α-SMA by 8.6 folds and 5.8 folds, respectively, as assessed by immunoblotting analysis. qRT-PCR detected similar magnitude of increases in mRNA expression of fibronectin, α-SMA, as well as collagen III, collagen I, and TGF-β in the obstructed kidneys. Similar results were obtained by Trichrome Masson staining. In parallel, the obstructed kidney exhibited 4-5-fold increases in renal medullary renin mRNA, renin activity, and active renin content contrasting to unaltered renin levels in the renal cortex. CD-specific deletion of PRR and renin induced comparable attenuation of various indices of renal fibrosis, inflammation, renal medullary renin levels so did PF treatment. UUO induced a 2-fold increase in plasma sPRR, which was completely blocked by PF treatment. Taken together, our results demonstrated that CD PRR/sPRR-dependent activation of renin represents a key determinant of the intrarenal RAS and thus obstruction-induced renal inflammation and fibrosis.

Endothelial Activin Receptor-Like Kinase 1 (ALK1) Regulates Myofibroblast Emergence and Peritubular Capillary Stability in the Early Stages of Kidney Fibrosis.

Renal tubulo-interstitial fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the tubular interstitium during chronic kidney disease. The main source of ECM proteins are emerging and proliferating myofibroblasts. The sources of myofibroblasts in the renal tubular interstitium have been studied during decades, in which the epithelial contribution of the myofibroblast population through the epithelial-to-mesenchymal (EMT) process was assumed to be the major mechanism. However, it is now accepted that the EMT contribution is very limited and other mechanisms such as the proliferation of local resident fibroblasts or the transdifferentiation of endothelial cells seem to be more relevant. Activin receptor-like kinase 1 (ALK1) is a type I receptor which belongs to the transforming growth factor beta (TGF-β) superfamily, with a key role in tissue fibrosis and production of ECM by myofibroblast. Predominantly expressed in endothelial cells, ALK1 also plays an important role in angiogenesis and vessel maturation, but the relation of these processes with kidney fibrosis is not fully understood. We show that after 3 days of unilateral ureteral obstruction (UUO), ALK1 heterozygous mice (Alk1 +/− ) display lower levels of kidney fibrosis associated to a lower number of myofibroblasts. Moreover, Alk1 +/− mice have a lower degree of vascular rarefaction, showing improved peritubular microvasculature after UUO. All these data suggest an important role of ALK1 in regulating vascular rarefaction and emergence of myofibroblasts.

MO434: Bemcentinib Targets Macrophage and Mesangial Cells in Renal Fibrosis

Abstract BACKGROUND AND AIMS Renal fibrosis, a progressive process of extracellular matrix accumulation leading to renal failure, lacks effective treatment. Expression of the AXL receptor tyrosine kinase has been implicated in kidney injury and mesangial proliferation [1]. Inhibition of AXL signalling with the selective AXL kinase inhibitor bemcentinib reduces fibrosis and inflammation in murine models of unilateral ureteral obstruction (UUO) and in glomerulonephritis [2, 3]. METHOD Male C57Bl/6 mice were subjected to UUO for 3 or 15 days and treated twice daily with vehicle or bemcentinib (50 mg/kg) by oral gavage. Kidneys were divided into pieces that were either dissociated into single cells for mass cytometry analysis or subjected to Sirius Red staining to evaluate collagen deposition. Samples for mass cytometry were stained with a 45-plex antibody panel, acquired on Helios, then cleaned and analysed using UMAP [4] for dimensionality reduction and PARC [5] for clustering. The effect of ligation was modelled with the least absolute shrinkage and selection operator (LASSO) using centered log-ratio transformed cluster compositions to predict the number of days of ligation. LASSO is a regression method that prevents overfitting by penalizing variables and is commonly used for variable selection. RESULTS Sirius Red staining confirmed, as previously published, reduced fibrosis development in kidneys from bemcentinib treated animals compared to the vehicle following 15 days of UUO. No significant effect of bemcentinib was observed after 3 days of obstruction. Mass cytometry analysis yielded 31 clusters representing immune, endothelial, pericyte, mesangial and epithelial cells from proximal tubules, loop of Henle, distal tubules and collecting duct. AXL was expressed by pericytes, endothelial cells, mesangial cells and macrophages. Modelling with LASSO suggested that the most important feature of ligation was the expansion of mesangial cells. Bemcentinib treatment did not result in any significant changes in non-ligated kidneys and kidneys ligated for 3 days. At 15 days, post-bemcentinib treatment, the number of mesangial cells and macrophages decreased, while the number of epithelial cells comprising the proximal tubules and loop of Henle increased. The LASSO model estimation corresponded to an apparent reduction in disease progression of 30% (corresponding to day 10 or 11 post-ligation). CONCLUSION AXL targeting macrophages and mesangial cells delays the progression of kidney fibrosis in the UUO model and represents a valid approach to treat kidney disease.

Abstract 07: Collecting Duct (Pro) Renin Receptor(PRR) Promotes Kidney Fibrosis Via Macrophage Alternative Activation Mediated By Soluble PRR (sPRR)-Dependent Activation Of Yap/Taz Axis

Renal collecting duct (CD), an important site for fine-tuning urinary Na + and water excretion, is not traditionally thought to play a role in pathogenesis of chronic kidney disease. In the present study, we examined a profibrotic role of CD PRR in a mouse model of unilateral ureteral obstruction (UUO) and further defined the underlying mechanism involving soluble PRR (sPRR)-dependent activation of alternative macrophages activation. We subjected mice with CD-specific deletion of PRR (CD PRR KO) and their floxed controls to UUO or sham surgery. After 7 days of UUO, CD PRR KO decreased the fibronectin (28.6±6.8%) and α-SMA (39.2±7.8%) protein expression in obstructed kidneys. The Masson’s trichrome staining data also showed that CD PRR KO significantly attenuated UUO-induced collagen deposition and histological damage in the kidney. In parallel, CD PRR KO reduced TGF-β1 (56.7±3.2%) and TGF-β2 (53±1.2%) mRNA expression in obstructed kidneys. Moreover, in macrophages sorted from obstructed kidneys of CD PRR KO mice showed a significantly reduced of M2 macrophage markers such as mannose receptor ( MR ) (58.7±2.1%), arginase-1 ( Arg-1 ) (50±1.5%), chitinase-like lectins ( YM-1 ) (59.3±1.9%), inflammatory zone-1 ( Fizz1 ) (39.1±4.1%) mRNA expression and Yes-associated protein ( Yap ) (77±6.8%)/ transcriptional coactivator with PDZ-binding motif ( Taz ) (54.5±1.8%) mRNA expression compared with macrophages sorted from obstructed kidneys of floxed mice. Meanwhile, plasma sPRR was elevated in floxed mice by UUO and this elevation was blunted by CD PRR KO (23.7±0.4%). Administration of site-1 protease (S1P) inhibitor PF-429242 to C57BL/6 mice with UUO almost completely recapitulated the antifibrotic action as well as the inhibitory effect on M2 activation of CD PRR KO. In bone marrow-derived macrophages, sPRR-His treatment promoted macrophage M2 polarization, fibrosis and Yap/Taz expression. Overall, these results suggest that activation of CD PRR releases sPRR that activates M2 polarization via Yap/Taz axis, leading to renal fibrosis during UUO.

Tamoxifen attenuates renal fibrosis in human kidney slices and rats subjected to unilateral ureteral obstruction

Background and PurposeRenal fibrosis plays an important role in the development and progression of chronic kidney disease (CKD). Clinical studies have shown that CKD progresses differently in males and females, which may be related to circulating levels of sex hormones. In this study, we investigated the effect of tamoxifen (TAM), a selective estrogen receptor modulator (SERM), on renal fibrosis in male and female rats subjected to unilateral ureteral obstruction (UUO) and human precision-cut kidney slices (PCKS). Experimental ApproachFemale, ovariectomized female (OVX), and male rats were subjected to 7 days of UUO and treated with TAM by oral gavage. Moreover, we studied individual responses to TAM treatment in PCKS prepared from female and male patients. In all models, the expression of fibrosis markers was examined by western blot, qPCR, and immunohistochemistry. Key ResultsTAM decreased the expression of fibronectin, α-smooth muscle actin, and collagen-1 and -3 in female, OVX, and male rats. In addition, TAM mitigated TGF‐β-induced fibrosis in human PCKS, irrespective of sex, yet interindividual differences in treatment response were observed. Conclusion and ImplicationsTAM ameliorates renal fibrosis in males and females, although we did observe sex differences in drug response. These findings warrant further research into the clinical applicability of TAM, or other SERMs, for the personalized treatment of renal disease.

Consistent alteration of chain length-specific ceramides in human and mouse fibrotic kidneys

BackgroundSeveral studies revealed alterations of single sphingolipid species, such as chain length-specific ceramides, in plasma and serum of patients with kidney diseases. Here, we investigated whether such alterations occur in kidney tissue from patients and mice suffering from renal fibrosis, the common endpoint of chronic kidney diseases. MethodsHuman fibrotic kidney samples were collected from nephrectomy specimens with hydronephrosis and/or pyelonephritis. Healthy parts from tumor nephrectomies served as nonfibrotic controls. Mouse fibrotic kidney samples were collected from male C57BL/6J mice treated with an adenine-rich diet for 14 days or were subjected to 7 days of unilateral ureteral obstruction (UUO). Kidneys of untreated mice and contralateral kidneys (UUO) served as respective controls. Sphingolipid levels were detected by LC-MS/MS. Fibrotic markers were analyzed by TaqMan® analysis and immunohistology. ResultsVery long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 were significantly downregulated in both fibrotic human kidney cortex and fibrotic murine kidney compared to respective control samples. These effects correlate with upregulation of COL1α1, COL3α1 and αSMA expression in fibrotic human kidney cortex and fibrotic mouse kidney. ConclusionWe have shown that very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 are consistently downregulated in fibrotic kidney samples from human and mouse. Our findings support the use of in vivo murine models as appropriate translational means to understand the involvement of ceramides in human kidney diseases. In addition, our study raises interesting questions about the possible manipulation of ceramide metabolism to prevent progression of fibrosis and the use of ceramides as potential biomarkers of chronic kidney disease.

P0734THE DELETION OF AKT1 PROMOTES THE RENAL FIBROSIS IN THE MURINE MODEL OF UNILATERAL URETERAL OBSTRUCTION

Abstract Background and Aims Previous studies have found the increased Akt activity in experimental renal fibrosis. We investigated the role of Akt1, one of the three Akt isoforms, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). Method In vivo, we subjected the wild type and Akt1−/− mice to UUO. In vitro, gene silencing of Akt1 was achieved using the short hairpin RNA delivered by the lentiviral vector in immortalized human proximal tubular cells (HK2 cells) and rat kidney fibroblasts (NRK-49F cells). Results In immunohistochemical stain, the expression of Akt1 was significantly higher in obstructed kidneys of wild type mice compared with control sham kidneys and increased gradually as UUO progressed. The fibronectin, type I collagen, and heat shock protein 47 (HSP47) were markedly more expressed in obstructed kidneys of Akt1−/− mice than in those of the wild type mice. Transforming growth factor β1 (TGFβ1) was highly induced within 1 day of UUO in obstructed kidneys of Akt1−/− mice and the expression of TGFβ1 was significantly higher in the Akt1−/− mice than in the wild type mice as UUO progressed. Western blot showed that silencing of Akt1 increased the expression of TGFβ1, which was enhanced by angiotensin II stimulation in HK2 cells, but not in NRK-49F cells. Immunohistochemical stain demonstrated that the expression of cleaved caspase-3 in renal tubules was significantly higher in the Akt1−/− mice than in the wild type mice. Western blot showed that silencing of Akt1 increased the expression of cleaved caspase-3 in HK2 cells, but not in NRK-49F cells. Conclusion Our findings suggest that the deletion of Akt1 might contribute to tubulointerstitial fibrosis and tubular apoptosis via TGFβ1 induction. We also showed that TGFβ1 upregulation by genetic deletion of Akt1 is associated with activation of STAT3 independently of the TGFβ1/Smad signaling pathway.

Brilliant blue G, a P2X7 receptor antagonist, attenuates early phase of renal inflammation, interstitial fibrosis and is associated with renal cell proliferation in ureteral obstruction in rats

BackgroundPrevious study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor.MethodsWe investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-β1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1β, procollagens type I, III, and IV) for mRNA quantification.ResultsThe group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- β1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1β mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1β mRNAs, as well as less immunoreactivity of HSP-47, TGF-β, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation.ConclusionBBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.

The effect of tamoxifen on gender differences in unilateral ureteral obstructed rats and human kidney slices

BACKGROUNDRenal fibrosis is regarded as the most damaging process in chronic kidney disease (CKD) and it is the most consistent predictor of irreversible loss of renal function. To date, there is no good treatment for renal fibrosis. Interestingly, females are less prone to CKD, which might be due to estrogen. We have previously shown that tamoxifen (TAM), a selective estrogen receptor modulator, plays a role for regulation of renal water and salt balance. In this study, we will investigate the effect of tamoxifen on a well‐established in vivo model using unilateral ureteral obstruction (UUO)‐induced fibrosis in female, male and ovariectomized (OVX) female rats. Furthermore, a recently develop model of human renal fibrosis was used to study multicellular pathological processes in human tissue.METHODSFibrosis was induced by 7 days of UUO. Female OVX rats had both their ovaries removed two weeks before UUO. Tamoxifen (25 mg/kg for female rats and 50 mg/kg for male rats; a higher dose of tamoxifen was lethal for female rats) was given by oral gavage starting 5 days before UUO. Tamoxifen treatment was continued for 7 days after UUO. Human precision‐cut kidney slices were exposed to TGF‐β for 48h in order to make the tissue fibrotic and afterwards treated with tamoxifen for 6h. Histological changes were examined by HE staining. Expression of α‐smooth muscle actin, fibronectin, and TGF‐β were evaluated by immunohistochemistry, quantitative PCR, and western blot analysis.RESULTSRenal fibrosis was increased after UUO in both male, female and OVX rats. Tamoxifen treatment reduced fibronectin, α‐SMA and TGF‐β gene expression in female UUO rats, but not in male and in OVX rats subjected to UUO. Yet, tamoxifen significantly reduced the protein level of fibrotic markers and the kidney weight in response to UUO in both female as well as OVX and male rats. Furthermore, our data demonstrated that sham operated female and female OVX rats had a higher plasma creatinine level compared to male rats. On the other hand, TAM treated male rats subjected to UUO had a significantly increased urea plasma level, which was not observed in female and female OVX rats. Importantly, treatment with tamoxifen has a tendency to reduce fibronectin in human kidney slices exposed to TGF‐β from women but not in men.CONCLUSIONThese findings indicate that tamoxifen reduces UUO‐induced fibrosis in both female, female OVX and male rats, but have different effects on females and males when it comes to gene levels of fibrotic markers and plasma data. Furthermore, our finding provide preclinical evidence that treatment with tamoxifen affects men and women differently and the concentration of the drug must be adapted to the gender.Support or Funding InformationThis study was kindly supported by Karen Elise Jensen Foundation, the Danish Council for Independent Research, Aarhus University Foundation and the AP Møller Foundation.

EP1 Receptor Antagonism as a Therapeutic Intervention Strategy in Renal Fibrosis

Renal fibrosis is a hallmark of Chronic Kidney Disease (CKD), which affects 10–16% of the world’s adult population. It has previously been shown that the cyclooxygenase/prostaglandin (COX/PG) system affects the progression of renal fibrosis. The downstream elements of this system, the eicosanoid prostaglandin (EP) receptors, EP1–4, have various physiological roles in health and disease. We therefore evaluated the effect of antagonizing one of these downstream receptors for prostaglandin, the EP1 receptor, on renal fibrosis.This was accomplished with a translational approach by utilizing three models of renal fibrosis; Transforming growth factor β (TGF‐β) treated Madin‐Darby Canine Kidney (MDCK) cells, mice that underwent seven days of unilateral ureteral obstruction (UUO), as well as TGF‐β treated Human Precision Cut Kidney Slices (HPCKS). We evaluated the extent of fibrosis in these models on the gene and protein level using QPCR as well as Western blotting and immunohistochemical analysis. Moreover, we determined the corresponding effects of the EP1 agonist 17‐phenyl trinor prostaglandin E2 (17‐phenyl PGE2) and the EP1 antagonist SC‐19220 on the intracellular Ca2+ concentration in MDCK cells measured by Flou‐4‐AM.We found, that as a whole, treatment with the EP1 receptor antagonist SC‐19220 successfully ameliorated renal fibrosis in all three models. Treatment with the EP1 receptor antagonist SC‐19220 (25mg/kg per day) diminished fibrosis in mice that underwent seven days of UUO. UUO‐induced protein expression of the fibrosis markers fibronectin (FN), α Smooth Muscle Actin (αSMA) and collagen measured by western blot, qPCR and immunohistochemical staining, was lowered by SC‐19220 treatment when compared to SHAM operated mice. Adding to this, treatment of HPCKS with SC‐19220 reduced TGF‐β‐mediated fibrosis as shown by decreased collagen 1A1, FN and αSMA gene expression as well as reduced staining intensity of Sirius Red and Masson’s Trichrome stains. In MDCK cells, fibrosis induced by TGF‐β was significantly reduced when treated with SC‐19220. This was evident by lower FN protein levels and possibly mediated by hampering of TGF‐β signalling. The specificity of SC‐19220 as an antagonist of the 17‐phenyl PGE2‐induced [Ca2+]i response was determined with standard concentration response experiments.Thus, this study shows that the EP1 receptor is a promising target for the treatment of renal fibrosis. However, further investigations into the mechanism underlying the anti‐fibrotic effect of EP1 receptor antagonism are necessary. It seems though, that intracellular calcium regulation is profoundly altered by the EP1 receptor and that this may alter fibrosis dynamics.Support or Funding InformationFunding for the project was provided by the Danish Research Council.

The effect of oleuropein on unilateral ureteral obstruction induced-kidney injury in rats: the role of oxidative stress, inflammation and apoptosis.

Unilateral ureteral obstruction (UUO) induces kidney injury. Oleuropein as a major compound of olive leaves modulates the inflammatory parameters and decreases oxidative stress. Accordingly, we evaluate the renoprotective effect of oleuropein against 3-day UUO rats. Forty rats were randomly divided into five groups (n = 8) including control, UUO and UUO + oleuropein groups (50, 100 and 200mg/kg). UUO model was induced by left ureter ligation and continued for 3-day. Rats were treated synchronic daily for 3-day, then mean arterial pressure (MAP), renal perfusion pressure (RPP), renal blood flow (RBF), serum creatinine level, and also superoxide dismutase (SOD), glutathione peroxidase (GPx) activity levels and malondialdehyde (MDA) concentration (in the obstructed kidney) were measured. The western blotting method was applied to evaluate the Bax, Bcl-2, cleaved caspase-3 and TNF-α proteins expression level. The hematoxylin and eosin method was applied to evaluate the kidney tissue damage score (KTDS). UUO significantly increased RVR, KTDS, and MDA, cleaved caspase-3, Bax, serum creatinine and TNF-α protein levels (P < 0.05), and also significantly decreased RBF, SOD, and GPx and Bcl-2 protein expression levels (P < 0.001) in the obstructed kidney and oleuropein (200mg/kg) significantly ameliorated the changes induced by UUO. Our findings showed that oleuropein has a renoprotective effect against 3-day UUO. The mechanisms underlying the observed effects may be related to its antioxidative stress, anti-apoptotic, and anti-inflammatory effects.

Vasopressin-Independent Regulation of Aquaporin-2 by Tamoxifen in Kidney Collecting Ducts.

Arginine vasopressin (AVP) mediates water reabsorption in the kidney collecting ducts through regulation of aquaporin-2 (AQP2). Also, estrogen has been known to regulate AQP2. Consistently, we previously demonstrated that tamoxifen (TAM), a selective estrogen receptor modulator, attenuates the downregulation of AQP2 in lithium-induced nephrogenic diabetes insipidus (NDI). In this study, we investigated the AVP-independent regulation of AQP2 by TAM and the therapeutic effect of TAM on the dysregulation of AQP2 and impaired urinary concentration in a unilateral ureteral obstruction (UUO) model. Primary cultured inner medullary collecting duct (IMCD) cells from kidneys of male Sprague-Dawley rats were treated with TAM. Rats subjected to 7 days of UUO were treated with TAM by oral gavage. Changes of intracellular trafficking and expression of AQP2 were evaluated by quantitative PCR, Western blotting, and immunohistochemistry. TAM induced AQP2 protein expression and intracellular trafficking in primary cultured IMCD cells, which were independent of the vasopressin V2 receptor (V2R) and cAMP activation, the critical pathways involved in AVP-stimulated regulation of AQP2. TAM attenuated the downregulation of AQP2 in TGF-β treated IMCD cells and IMCD suspensions prepared from UUO rats. TAM administration in vivo attenuated the downregulation of AQP2, associated with an improvement of urinary concentration in UUO rats. In addition, TAM increased CaMKII expression, suggesting that calmodulin signaling pathway is likely to be involved in the TAM-mediated AQP2 regulation. In conclusion, TAM is involved in AQP2 regulation in a vasopressin-independent manner and improves urinary concentration by attenuating the downregulation of AQP2 and maintaining intracellular trafficking in UUO.

The contralateral kidney presents with impaired mitochondrial functions and disrupted redox homeostasis after 14 days of unilateral ureteral obstruction in mice.

In unilateral ureteral obstruction (UUO), both oxidative stress and mitochondrial dysfunction are related to cell death. The aim of this study has been to characterize profiles of enzyme antioxidant activities and mitochondrial functioning of the contralateral (CL) compared to UUO and Sham (false-operated) kidneys of Balb/c mice. Kidneys were resected 14 days after obstruction for immunohistochemical and cortical mitochondrial functioning assays. Antioxidant enzymes activities were investigated in mitochondria and cytosol. Oxygen consumption (QO2) and formation of O2 reactive species (ROS) were assessed with pyruvate plus malate or succinate as the respiratory substrates. QO2 decreased in CL and UUO in all states using substrates for complex II, whereas it was affected only in UUO when substrates for complex I were used. Progressive decrease in mitochondrial ROS formation–in the forward and reverse pathway at complex I–correlates well with the inhibition of QO2 and, therefore, with decreased electron transfer at the level of complexes upstream of cytochrome c oxidase. CL and UUO transmembrane potential responses to ADP were impaired with succinate. Intense Ca2+-induced swelling was elicited in CL and UUO mitochondria. Important and selective differences exist in CL antioxidant enzymes with respect to either Sham or UUO kidneys: CL kidneys had increased mitochondrial glutathione peroxidase and cytosolic catalase activities, indicative of compensatory responses in the face of an early altered ROS homeostasis (as detected by 4-hydroxynonenal), and of a significant tendency to apoptosis. In CL and UUO, upregulation of nuclear (erythroid-derived 2)-like 2 transcription factor (Nrf2), as well as of cytoplasmic and nuclear Kelch-like ECH-associated protein 1 (Keap1) in opposition to decreased heme oxygenase-1 (HO-1), suggest impairment of the Nrf2/Keap1/HO-1 system. It is concluded that chronic obstruction impairs mitochondrial function in CL and UUO, preferentially affecting complex II.

Periplaneta Americana Extract May Attenuate Renal Fibrosis through Inhibiting Janus Tyrosine Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway

Background: Periplaneta americana is one of the ancient insect groups with the strongest vitality. Periplaneta americana extract (PAE) has been explored as an alternative remedy for many diseases. Although much progress has been made in the study about PAE, the role of the drug in renal disease is rarely reported, especially in renal fibrosis. This study was designed to evaluate the renoprotective effect of PAE treatment to renal fibrosis. Method: An in vivo, unilateral ureteral obstruction (UUO) mouse model was built. Then the mice were treated with PAE (100 mg/kg body weight) once daily by oral gavage, again starting on the day of UUO and continued for 1 week. At the end of 1 week, the mice were sacrificed; kidney samples were collected for further analysis. In vitro, Boston University mouse proximal tubular cells were plated in 35-mm dishes at a density of 0.3 * 10⁶ cells/dish. Then the cells were treated with 5-ng/mL TGF-β1 in serum-free DMEM medium for an indicated length of time. The experimental groups were pretreated with the indicated concentrations of PAE (0.3125 mg/mL). The cells were further cultured for 24 h, and then cells were monitored morphologically or collected for biochemical analyses. Results: Both in vivo and vitro PAE inhibits the expression of FN and alpha-smooth muscle actin and suppresses renal fibrosis. Importantly, PAE protects against renal fibrosis by inhibiting Janus tyrosine kinase 2 (JAK)/signal transducer and activator of transcription 3 (STAT) tyrosine phosphorylation. Conclusion: PAE attenuates renal fibrosis through the suppression of the JAK2/STAT3 pathway.

The Role of Mas Receptor on Renal Hemodynamic Responses to Angiotensin 1-7 in Both Irreversible and Reversible Unilateral Ureteral Obstruction Rats.

Background:Unilateral ureteral obstruction (UUO) alters the expression of renin-angiotensin system (RAS) components and angiotensin 1-7 (Ang 1-7) as a main arm of RAS is affected by UUO. The role of Mas receptor antagonist (A779) was examined in renal hemodynamic responses to Ang 1-7 in 3-day UUO and UUO removal (RUUO) in rats.Materials and Methods:Forty-five male Wistar rats were randomly divided into three groups of sham operated, UUO, and RUUO, while each group was divided into two subgroups treated with vehicle or A779. Renal blood flow (RBF) and renal vascular resistance (RVR) responses to graded Ang 1-7 infusion were measured at controlled renal perfusion pressure.Results:Mean arterial pressure response to Ang 1-7 was increased in vehicle-treated subgroup significantly (P < 0.05) when compared with A779-treated subgroup. However, such observation was not seen in UUO and RUUO rats. The graded Ang 1-7 infusion increased RBF and decreased RVR significantly in vehicle-treated rats (P < 0.005). Furthermore, a significant difference was found between vehicle and A779-treated subgroups in sham, UUO, and RUUO groups (P < 0.005).Conclusion:Ang 1-7 could alter the kidney hemodynamics responses in ureteral obstruction models.

Renal blood flow and vascular resistance responses to angiotensin II in irreversible and reversible unilateral ureteral obstruction rats; the role of angiotensin II type 1 &amp; 2 receptors

Background: Unilateral ureteral obstruction (UUO) alters the expression of renin-angiotensin system (RAS) components and angiotensin II (Ang II) as the main arm of RAS is affected by UUO. Objectives: In this study the role of Ang II subtypes 1 and 2 receptors (AT1R and AT2R) antagonists (losartan and PD123319) was examined in renal hemodynamic responses to graded Ang II infusion in sham, 3-day UUO and removal UUO (RUUO) models in rats. Materials and Methods: Seventy-one male Wistar rats randomly divided into three different sets of animal models; sham-operated, UUO and RUUO that each set contains three groups treated with vehicle, losartan, and PD123319. Renal vascular responses to Ang II infusion were measured at controlled renal perfusion pressure (RPP). Results: The graded Ang II infusion decreased renal blood flow (RBF), increased renal vascular resistance (RVR) and mean arterial pressure (MAP) in vehicle or PD123319 treated groups significantly (P<0.005), but no significant difference was found between these treated groups. However, RBF, RVR and MAP responses to graded Ang II infusion in losartan-treated rats were attenuated significantly when compared with vehicle or PD123319 treated groups (P<0.05). In addition, the RBF, RVR and MAP responses to Ang II were not similar in sham, UUO and RUUO rats treated with losartan. Conclusions: Vascular responses to Ang II in UUO and RUUO rat model treated with losartan is not as the normal pattern.

Inhibition of IκB Kinase at 24 Hours After Acute Kidney Injury Improves Recovery of Renal Function and Attenuates Fibrosis

BackgroundAcute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease. Nuclear factor‐κB is a nuclear transcription factor activated post‐ischemia, responsible for the transcription of proinflammatory proteins. The role of nuclear factor‐κB in the renal fibrosis post‐AKI is unknown.Methods and ResultsWe used a rat model of AKI caused by unilateral nephrectomy plus contralateral ischemia (30 minutes) and reperfusion injury (up to 28 days) to show impairment of renal function (peak: 24 hours), activation of nuclear factor‐κB (peak: 48 hours), and fibrosis (28 days). In humans, AKI is diagnosed by a rise in serum creatinine. We have discovered that the IκB kinase inhibitor IKK16 (even when given at peak serum creatinine) still improved functional and structural recovery and reduced myofibroblast formation, macrophage infiltration, transforming growth factor‐β expression, and Smad2/3 phosphorylation. AKI resulted in fibrosis within 28 days (Sirius red staining, expression of fibronectin), which was abolished by IKK16. To confirm the efficacy of IKK16 in a more severe model of fibrosis, animals were subject to 14 days of unilateral ureteral obstruction, resulting in tubulointerstitial fibrosis, myofibroblast formation, and macrophage infiltration, all of which were attenuated by IKK16.ConclusionsInhibition of IκB kinase at peak creatinine improves functional recovery, reduces further injury, and prevents fibrosis.

MP19-09 DAILY HYDROGEN SULFIDE THERAPY DURING PROLONGED URETERIC OBSTRUCTION ENABLES EARLY RENAL RECOVERY FOLLOWING DECOMPRESSION

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology II1 Apr 2017MP19-09 DAILY HYDROGEN SULFIDE THERAPY DURING PROLONGED URETERIC OBSTRUCTION ENABLES EARLY RENAL RECOVERY FOLLOWING DECOMPRESSION Shouzhe Lin, Dameng Lian, Weihua Liu, Aaron Haig, Ian Lobb, Ahmed Hijazi, Matthew Whiteman, and Alp Sener Shouzhe LinShouzhe Lin More articles by this author , Dameng LianDameng Lian More articles by this author , Weihua LiuWeihua Liu More articles by this author , Aaron HaigAaron Haig More articles by this author , Ian LobbIan Lobb More articles by this author , Ahmed HijaziAhmed Hijazi More articles by this author , Matthew WhitemanMatthew Whiteman More articles by this author , and Alp SenerAlp Sener More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3251AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal injury acquired during prolonged ureteral obstruction (UO) can lead to permanent renal dysfunction. Treatments aimed at mitigating renal injury during UO are limited. Hydrogen sulfide (H2S), an endogenous gasotransmitter, has been shown to ameliorate tissue injury. We have previously demonstrated that daily H2S treatment can reduce the histopathological markers of renal injury following 30 days of unilateral ureteral obstruction (UUO). The current study employs UUO followed by reimplantation to investigate the effects of daily H2S on renal function following the relief of obstruction. METHODS The left ureter of male Lewis rats were ligated at the ureterovesical junction. Starting on post-operative day (POD) 1 and every day for 13 days, rats received either daily intraperitoneal (IP) injection of phosphate buffered saline (PBS) vehicle or 200µmol/kg of GYY4137 (slow-releasing H2S donor) in PBS. On POD 14, the ligature was removed and the left ureter was reimplanted into the bladder, and a right nephrectomy performed so that the rat is solely dependent on the left kidney. Urine and serum samples were collected to monitor renal function. On POD 30, the left kidney was removed and tissue sections were stained with hematoxylin and eosin (H&E) to measure cortical thickness. RESULTS 67% of control rats, compared to 100% of H2S-treated rats, lived until POD 30. Histological analysis showed a significant decrease in cortical thickness (P<0.0001) in control rats when compared to Sham rats, which was significantly rescued upon treatment with H2S (P<0.0001). Serum creatinine (SCr) in control rats, but not H2S-treated rats, was significantly increased when compared to Sham animals on POD 3 and 7 (P<0.05). Following reimplantation (PODs 17, 21, 24 and 30), SCr in both control and treatment groups were significantly elevated when compared to Sham. Interestingly, H2S treatment drastically decreased SCr levels when compared to control animals on all PODs after reimplantation. Proteinuria continued to be higher in both control and treatment groups following reimplantation compared to Sham (P<0.05), however, H2S treatment group showed markedly reduced proteinuria compared to the control group. CONCLUSIONS We show, for the first time, that daily H2S therapy rescues renal function and preserves renal architecture following UO. H2S may one day become a clinically viable pre-emptive therapy against renal injury associated with UO and contribute to improved renal function in patients with UO. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e232-e233 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Shouzhe Lin More articles by this author Dameng Lian More articles by this author Weihua Liu More articles by this author Aaron Haig More articles by this author Ian Lobb More articles by this author Ahmed Hijazi More articles by this author Matthew Whiteman More articles by this author Alp Sener More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-382 Contributes to Renal Tubulointerstitial Fibrosis by Downregulating HSPD1.

Redox imbalance plays an important role in the pathogenesis of CKD progression. Previously, we demonstrated that microRNA-382 (miR-382) contributed to TGF-β1-induced loss of epithelial polarity in human kidney epithelial cells, but its role in the development of renal tubulointerstitial fibrosis remains unknown. In this study, we found that with 7 days of unilateral ureteral obstruction (UUO) in mice, the abundance of miR-382 in the obstructed kidney was significantly increased. Meanwhile, the protein expression of heat shock protein 60 (HSPD1), a predicted target of miR-382, was reduced after 7 days of UUO. Expression of 3-nitrotyrosine (3-NT) was upregulated, but expression of thioredoxin (Trx) was downregulated. Anti-miR-382 treatment suppressed the upregulation of miR-382, attenuated renal interstitial fibrosis in the obstructed kidney, and reversed the downregulation of HSPD1/Trx and upregulation of 3-NT after UUO. Furthermore, in vitro study revealed that overexpression of HSPD1 significantly restored Trx expression and reversed TGF-β1-induced loss of E-cadherin, while in vivo study found that direct siRNA-mediated suppression of HSPD1 in the UUO kidney promoted oxidative stress despite miR-382 blockade. Our clinical data showed that upregulation of miR-382/3-NT and downregulation of HSPD1/Trx were also observed in IgA nephropathy patients with renal interstitial fibrosis. These data supported a novel mechanism in which miR-382 targets HSPD1 and contributes to the redox imbalance in the development of renal fibrosis.

15-Deoxy-Δ12,14-prostaglandin J2 Exerts Antioxidant Effects While Exacerbating Inflammation in Mice Subjected to Ureteral Obstruction.

Urinary obstruction is associated with inflammation and oxidative stress, leading to renal dysfunction. Previous studies have shown that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has both antioxidant and anti-inflammatory effects. Using a unilateral ureteral obstruction (UUO) mouse model, we examined the effects of 15d-PGJ2 on oxidative stress and inflammation in the kidney. Mice were subjected to UUO for 3 days and treated with 15d-PGJ2. Protein and RNA expression were examined using immunoblotting and qPCR. 15d-PGJ2 increased NF-E2-related nuclear factor erythroid-2 (Nrf2) protein expression in response to UUO, and heme oxygenase 1 (HO-1), a downstream target of Nrf2, was induced by 15d-PGJ2. Additionally, 15d-PGJ2 prevented protein carbonylation, a UUO-induced oxidative stress marker. Inflammation, measured by nuclear NF-κB, F4/80, and MCP-1, was increased in response to UUO and further increased by 15d-PGJ2. Renal injury was aggravated by 15d-PGJ2 treatment as measured by kidney injury molecule-1 (KIM-1) and cortical caspase 3 content. No effect of 15d-PGJ2 was observed on renal function in mice subjected to UUO. This study illustrates differentiated functioning of 15d-PGJ2 on inflammation and oxidative stress in response to obstructive nephropathy. High concentrations of 15d-PGJ2 protects against oxidative stress during 3-day UUO in mice; however, it aggravates the associated inflammation.