Postischemic Acute Kidney Injury Research Articles

#185 The role of angiotensin 2 type 1 receptor and type 2 receptor after postischemic acute kidney injury in spontaneously hypertensive rats

Abstract Background and Aims Renal ischemia/reperfusion injury is a common cause of acute kidney injury (AKI), and hypertension might contribute to the increased incidence of AKI. AKI has a multifactorial causality, but the mechanism of pathogenesis and the development of this disease are still incompletely defined. The purpose of this study was to compare the effects of AT1R blockade and AT2R simulation on AKI development in spontaneously hypertensive rats (SHR). Method We used male 24 weeks old SHR. Animals were divided into following experimental groups: sham operated rats (SHAM); rats with induced postischemic AKI (AKI); a group which received AT1R antagonist Losartan (10 mg/kg b.w.)(AKI+LOS) and group which received AT2R agonist novokinin (0.3 mg/kg b.w.)(AKI+NOVO) in the moment of kidney reperfusion. AKI was induced by surgical removal of right kidney followed by atraumatic clamp occlusion of the left renal artery for 40 minutes. Hemodynamic and biochemical measurements were done 24 hours after reperfusion. Results Conclusion Our results confirm the dominant role of AT1R on AKI development, but AT2R stimulation failed to buffer deleterious effects of AT1R hyperactivity on renal vasoconstriction and glomerular filtration. However, the role of AT2R during AKI development in hypertension will be in focus in our further studies.

#2170 BET inhibitor nanotherapy reduces chronic kidney disease progression after ischemia-reperfusion injury

Abstract Background and Aims Targeting epigenetic mechanisms offer a potential breakthrough in addressing kidney diseases. Thus, inhibition of the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1, has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, clinical translation faces hurdles like poor pharmacokinetics and side effects. To tackle this, we engineered liposomes loaded with JQ1 to enhance kidney drug delivery and minimize side effects. By optimizing the encapsulation of JQ1, our aim was to improve its efficacy compared to freely delivered JQ1 and reduce its cytotoxicity. Moreover, the therapeutic potential of this new nanoformulation to halt the acute renal ischemic injury and its transition to CKD will be explored. Method JQ1 was encapsulated in different types of lipid nanoparticles (NPs), and its effect was analyzed in vitro on the HK2 tubular epithelial line. Additionally, in vivo administration was carried out 1 hour after the induction of 45 minutes bilateral renal ischemia, and its effect was further analyzed at 24 h in the AKI setting or at 21 days for CKD. Non-encapsulated JQ1 was used as a control, and in vivo biodistribution studies were conducted using IVIS and fluorescent Cy5.5-labelled NPs. Damage markers, inflammatory burden and fibrosis development were analyzed through conventional techniques (Western blot, RT-PCR, IHC, flow cytometry), and functional parameters (BUN, Creatinine) were assessed through colorimetric assays. Results Three types of liposomal NPs were assayed to optimize JQ1 encapsulation, being the combined encapsulation of JQ1 in both the core and lipid layer the ones proving the best effectiveness. With an average hydrodynamic diameter of 78 nm, these NPs effectively reached the kidneys as assayed by in vivo biodistribution assays. In vitro assays performed on the HK2 cell line reveal their potential to inhibit TNF-α-induced inflammation. Importantly, in vivo dose-dependent studies determined that a 40 mg/kg dose was effective in post-ischemic acute kidney injury, showing a reduction in inflammatory (Il6, Csf2, Ccl2, Ccl5) and renal damage markers (Ngal), as well as reduced immune cell infiltration into the renal tissue of monocytes (CD45+CD11b+Ly6G−Ly6ChiF4/80low) and neutrophils (CD45+CD11b+Ly6GhiLy6Clow) quantified by flow cytometry. In accordance, decreased levels of serum BUN and Creatinine were detected in the group of mice treated with NPs-JQ1, indicating a better renal function. Of note, none of these effects were observed when the same dose of the unencapsulated inhibitor was used, demonstrating no effectiveness whatsoever. In the long term, by establishing an AKI to CKD transition model analysing fibrosis at 21 days post-ischemia induction, mice receiving a single dose of NPs-JQ1 early after damage displayed reduced fibrosis markers expression (α-SMA and Fibronectin), decreased collagen deposits quantified by Masson's Trichrome tissue staining and a partial recovery of the renal function. Conclusion The encapsulation of JQ1 in lipid nanoparticles is an effective strategy that allows for a decrease in the minimum effective dose of the drug. Due to its higher concentration and stability in the kidney, this therapy could be relevant in the treatment of renal pathologies and represents a significant step forward in the development of an innovative therapy for AKI-to-CKD transition. Our pioneering liposomal formulation not only expands the therapeutic potential of JQ1 but also addresses a significant barrier to its clinical translation in the nephrology field.

Hyperbaric Oxygen Reduces Oxidative Stress Impairment and DNA Damage and Simultaneously Increases HIF-1α in Ischemia-Reperfusion Acute Kidney Injury.

The central exacerbating factor in the pathophysiology of ischemic-reperfusion acute kidney injury (AKI) is oxidative stress. Lipid peroxidation and DNA damage in ischemia are accompanied by the formation of 3-nitrotyrosine, a biomarker for oxidative damage. DNA double-strand breaks (DSBs) may also be a result of postischemic AKI. γH2AX(S139) histone has been identified as a potentially useful biomarker of DNA DSBs. On the other hand, hypoxia-inducible factor (HIF) is the "master switch" for hypoxic adaptation in cells and tissues. The aim of this research was to evaluate the influence of hyperbaric oxygen (HBO) preconditioning on antioxidant capacity estimated by FRAP (ferric reducing antioxidant power) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, as well as on oxidative stress parameter 3-nitrotyrosine, and to assess its effects on γH2AX(S139), HIF-1α, and nuclear factor-κB (NF-κB) expression, in an experimental model of postischemic AKI induced in spontaneously hypertensive rats. The animals were divided randomly into three experimental groups: sham-operated rats (SHAM, n = 6), rats with induced postischemic AKI (AKI, n = 6), and group exposed to HBO preconditioning before AKI induction (AKI + HBO, n = 6). A significant improvement in the estimated glomerular filtration rate, eGFR, in AKI + HBO group (p < 0.05 vs. AKI group) was accompanied with a significant increase in plasma antioxidant capacity estimated by FRAP (p < 0.05 vs. SHAM group) and a reduced immunohistochemical expression of 3-nitrotyrosine and γH2AX(S139). Also, HBO pretreatment significantly increased HIF-1α expression (p < 0.001 vs. AKI group), estimated by Western blot and immunohistochemical analysis in kidney tissue, and decreased immunohistochemical NF-κB renal expression (p < 0.01). Taking all of these results together, we may conclude that HBO preconditioning has beneficial effects on acute kidney injury induced in spontaneously hypertensive rats.

Clinicopathological Relevance of PAX8 Expression Patterns in Acute Kidney Injury and Chronic Kidney Diseases.

Transcription factor PAX8, expressed during embryonic kidney development, has been previously detected in various kidney tumors. In order to investigate expression of PAX8 transcription factor in acute kidney injury (AKI) and chronic kidney diseases (CKD), immunohistochemical analysis was performed. Presence, location and extent of PAX8 expression were analyzed among 31 human kidney samples of AKI (25 autopsy cases, 5 kidney biopsies with unknown etiology and 1 AKI with confirmed myoglobin cast nephropathy), as well as in animals with induced postischemic AKI. Additionally, expression pattern was analyzed in 20 kidney biopsy samples of CKD. Our study demonstrates that various kidney diseases with chronic disease course that results in the formation of tubular atrophy and interstitial fibrosis, lead to PAX8 expression in the nuclei of proximal tubules. Furthermore, patients with PAX8 detected within the damaged proximal tubuli would be carefully monitored, since deterioration in kidney function was observed during follow-up. We also showed that myoglobin provoked acute kidney injury followed with large extent of renal damage, was associated with strong nuclear expression of PAX8 in proximal tubular cells. These results were supported and followed by data obtained in experimental model of induced postischemic acute kidney injury. Considering these findings, we can assume that PAX8 protein might be involved in regeneration process and recovery after acute kidney injury. Thus, accordingly, all investigation concerning PAX8 immunolabeling should be performed on biopsy samples of the living individuals.

EFFECTS OF APOCYNIN TREATMENT AND HBO PRECONDITIONING ON IMMUNOHISTOCHEMICAL 4-HNE, HIF1 ALPHA, HIF2 ALPHA AND NGAL TISSUE EXPRESSION IN POSTISCEMIC ACUTE KIDNEY INJURY IN SHR RATS

Objective: Oxidative stress has been considered as a central aggravating factor in the development of postischemic acute kidney injury (AKI). 4-hydroxynonenal (4-HNE) is a product of lipid peroxidation and may serve as non-invasive biomarker of oxidative stress while neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker of AKI. Hypoxia-inducible factors (HIFs) are the “master’’ transcription factors responsible for gene expression in hypoxia, which play an important role in kidney injury and repair by regulating HIF target genes, including microRNAs. Design and method: The study was conducted on male SHR rats, randomly divided into following groups: sham-operated group (SHAM) AKI control group, AKI group with apocynin treatment (AKI+APO), group with HBO preconditioning before AKI induction (AKI+HBO) and group with HBO preconditioning before and apocynin treatment after AKI induction (AKI+APO+HBO). HBO preconditioning was performed by exposing to pure oxygen (2.026 bar) twice a day for two consecutive days for 60 minutes, and 24 h before AKI induction. Apocynin, NADPH oxidase inhibitor was injected 5 minutes before reperfusion. Results: AKI induction significantly decreased creatinine (CCr), urea (CU) and phosphate (CPhos) clearances when compared to SHAM group. Remarkable increase in CCr, CU, CPhos levels were observed in all treated groups (AKI+APO, AKI+HBO, AKI+APO+HBO) in comparison to AKI group. Sham – operated rats did not express 4-HNE and NGAL in any parenchymal structure. AKI induced abundant and strong glomerular expression of 4-HNE along with expression in interstitial compartment. All treatments (AKI+APO, AKI+HBO, AKI+APO+HBO) significantly decreased 4-HNE expression both in glomeruli and interstitium. Also, AKI stimulated widespread NGAL expression in renal epithelial tubular cells with fine granular appearance on the apical surface of the cells, affecting entire circumference of tubular cross-sectioning, which was decreased in all treated groups. In SHAM group, the expression of HIF1 alpha and HIF2 alpha was focal with weak intensity in glomeruli and interstitium. In AKI group, the expression was diffuse and strong, with remarkable improvement in all treated groups. Conclusions: Considering our results, we can conclude that apocynin treatment, as well as HBO preconditioning may have protective effects in postischemic acute kidney injury induced in spontaneously hypertensive rats.

Immunohistochemical Analysis of 4-HNE, NGAL, and HO-1 Tissue Expression after Apocynin Treatment and HBO Preconditioning in Postischemic Acute Kidney Injury Induced in Spontaneously Hypertensive Rats.

Oxidative stress has been considered as a central aggravating factor in the development of postischemic acute kidney injury (AKI). The aim of this study was to perform the immunohistochemical analysis of 4-hydroxynonenal (4-HNE), neutrophil gelatinase-associated lipocalin (NGAL), and heme oxygenase-1 (HO-1) tissue expression after apocynin (APO) treatment and hyperbaric oxygenation (HBO) preconditioning, applied as single or combined protocol, in postischemic acute kidney injury induced in spontaneously hypertensive rats (SHR). Twenty-four hours before AKI induction, HBO preconditioning was carried out by exposing to pure oxygen (2.026 bar) twice a day, for 60 min in two consecutive days. Acute kidney injury was induced by removal of the right kidney while the left renal artery was occluded for 45 min by atraumatic clamp. Apocynin was applied in a dose of 40 mg/kg body weight, intravenously, 5 min before reperfusion. We showed increased 4-HNE renal expression in postischemic AKI compared to Sham-operated (SHAM) group. Apocynin treatment, with or without HBO preconditioning, improved creatinine and phosphate clearances, in postischemic AKI. This improvement in renal function was accompanied with decreased 4-HNE, while HO-1 kidney expression restored close to the control group level. NGAL renal expression was also decreased after apocynin treatment, and HBO preconditioning, with or without APO treatment. Considering our results, we can say that 4-HNE tissue expression can be used as a marker of oxidative stress in postischemic AKI. On the other hand, apocynin treatment and HBO preconditioning reduced oxidative damage, and this protective effect might be expected even in experimental hypertensive condition.

IRF8-Dependent Type I Conventional Dendritic Cells (cDC1s) Control Post-Ischemic Inflammation and Mildly Protect Against Post-Ischemic Acute Kidney Injury and Disease.

Post-ischemic acute kidney injury and disease (AKI/AKD) involve acute tubular necrosis and irreversible nephron loss. Mononuclear phagocytes including conventional dendritic cells (cDCs) are present during different phases of injury and repair, but the functional contribution of this subset remains controversial. Transcription factor interferon regulatory factor 8 (IRF8) is required for the development of type I conventional dendritic cells (cDC1s) lineage and helps to define distinct cDC1 subsets. We identified one distinct subset among mononuclear phagocyte subsets according to the expression patterns of CD11b and CD11c in healthy kidney and lymphoid organs, of which IRF8 was significantly expressed in the CD11blowCD11chigh subset that mainly comprised cDC1s. Next, we applied a Irf8-deficient mouse line (Irf8 fl/fl Clec9a cre mice) to specifically target Clec9a-expressing cDC1s in vivo. During post-ischemic AKI/AKD, these mice lacked cDC1s in the kidney without affecting cDC2s. The absence of cDC1s mildly aggravated the loss of living primary tubule and decline of kidney function, which was associated with decreased anti-inflammatory Tregs-related immune responses, but increased T helper type 1 (TH1)-related and pro-inflammatory cytokines, infiltrating neutrophils and acute tubular cell death, while we also observed a reduced number of cytotoxic CD8+ T cells in the kidney when cDC1s were absent. Together, our data show that IRF8 is indispensable for kidney cDC1s. Kidney cDC1s mildly protect against post-ischemic AKI/AKD, probably via suppressing tissue inflammation and damage, which implies an immunoregulatory role for cDC1s.

MO333TARGETING HIGH MOBILITY GROUP BOX 1 WITH INHIBITORS ATTENUATE ACUTE KIDNEY INJURY IN POSTISCHEMIC KIDNEYS

Abstract Background and Aims HMGB1 (high mobility group box 1) is released from dying cells and hence works as a danger signal, which amplifies necroinflammation in postischemic acute kidney injury. If and to what extend small molecule HMGB1 inhibitors are capable to ameliorate the acute kidney injury is still unknown. Method Mice underwent 17 min unilateral renal ischemia followed by reperfusion (IR) for 4 or 7 days and were administered either 50 mg/kg glycyrrhizic acid, 80 mg/kg ethyl pyruvate, or PBS i.p. 1h before surgery and once daily until day 7. Results Administration of HMGB1 inhibitors consistently increased the glomerular infiltration rate at day 4 and day 7, in parallel with a reduction of plasma creatinine and circulating HMGB1 at day 7 of reperfusion. Histopathological analysis confirmed that HMGB1 inhibitors consistently attenuated tubule injury. In vitro, HMGB1 inhibitors protected primary murine tubular epithelial cells against H2O2-induced cell death. Conclusion Our data indicate that the treatment with glycyrrhizic acid or ethyl pyruvate ameliorates post-ischemic renal injury. This serves as a rationale to further study the role of extracellular HMGB1 and its inhibition in different models of acute kidney injury.

POS-085 HMGB1 IN MYELOID CELLS ATTENUATES POSTISCHEMIA ACUTE KIDNEY INJURY

HMGB1 released from necrotic cells acts as danger signal (DAMP) as it activates immune cells, which amplifies necroinflammation in postischemic acute kidney injury (IRI-AKI). The contribution of HMGB1 inside and released from resident and infiltrating myeloid cells in this context is unknown.

Hyperbaric Oxygen Preconditioning Upregulates Heme OxyGenase-1 and Anti-Apoptotic Bcl-2 Protein Expression in Spontaneously Hypertensive Rats with Induced Postischemic Acute Kidney Injury.

Renal ischemia and reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI). Pathogenesis of postischemic AKI involves hemodynamic changes, oxidative stress, inflammation process, calcium ion overloading, apoptosis and necrosis. Up to date, therapeutic approaches to treat AKI are extremely limited. Thus, the aim of this study was to evaluate the effects of hyperbaric oxygen (HBO) preconditioning on citoprotective enzyme, heme oxygenase-1 (HO-1), pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins expression, in postischemic AKI induced in normotensive Wistar and spontaneously hypertensive rats (SHR). The animals were randomly divided into six experimental groups: SHAM-operated Wistar rats (W-SHAM), Wistar rats with induced postischemic AKI (W-AKI) and Wistar group with HBO preconditioning before AKI induction (W-AKI + HBO). On the other hand, SHR rats were also divided into same three groups: SHR-SHAM, SHR-AKI and SHR-AKI + HBO. We demonstrated that HBO preconditioning upregulated HO-1 and anti-apoptotic Bcl-2 protein expression, in both Wistar and SH rats. In addition, HBO preconditioning improved glomerular filtration rate, supporting by significant increase in creatinine, urea and phosphate clearances in both rat strains. Considering our results, we can also say that even in hypertensive conditions, we can expect protective effects of HBO preconditioning in experimental model of AKI.

Pre-ischemic renal lavage protects against renal ischemia-reperfusion injury by attenuation of local and systemic inflammatory responses.

Postischemic acute kidney injury (AKI) is a common clinical complication and often fatal, with no effective treatment available. Little is known about the role of leukocytes trapped in renal vessels during ischemia-reperfusion injury (IRI) in the postischemic AKI. We designed a new animal model in rats with preforming renal artery lavage prior to IRI to investigate the effect of diminishing the residual circulating leukocytes on kidney damage and inflammation. Moreover, the functional changes of macrophages in hypoxia reoxygenation condition were also analyzed. We found pre-ischemic renal lavage significantly decreased the serum creatinine and blood urea nitrogen levels, and downregulated the mRNA and protein expressions in kidneys and urinary secretion of kidney injury molecule-1 of rats after IRI. The renal pathological damage caused by IRI was also ameliorated by pre-ischemic renal lavage, as evidenced by fewer cast formation, diminished morphological signs of AKI in the tissue at 24hours after IRI. Pre-ischemic renal lavage reduced the numbers of infiltrating CD68+ macrophages and MPO+ neutrophils. The mRNA expression of pro-inflammatory mediator in IRI kidneys and the levels of pro-inflammatory cytokines in circulatory system and urine were also reduced due to pre-ischemic lavage. Compared with nontreated rats with IRI, pre-ischemic renal lavage significantly reduced the phosphorylation levels of ERK and p65 subunit of NF-κB in the kidney after IRI. In addition, we found hypoxia/reoxygenation could promote the expression of pro-inflammatory mediators and inhibit the expression of anti-inflammatory factors by regulating ERK/NF-κB signaling pathway. Thus, pre-ischemic renal lavage could clearly reduce the renal damage after IRI by attenuating inflammation, and macrophages trapped in renal vessels during IRI could be important pathogenic factors driving tissue injury.

Hyperbaric oxygen preconditioning and the role of NADPH oxidase inhibition in postischemic acute kidney injury induced in spontaneously hypertensive rats.

Renal ischemia/reperfusion injury is a common cause of acute kidney injury (AKI) and hypertension might contribute to the increased incidence of AKI. The purpose of this study was to investigate the effects of single and combined hyperbaric oxygen (HBO) preconditioning and NADPH oxidase inhibition on oxidative stress, kidney function and structure in spontaneously hypertensive rats (SHR) after renal ischemia reperfusion injury. HBO preconditioning was performed by exposing to pure oxygen (2.026 bar) twice a day for two consecutive days for 60 minutes, and 24h before AKI induction. For AKI induction, the right kidney was removed and ischemia was performed by clamping the left renal artery for 45 minutes. NADPH oxidase inhibition was induced by apocynin (40 mg/kg b.m., intravenously) 5 minutes before reperfusion. AKI significantly increased renal vascular resistance and reduced renal blood flow, which were significantly improved after apocynin treatment. Also, HBO preconditioning, with or without apocynin treatment showed improvement on renal hemodynamics. AKI significantly increased plasma creatinine, urea, phosphate levels and lipid peroxidation in plasma. Remarkable improvement, with decrease in creatinine, urea and phosphate levels was observed in all treated groups. HBO preconditioning, solitary or with apocynin treatment decreased lipid peroxidation in plasma caused by AKI induction. Also, combined with apocynin, it increased catalase activity and solitary, glutathione reductase enzyme activity in erythrocytes. While AKI induction significantly increased plasma KIM– 1 levels, HBO preconditioning, solitary or with apocynin decreased its levels. Considering renal morphology, significant morphological alterations present after AKI induction were significantly improved in all treated groups with reduced tubular dilatation, tubular necrosis in the cortico-medullary zone and PAS positive cast formation. Our results reveal that NADPH oxidase inhibition and hyperbaric oxygen preconditioning, with or without NADPH oxidase inhibition may have beneficial effects, but their protective role should be evaluated in further studies.

FP251HMGB1 in myeloid cells drives postischemic acute kidney injury by promoting neutrophil infiltration

FP251HMGB1 in myeloid cells drives postischemic acute kidney injury by promoting neutrophil infiltration

Erythropoietin modulates macrophages but not post-ischemic acute kidney injury in mice.

Substantial progress was made in acute kidney injury (AKI) over the past 10 years, but no therapeutic interventions have been shown to prevent AKI or accelerate functional recovery after injury. A large number of preclinical studies supports the use of recombinant human erythropoietin (rHuEPO) to prevent AKI, but the clinical trial data are inconclusive. To address concerns about preclinical study design and reporting in AKI, we here presented our rigorous experiments on the use of rHuEPO in a mouse model simulating the most common post-ischemic AKI in patients. Use of saline vehicle or rHuEPO (100 or 1000U/KgBW) in mice subjected to AKI induced by ischemia-reperfusion injury of left kidney 2 weeks after right nephrectomy (NX+IRI). NX+IRI resulted in a reproducible AKI model. Use of rHuEPO as a pretreatment or posttreatment did not affect AKI severity, functional recovery, and mouse survival regardless of gender, injury severity, or doses of rHuEPO. Administering rHuEPO with 1000U/KgBW did increase hematocrit and modulate AKI kidney macrophages by Nos2 downregulation and Ccl17 upregulation. Active expression of erythropoietin receptor (EPOR) was not identified in renal cells by lineage tracing study, whereas expression of colony-stimulating factor 2 receptor β (CSF2Rβ) was identified in kidney macrophages and upregulated after AKI. Both EPOR and CSF2Rβ were identified in cultured bone marrow derived macrophages, possibly mediated the robust inhibition of cytokine-induced phenotype switching by rHuEPO. Use of rHuEPO can modulate macrophage function but not the post-ischemic AKI severity, functional recovery and survival in mice.

Autophagy activation in circulating proangiogenic cells aggravates AKI in type I diabetes mellitus.

Acute kidney injury (AKI) occurs frequently in hospitals worldwide, but the therapeutic options are limited. Diabetes mellitus (DM) affects more and more people around the globe. The disease worsens the prognosis of AKI even further. In recent years, cell-based therapies have increasingly been applied in experimental AKI. The aim of the study was to utilize two established autophagy inducers for pharmacological preconditioning of so-called proangiogenic cells (PACs) in PAC treatment of diabetic AKI. Insulin-dependent DM was induced in male C57/Bl6N mice by intraperitoneal injections of streptozotocine. Six weeks later, animals underwent bilateral renal ischemia for 45 min, followed by intravenous injections of either native or zVAD (benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone)- or Z-Leu-Leu-Leu-al (MG132)-pretreated syngeneic murine PACs. Mice were analyzed 48 h (short term) and 6 wk (long term) later, respectively. DM worsened postischemic AKI, and PAC preconditioning with zVAD and MG132 resulted in a further decline of excretory kidney function. Injection of native PACs reduced fibrosis in nondiabetic mice, but cell preconditioning promoted interstitial matrix accumulation significantly. Both substances aggravated endothelial-to-mesenchymal transition (EndoMT) under diabetic conditions; these effects occurred either exclusively in the short (zVAD) or in the short and long term (MG132). Preconditioned cells stimulated the autophagocytic flux in intrarenal endothelial cells, and all experimental groups displayed increased endothelial abundances of senescence-associated β-galactosidase, a marker of premature cell senescence. Pharmacological autophagy activation may not serve as an effective strategy for improving PAC competence in diabetic AKI in general. On the contrary, several outcome parameters (excretory function, fibrosis, EndoMT) may even be worsened.

Combined Angiotensin II Type-1 Receptor Blockade and Superoxide Anion Scavenging Affect the Post-Ischemic Kidney in Hypertensive Rats

Abstract Ischemic acute kidney injury is characterized by renal vasoconstriction, filtration failure, tubular obstruction, tubular backleak and overproduction of angiotensin II and reactive oxygen species. Considering this complexity, the aim of our study was to investigate the effects of angiotensin II type-1 receptor blocker - Losartan and superoxide anion scavenger - Tempol, in a combined treatment on acute kidney injury in postischemic hypertensive rats. The experiment was performed in anesthetized, adult male spontaneously hypertensive rats. The right kidney was removed and the left renal artery was occluded for 40 minutes. Experimental groups received combined treatment (Losartan + Tempol) or saline in the femoral vein 5 minutes before, during and 175 minutes after clamp removal. Hemodynamics and biochemical parameters were measured and kidney specimens were collected 24h after reperfusion. Histological examination was performed by optical microscopy. Combined treatment improves renal haemodynamics parameters which were exacerbated due to acute kidney injury. Acute kidney injury significantly decreased creatinine and urea clearance and increased lipid peroxidation in the plasma. Treatment with Losartan and Tempol induced a significant increase of creatinine and urea clearance. Lipid peroxidation in the plasma decreased and glutathione peroxidase enzyme activity in the erythrocytes increased after Losartan + Tempol treatment. This combined treatment reduced cortico-medullary necrosis and tubular dilatation in the kidney. Our results indicate that synergism of Losartan and Tempol treatment could have beneficial effects on blood pressure and kidney function, during postischemic acute kidney injury development in experimental hypertension.

17β-Estradiol Accelerated Renal Tubule Regeneration in Male Rats After Ischemia/Reperfusion-Induced Acute Kidney Injury.

Ischemic/reperfusion injury (IRI) is the most common cause of acute kidney injury (AKI). Murine studies report that pretreatment with 17β-estradiol protects against AKI using multiple mechanisms, but how 17β-estradiol is involved in regenerating tubular cells is unknown. To visualize the kidney injury and repair, we used 17β-estradiol to treat rats with postischemic acute kidney injury. AKI was induced by clamping the renal pedicle for 90 minutes 2 weeks after a unilateral nephrectomy. Rats were treated with an intravenous injection of 17β-estradiol or vehicle immediately after reperfusion. Kidney injury was assessed by measuring biochemical and histopathological changes. Immunohistochemical staining of vimentin, proliferating cell nuclear antigen (PCNA), and E-cadherin were used to assess dedifferentiation, proliferation, and redifferentiation. Rats treated with 17β-estradiol had less kidney injury than did vehicle-treated rats post-IRI day 1. The number of PCNA-positive (PCNA) cells was significantly higher in post-IRI kidneys on day 1 in 17β-estradiol-treated rats. Moreover, vimentin and E-cadherin cells, which were interpreted as regeneration markers, were expressed earlier and significantly more copiously in 17β-estradiol-treated rats. We hypothesize that 17β-estradiol attenuates IRI-induced AKI by reducing inflammation and accelerating injured tubular cell regeneration.

Optimizing Mouse Surgery with Online Rectal Temperature Monitoring and Preoperative Heat Supply. Effects on Post-Ischemic Acute Kidney Injury.

Body temperature affects outcomes of tissue injury. We hypothesized that online body core temperature recording and selective interventions help to standardize peri-interventional temperature control and the reliability of outcomes in experimental renal ischemia reperfusion injury (IRI). We recorded core temperature in up to seven mice in parallel using a Thermes USB recorder and ret-3-iso rectal probes with three different protocols. Setup A: Heating pad during ischemia time; Setup B: Heating pad from incision to wound closure; Setup C: A ventilated heating chamber before surgery and during ischemia time with surgeries performed on a heating pad. Temperature profile recording displayed significant declines upon installing anesthesia. The profile of the baseline experimental setup A revealed that <1% of the temperature readings were within the target range of 36.5 to 38.5°C. Setup B and C increased the target range readings to 34.6 ± 28.0% and 99.3 ± 1.5%, respectively. Setup C significantly increased S3 tubular necrosis, neutrophil influx, and mRNA expression of kidney injury markers. In addition, using setup C different ischemia times generated a linear correlation with acute tubular necrosis parameters at a low variability, which further correlated with the degree of kidney atrophy 5 weeks after surgery. Changing temperature control setup A to C was equivalent to 10 minutes more ischemia time. We conclude that body temperature drops quickly in mice upon initiating anesthesia. Immediate heat supply, e.g. in a ventilated heating chamber, and online core temperature monitoring can help to standardize and optimize experimental outcomes.

Progressive endothelin-1 gene activation initiates chronic/end-stage renal disease following experimental ischemic/reperfusion injury

This study assessed whether endothelin-1 (ET1) helps mediate post-ischemic acute kidney injury (AKI) progression to chronic kidney disease (CKD). The impact(s) of potent ETA or ETB receptor-specific antagonists (Atrasentan and BQ-788, respectively) on disease progression were assessed 24 hours or 2 weeks following 30 minutes of unilateral ischemia in CD-1 mice. Unilateral ischemia caused progressive renal ET-1 protein/mRNA increases with concomitant ETA, but not ETB, mRNA elevations. Extensive histone remodeling consistent with gene activation and increased RNA polymerase II binding occurred at the ET-1 gene. Unilateral ischemia produced progressive renal injury as indicated by severe histologic injury and a 40% loss of renal mass. Pre- and post-ischemia or just post-ischemic treatment with Atrasentan conferred dramatic protective effects such as decreased tubule/microvascular injury, normalized tissue lactate, and total preservation of renal mass. Nuclear KI-67 staining was not increased by Atrasentan, implying that increased tubule proliferation was not involved. Conversely, ETB blockade had no protective effect. Thus, our findings provide the first evidence that ET-1 operating through ETA can play a critical role in ischemic AKI progression to CKD. Blockade of ETA provided dramatic protection, indicating the functional significance of these results.

Endogenous and exogenous pentraxin-3 limits postischemic acute and chronic kidney injury

Ischemia-reperfusion activates innate immunity and sterile inflammation, resulting in acute kidney injury. Since pentraxin 3 (PTX3) regulates multiple aspects of innate immunity and tissue inflammation, we tested whether PTX3 would be involved in renal ischemia-reperfusion injury. Renal pedicle clamping increased PTX3 serum levels, as well as PTX3 expression, inside the kidney but predominantly in CD45/CD11c(+) cells, a subpopulation of intrarenal mononuclear phagocytes. Lack of PTX3 aggravated postischemic acute kidney injury as evidenced by massive tubular necrosis, and TNF and IL-6 release, as well as massively increased neutrophil and macrophage infiltrates at 24 h. This was followed by tubular atrophy, interstitial fibrosis, and kidney shrinking 10 weeks later. In vivo microscopy uncovered increased leukocyte adhesion and transmigration in postischemic microvessels of Ptx3-deficient mice. Furthermore, injection of recombinant PTX3 up to 6 h after reperfusion prevented renal leukocyte recruitment and postischemic kidney injury. Thus, local PTX3 release from a subpopulation of intrarenal mononuclear phagocytes or delayed PTX3 treatment limits postischemic renal inflammation. Conversely, Ptx3 loss-of-function mutations predispose to postischemic acute kidney injury and subsequent chronic kidney disease.