IR-induced Acute Kidney Injury Research Articles

Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

Urotensin II (UII) and UII-related peptide (URP) and their receptor, UT, comprise the UII system. Although UII is conserved across all vertebrates and shares a cyclic hexapeptide core-sequence motif of CFWKYC, there are differences in N-terminal amino acid sequences between species. The kidney is a significant source of UII, the level of which is amplified in infants with chronic kidney disease. We have previously shown that human UII increases renal vascular resistance (RVR) and reduces renal blood flow in neonatal pigs. However, the effect of pig UII (pUII) on renal microcirculation remains unclear. Since the mechanisms of renal ischemia-reperfusion (IR) injury include kidney hypoperfusion, increased activity of the UII system may contribute to ischemic acute kidney injury (AKI). Here, we demonstrate that kidney tissue UII, URP, and UT and renal vascular UT expression levels are higher in neonatal pigs than in adults. Intrarenal artery infusion of pUII reduced kidney perfusion and increased RVR, which was reversed by urantide, a peptide UT inhibitor. One hour of renal ischemia, followed by 8 hours of reperfusion, did not alter kidney tissue UT but upregulated kidney tissue UII and URP and renal vascular UT expression levels. Urantide mitigated renal IR-induced increase in AKI biomarkers. Urantide also attenuated renal IR-induced increases in serum interleukin-1beta, interleukin-10, interferon-α, and morphological kidney injury. The enzyme that converts prepro UII to active UII has been proposed to be serine protease furin. Kidney tissue expression of furin is higher in neonatal pigs compared with adults. Renal IR also upregulated furin expression in neonatal pig kidneys. Exposure of primary neonatal pig renal epithelial cells to the cellular model of IR (cIR) increased furin levels. cIR also increased UII production in the cells, an effect attenuated by furin inhibitor SS3. These findings suggest that in pigs, 1) the UII system is upregulated in immature kidneys, 2) renal IR upregulates furin, UII, and URP in kidney tissues and UT in renal blood vessels of neonates, 3) ischemic activation of furin promotes UII biosynthesis by renal epithelial cells, and 4) pharmacological inhibition of UT mitigates renal IR-induced AKI. We conclude that the UII systems may contribute to IR-induced neonatal renal insuffciency. NIH R01DK120595 R01DK127625. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia-Reperfusion-Induced Kidney Injury.

Background: Acute kidney injury (AKI) is a sudden episode of kidney failure which is frequently observed at intensive care units and related to high morbidity/mortality. Although AKI can have many different causes, ischemia-reperfusion (IR) injury is the main cause of AKI. Mechanistically, NADPH oxidases (NOXs) are involved in the pathophysiology contributing to oxidative stress following IR. Previous reports have indicated that knockout of NOX4 may offer protection in cardiac and brain IR, but there is currently less knowledge about how this could be exploited therapeutically and whether this could have significant protection in IR-induced AKI. Aim: To investigate the hypothesis that a novel and specific NOX4 inhibitor (GLX7013114) may have therapeutic potential on kidney and mitochondrial function in a mouse model of IR-induced AKI. Methods: Kidneys of male C57BL/6J mice were clamped for 20 min, and the NOX4 inhibitor (GLX7013114) was administered via osmotic minipump during reperfusion. Following 3 days of reperfusion, kidney function (i.e., glomerular filtration rate, GFR) was calculated from FITC-inulin clearance and mitochondrial function was assessed by high-resolution respirometry. Renal histopathological evaluations (i.e., hematoxylin-eosin) and TUNEL staining were performed for apoptotic evaluation. Results: NOX4 inhibition during reperfusion significantly improved kidney function, as evidenced by a better-maintained GFR (p < 0.05) and lower levels of blood urea nitrogen (p < 0.05) compared to untreated IR animals. Moreover, IR caused significant tubular injuries that were attenuated by simultaneous NOX4 inhibition (p < 0.01). In addition, the level of renal apoptosis was significantly reduced in IR animals with NOX4 inhibition (p < 0.05). These favorable effects of the NOX4 inhibitor were accompanied by enhanced Nrf2 Ser40 phosphorylation and conserved mitochondrial function, as evidenced by the better-preserved activity of all mitochondrial complexes. Conclusion: Specific NOX4 inhibition, at the time of reperfusion, significantly preserves mitochondrial and kidney function. These novel findings may have clinical implications for future treatments aimed at preventing AKI and related adverse events, especially in high-risk hospitalized patients.

LXA4 protected mice from renal ischemia/reperfusion injury by promoting IRG1/Nrf2 and IRAK-M-TRAF6 signal pathways

Excessive inflammatory response and increased oxidative stress play an essential role in the pathophysiology of ischemia/reperfusion (I/R)-induced acute kidney injury (IRI-AKI). Emerging evidence suggests that lipoxin A4 (LXA4), as an endogenous negative regulator in inflammation, can ameliorate several I/R injuries. However, the mechanisms and effects of LXA4 on IRI-AKI remain unknown. In this study, A bilateral renal I/R mouse model was used to evaluate the role of LXA4 in wild-type, IRG1 knockout, and IRAK-M knockout mice. Our results showed that LXA4, as well as 5-LOX and ALXR, were quickly induced, and subsequently decreased by renal I/R. LXA4 pretreatment improved renal I/R-induced renal function impairment and renal damage and inhibited inflammatory responses and oxidative stresses in mice kidneys. Notably, LXA4 inhibited I/R-induced the activation of TLR4 signal pathway including decreased phosphorylation of TAK1, p36, and p65, but did not affect TLR4 and p-IRAK-1. The analysis of transcriptomic sequencing data and immunoblotting suggested that innate immune signal molecules interleukin-1 receptor-associated kinase-M (IRAK-M) and immunoresponsive gene 1 (IRG1) might be the key targets of LXA4. Further, the knockout of IRG1 or IRAK-M abolished the beneficial effects of LXA4 on IRI-AKI. In addition, IRG1 deficiency reversed the up-regulation of IRAK-M by LXA4, while IRAK-M knockout had no impact on the IRG1 expression, indicating that IRAK-M is a downstream molecule of IRG1. Mechanistically, we found that LXA4-promoted IRG1-itaconate not only enhanced Nrf2 activation and increased HO-1 and NQO1, but also upregulated IRAK-M, which interacted with TRAF6 by competing with IRAK-1, resulting in deactivation of TLR4 downstream signal in IRI-AKI. These data suggested that LXA4 protected against IRI-AKI via promoting IRG1/Itaconate-Nrf2 and IRAK-M-TRAF6 signaling pathways, providing the rationale for a novel strategy for preventing and treating IRI-AKI.

Comprehensive overview of the role of mitochondrial dysfunction in the pathogenesis of acute kidney ischemia-reperfusion injury: a narrative review.

Acute kidney ischemia-reperfusion (IR) injury is a life-threatening condition that predisposes individuals to chronic kidney disease. Since the kidney is one of the most energy-demanding organs in the human body and mitochondria are the powerhouse of cells, mitochondrial dysfunction plays a central role in the pathogenesis of IR-induced acute kidney injury. Mitochondrial dysfunction causes a reduction in adenosine triphosphate production, loss of mitochondrial dynamics (represented by persistent fragmentation), and impaired mitophagy. Furthermore, the pathological accumulation of succinate resulting from fumarate reduction under oxygen deprivation (ischemia) in the reverse flux of the Krebs cycle can eventually lead to a burst of reactive oxygen species driven by reverse electron transfer during the reperfusion phase. Accumulating evidence indicates that improving mitochondrial function, biogenesis, and dynamics, and normalizing metabolic reprogramming within the mitochondria have the potential to preserve kidney function during IR injury and prevent progression to chronic kidney disease. In this review, we summarize recent advances in understanding the detrimental role of metabolic reprogramming and mitochondrial dysfunction in IR injury and explore potential therapeutic strategies for treating kidney IR injury.

Melatonin Reduces Aggravation of Renal Ischemia-Reperfusion Injury in Obese Rats by Maintaining Mitochondrial Homeostasis and Integrity through AMPK/PGC-1α/SIRT3/SOD2 Activation.

This study examined the potential benefits of melatonin against renal ischemia and reperfusion (IR) injury in obesity and explored the underlying mechanisms. Obesity was induced in Wistar rats by feeding a high-fat diet for 16 weeks. Three obese groups that underwent renal IR induction (30-min renal ischemia followed by 24-h reperfusion) were randomly assigned to receive melatonin at ischemic onset, reperfusion onset, or pretreatment for 4 weeks before IR induction. Groups of vehicle-treated obese and normal-diet-fed rats that underwent sham or IR induction were also included in the study. The results showed that renal functional and structural impairments after IR incidence were aggravated in obese rats compared to normal-diet-fed rats. The obese-IR rats also exhibited oxidative stress, mitochondrial dysfunction, apoptosis, and mitochondrial dynamics and mitophagy imbalances, which were all considerably improved upon melatonin treatment, irrespective of the treatment time. This study suggests the prophylactic and therapeutic efficacy of melatonin in IR-induced acute kidney injury (AKI) in obese individuals, which may improve the prognosis of AKI in these populations. The benefits of melatonin are likely mediated by the modification of various signaling molecules within the mitochondria that maintain mitochondrial redox balance and lead to the protection of mitochondrial homeostasis and integrity.

Inhibition of ACSF2 protects against renal ischemia/reperfusion injury via mediating mitophagy in proximal tubular cells.

Acute kidney injury (AKI) is a prevalent clinical condition caused by sepsis and ischemia reperfusion (IR) injury. The principal driver of IR-induced AKI involves renal tubular structural changes triggered by the impairment of function in renal tubular cells. The target gene, Acyl-CoA Synthetase Family Member 2 (ACSF2), was retrieved from the GEO database based on high specific expression in renal tubular cells and location in mitochondria. Here, we substantiate that ACSF2 is specifically localized in the mitochondria of the renal tubular epithelium. Functionally silencing ACSF2 in HK2 cells enhanced hypoxia-reoxygenation (HR)-induced mitophagy, restored mitochondrial function and decreased the production of mitochondrial superoxide. Our study demonstrated that these effects were reversed by silencing Bcl-2 19-kDa interacting protein 3 (BNIP3), a receptor regulating mitophagy. In vivo, ACSF2 knockdown significantly enhanced IR-induced mitophagy and improved renal function in mice with IR injury. Conversely, BNIP3 knockdown inhibited mitophagy and exacerbated renal damage in ACSF2-knockdown mice with IR injury. In conclusion, our study demonstrated that inhibition of ACSF2 enhances mitophagy, restoring mitochondrial function and protects against IR-induced AKI, providing a new target and potential strategy for therapy.

Sufentanil promotes autophagy and improves ischemia-reperfusion-induced acute kidney injury via up-regulating microRNA-145.

Sufentanil has a good protective effect on myocardial and liver injury caused by ischemia reperfusion (IR), but its protective effect on kidney is still unclear. This study aims to investigate whether sufentanil can prevent IR-induced acute kidney injury (AKI) and to determine whether its efficacy is related to miR-145-mediated autophagy. A total of 40 rats were randomly divided into 5 groups (n=8 in each group): A sham group, an IR group, a sufentanil group, a sufentanil+miR-145 inhibitor control group (an anti-NC group) and a sufentanil+miR-145 inhibitor group (an anti-miR-145 group). Except for the sham group, the other groups established a rat AKI model induced by IR. The sufentanil group, the sufentanil+anti-NC group, and the sufentanil+anti-miR-145 were injected with sufentanil (1 μg/kg) through femoral vein 30 min before ischemia. The sufentanil+anti-NC group and the sufentanil+anti-miR-145 group were injected with miR-145 inhibitor control or anti-miR-145 (80 mg/kg) through the tail vein before sufentanil pretreatment. The structure and function of kidneys harvested from the rats were evaluated, and the protein levels of autophagy-related proteins, oxidative stress levels, and apoptosis levels were measured. Compared with the IR group, the renal structure and function were improved in the sufentanil group. The levels of blood urea nitrogen (BUN), creatinine (Cr), urinary kidney injury molecule 1 (KIM-1), neutrophil gelatinase related lipid transporter (NGAL), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and ROS were significantly decreased (all P<0.05). In addition, compared with the IR group, the levels of Beclin-1 and LC3 in renal tissues in the sufentanil group were significantly increased (both P<0.05), and the apoptosis in renal tissues was significantly reduced (P<0.05). Compared with the sufentanil+anti-NC group, the levels of BUN, Cr, KIM-1, NGAL, TNF-α, IL-1β, IL-6 and ROS in the sufentanil+anti-miR-145 group were significantly increased (all P<0.05), the levels of Beclin-1 and LC3 in renal tissues were significantly decreased (both P<0.05), and the apoptosis in renal tissues was significantly increased (P<0.05). Sufentanil can prevent the AKI induced by IR, which is related to the up-regulation of miR-145-mediated autophagy.

Disulfiram ameliorates ischemia/reperfusion-induced acute kidney injury by suppressing the caspase-11-GSDMD pathway

Acute kidney injury (AKI) is a serious condition with high mortality. The most common cause is kidney ischemia/reperfusion (IR) injury, which is thought to be closely related to pyroptosis. Disulfiram is a well-known alcohol abuse drug, and recent studies have shown its ability to mitigate pyroptosis in mouse macrophages. This study investigated whether disulfiram could improve IR-induced AKI and elucidated the possible molecular mechanism. We generated an IR model in mouse kidneys and a hypoxia/reoxygenation (HR) injury model with murine tubular epithelial cells (MTECs). The results showed that IR caused renal dysfunction in mice and triggered pyroptosis in renal tubular epithelial cells, and disulfiram improved renal impairment after IR. The expression of proteins associated with the classical pyroptosis pathway (Nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3), apoptosis-related specific protein (ASC), caspase-1, N-GSDMD) and nonclassical pyroptosis pathway (caspase-11, N-GSDMD) were upregulated after IR. Disulfiram blocked the upregulation of nonclassical but not all classical pyroptosis pathway proteins (NLRP3 and ASC), suggesting that disulfiram might reduce pyroptosis by inhibiting the caspase-11-GSDMD pathway. In vitro, HR increased intracellular ROS levels, the positive rate of PI staining and LDH levels in MTECs, all of which were reversed by disulfiram pretreatment. Furthermore, we performed a computer simulation of the TIR domain of TLR4 using homology modeling and identified a small molecular binding energy between disulfiram and the TIR domain. We concluded that disulfiram might inhibit pyroptosis by antagonizing TLR4 and inhibiting the caspase-11-GSDMD pathway.

Methyl eugenol protects the kidney from oxidative damage in mice by blocking the Nrf2 nuclear export signal through activation of the AMPK/GSK3β axis.

Disrupted redox homeostasis contributes to renal ischemia-reperfusion (IR) injury. Abundant natural products can activate nuclear factor erythroid-2-related factor 2 (Nrf2), thereby providing therapeutic benefits. Methyl eugenol (ME), an analog of the phenolic compound eugenol, has the ability to induce Nrf2 activity. In this study, we investigated the protective effects of ME against renal oxidative damage in vivo and in vitro. An IR-induced acute kidney injury (AKI) model was established in mice. ME (20 mg·kg-1·d-1, i.p.) was administered to mice on 5 consecutive days before IR surgery. We showed that ME administration significantly attenuated renal destruction, improved the survival rate, reduced excessive oxidative stress and inhibited mitochondrial lesions in AKI mice. We further demonstrated that ME administration significantly enhanced Nrf2 activity and increased the expression of downstream antioxidative molecules. Similar results were observed in vitro in hypoxia/reoxygenation (HR)-exposed proximal tubule epithelial cells following pretreatment with ME (40 μmol·L-1). In both renal oxidative damage models, ME induced Nrf2 nuclear retention in tubular cells. Using specific inhibitors (CC and DIF-3) and molecular docking, we demonstrated that ME bound to the binding pocket of AMPK with high affinity and activated the AMPK/GSK3β axis, which in turn blocked the Nrf2 nuclear export signal. In addition, ME alleviated the development of renal fibrosis induced by nonfatal IR, which is frequently encountered in the clinic. In conclusion, we demonstrate that ME modulates the AMPK/GSK3β axis to regulate the cytoplasmic-nuclear translocation of Nrf2, resulting in Nrf2 nuclear retention and thereby enhancing antioxidant target gene transcription that protects the kidney from oxidative damage.

A bioinspired carbon monoxide delivery system prevents acute kidney injury and the progression to chronic kidney disease

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-β1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.

LncRNA IRAR regulates chemokines production in tubular epithelial cells thus promoting kidney ischemia-reperfusion injury

Increasing evidence demonstrates that long noncoding RNAs (lncRNAs) play an important role in several pathogenic processes of the kidney. However, functions of lncRNAs in ischemic acute kidney injury (AKI) remain undefined. In this study, global lncRNA profiling indicated that many lncRNA transcripts were deregulated in kidney after ischemia reperfusion (IR). Among them, we identified IRAR (ischemia-reperfusion injury associated RNA) as a potential lncRNA candidate, which was mostly expressed by the tubular epithelial cells (TECs) after IR, involved in the development of AKI. GapmeR-mediated silencing and viral-based overexpression of IRAR were carried out to assess its function and contribution to IR-induced AKI. The results revealed that in vivo silencing of IRAR significantly reduced IR-induced proinflammatory cells infiltration and AKI. IRAR overexpression induced chemokine CCL2, CXCL1 and CXCL2 expression both in mRNA and protein levels in TECs, while, silencing of IRAR resulted in downregulation of these chemokines. RNA immunoprecipitation and RNA pulldown assay validated the association between IRAR and CCL2, CXCL1/2. Further examination revealed that specific ablation of CCL2 in TECs reduced macrophages infiltration and proinflammatory cytokine production, attenuated renal dysfunction in IR mice. Inhibition of CXC chemokine receptor 2 (receptor of CXCL1/2) reduced neutrofils infiltration, but had no overt effect on kidney function. To explore the mechanism of IRAR upregulation in kidney during IR, we analyzed promoter region of IRAR and predicted a potential binding site for transcription factor C/EBP β on IRAR promoter. Silencing of C/EBP β reduced IRAR expression in TECs. A dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) confirmed that IRAR was a transcriptional target of the C/EBP β. Altogether, our findings identify IRAR as a new player in the development of ischemic AKI through regulating chemokine production and immune cells infiltration, suggesting that IRAR is a potential target for prevention and/or attenuation of AKI.

Sex Difference in Autoregulatory Responses of Juxtamedullary Afferent Arterioles Following Ischemia‐Reperfusion in Rats

Renal ischemia‐reperfusion (IR) is a major cause of acute kidney injury (AKI). Renal autoregulation protects the kidney from ischemia at low arterial pressure and hyperperfusion at high arterial pressure. Female rats are more protected against AKI than male rats. We reported that IR impaired autoregulatory behavior of afferent arterioles (AA) in male rats 24 hours and 1‐week post‐IR. We hypothesize that AA autoregulatory control in IR‐induced AKI (IR‐AKI) is better preserved in female rats compared to males. IR was induced in female rats by bilateral renal arterial clamping for 60 minutes followed by reperfusion for 24 hours or 1 week. Autoregulatory responses were assessed using the in vitro blood‐perfused juxtamedullary nephron preparation during step increases in perfusion pressure (PP) from 65 to 170 mmHg at 15 mmHg intervals in sham‐operated and IR rats 24 hours and 1‐week post‐IR. AA from sham rats exhibited pressure‐dependent vasoreactivity. Baseline AA diameter averaged 12.1±1.4 µm in shams (n=3) 24 hours post‐surgery. Diameter increased to 112±2% of baseline when PP decreased from 100 to 65 mmHg and decreased to 70±6% of baseline when PP increased to 170 mmHg (P&lt;0.05), indicating intact autoregulation. Conversely, baseline diameter in IR rats (n=5) averaged 10.0±1.4 µm and remained relatively unaltered during PP changes averaging between 98‐102% (P&gt;0.05) of baseline, indicating impaired autoregulation. Shams 1 week post‐IR (n=3) exhibited autoregulatory responses similar to 24‐hour shams. Intriguingly, autoregulatory responses normalized within 1 week in female IR rats (n=3) though these rats still maintained increased urine output over 1 week (P&lt;0.05). Diameter increased to 110±4% of baseline at 65 mmHg and decreased to 82±4% at 170 mmHg (P&lt;0.05) of baseline (9.4±1.2 µm, n=3). mRNA expression of NADPH oxidase (NOX) subunits and the inflammatory chemokine (C‐C motif) Ligand 2 (CCL2) were measured in isolated preglomerular microvessels (PGMV, n=4‐8/group). Only p67phox expression in PGMV increased in female IR rats 24 hours post‐IR compared to elevation of all three of the NOX subunits (gp91phox, p67phox, and p47phox) to male IR rats. IR significantly increased CCL2 mRNA expression in female PGMV 7‐fold and 3‐fold at 24 hours and 1‐week post‐IR, respectively, compared to shams (P&lt;0.05). mRNA expression increased less in females than males (9‐fold at 24 hours and 6‐fold at 1 week after IR, respectively). In conclusion, autoregulatory behavior in female rats is significantly blunted, 24 hours post‐IR and normalized 1‐week post‐IR, in contrast, all male IR rats exhibited blunted autoregulatory behavior 24 hours and 1‐week post‐IR. Increased CCL2 mRNA expression parallels the dimorphism of AA autoregulatory reactivity following IR, suggesting that elevated CCL2 could mediate IR‐induced AA autoregulatory dysfunction. Overall, these results suggest a sexual dimorphism in the renal microvascular response to IR injury and that female rats may possess innate protection against IR‐AKI.

Meprin β Regulates Protein Kinase A Mediated TGF‐β Signaling in Ischemia Reperfusion‐Induced Kidney Injury

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and is associated with high morbidity and mortality rates. The underlying mechanisms involved in IR‐induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically processing the catalytic subunit of protein kinase A (PKA‐C) and several mediators of inflammation. The protein kinase A (PKA) signaling pathway plays an important role in renal fibrosis, promoting the production of transforming growth factor‐β (TGF‐β) and/or TGF‐β‐dependent molecules. The TGF‐β signaling is involved in wound healing and tissue repair of injured kidneys. Activation of Smad2 and Smad3 is the major downstream event of the TGF‐β signaling pathway. The objective of the current study was to determine how meprin β expression impacts PKA C‐mediated TGF‐β signaling in IR‐induced kidney injury. To this end, we used surgical procedures for to achieve unilateral IR with contralateral nephrectomy in wild‐type (WT) and meprin β knockout (βKO) mice. Meprin βKO mice are deficient in the meprin B (β‐β) and the heterodimeric isoform of meprin A (α‐β). The mice were sacrificed at 96 h post‐IR and kidney tissues obtained for analysis. Real‐time PCR, and immunohistochemical staining, were used to evaluate the expression levels of PKA C and TGF‐β. Statistical analysis utilized 2‐way ANOVA. Real‐time PCR data showed significant increases in mRNA levels of PKA C only in βKO kidneys after IR (p&lt; 0.05). In contrast, the mRNA levels for TGF‐β were higher in both WT and βKO at 96 h post‐IR (p&lt; 0.01). Interestingly, the baseline mRNA levels for TGF‐β were higher in meprin βKO mice (p&lt; 0.001). Immunohistochemical staining for PKA C and TGF‐β was quantified using Image J software. The data showed significant increases in the levels of PKA C proteins in select kidney tubules of both genotypes (p≤ 0.001) after IR. Similarly, staining intensity for TGF‐β were higher in select kidney tubules of both WT (p=0.01) and meprin β deficient mice at 96 h IR (p=0.001). Immunofluorescence counterstaining of kidney tissues with anti‐meprin β, anti‐PKA C and anti‐TGF‐β antibodies showed a positive correlation between meprin β expression and tubular PKA C and TGF‐β levels in kidneys subjected to IR. These findings suggest that meprin β regulates PKA C‐mediated TGF‐β signaling pathway in IR‐induced kidney injury and provides new insights on the mechanisms underlying meprin β modulation of the pathophysiology of IR‐induced renal injury.

Meprin β mediates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) causes acute kidney injury (AKI) and is associated with high morbidity and mortality rates. Meprins metalloproteases are expressed abundantly in the brush border membranes of proximal kidney tubules and implicated in the progression of AKI. However, the underlying mechanisms are not fully understood. Meprins are capable to proteolytically processing osteopontin (OPN), a highly phosphorylated multifunctional and matricellular glycophosphoprotein. OPN mediates cell signaling pathways involved in apoptosis and extracellular matrix metabolism. The objective of the present study was to determine the effect of meprin β expression on OPN and downstream mediators of the OPN‐signaling pathway in IR‐induced renal injury. Surgical procedures were used to induce unilateral IR in wild‐type (WT) and meprin β knockout (βKO) mice. The mice were sacrificed at 24h post‐IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real‐time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase‐3, Bcl‐2, and NFκB. Statistical analysis utilized 2‐way ANOVA. Our data show that NGAL levels were increased significantly in the kidney of WT (P=0.0001) and βKO (P=0.01) mice at 24h post‐IR when compared to their control counterparts. OPN mRNA increased in both genotypes at 24h post‐IR. Western blot analysis detected a full length OPN protein band at 66 kDa, with significant increases (p=0.001) in protein levels in WT kidney at 24h post‐IR when compared to levels in control kidneys. This increase in OPN was not observed in meprin βKO mice subjected to IR. Additionally, we detected a cleaved OPN fragment at 32 kDa, for which there were no significant changes in either genotype at 24h IR post‐IR. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules and in renal corpuscles. To determine whether meprin β expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in both proximal and distal kidney tubules. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is partly responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti‐apoptotic protein in different diseases, therefore we evaluated its impact on caspase‐3, Bcl‐2, and NFκB. The mRNA levels for caspase‐3, Bcl‐2 and NFκB decreased in WT but increased in βKO mice at 24h post‐IR. Western blot detected higher levels of caspase‐3 and Bcl‐2 only in WT kidneys. Immunohistochemical staining for caspase‐3, Bcl‐2 and NFκB also showed higher levels in tubules and renal corpuscle of WT kidneys when compared to βKO kidneys at 24h post‐IR. Taken together, our data show that meprin β regulates OPN and down‐stream mediators of OPN‐signaling pathway in IR‐induced AKI.

Meprin β Modulates Cellular Proliferation Through The IL‐6 Signaling Pathway In IRInduced Kidney Injury

Inflammation plays a central role in the progression of kidney injury induced by ischemia/reperfusion (IR). Interleukin‐6 (IL‐6) trans‐signaling has been shown to play a protective role by promoting repair processes in IR‐induced renal injury. Meprin metalloproteases have been implicated in inflammation‐induced kidney injury. Meprins proteolytically process the proinflammatory cytokine, IL‐6, causing IL‐6 inactivation. It was recently reported that meprins also cleave the IL‐6 receptor (IL‐6R). IL‐6 binds to its receptor (IL‐6R) in two ways, membrane bound (mbIL‐6R), activating the classic IL‐6 signaling pathway, or the soluble form (sIL‐6R), activating the IL‐6 trans‐signaling pathway. IL‐6 trans‐signaling induces proliferation through either MAPK/ERK or PI3K/AKT pathway or in crosstalk with AKT/ERK. We previously showed that meprin β modulates cellular survival (BCL‐2) through IL‐6/JAK/STAT signaling pathway in IRinduced kidney injury. However, it’s not known how meprin β modulation of the IL‐6 signaling pathway impacts the cellular proliferation in IR‐induced acute kidney injury. Previous studies have shown that IL‐6 binds to its receptor forming IL‐6/IL6R complex, which binds to the membranebound gp130 dimer. This leads to activation phosphorylation of ERK and AKT pathways, which in turn leads to induction of cellular proliferation. PCNA is a cellular proliferation marker that is induced through activation of the IL‐6 signaling pathway. The goal of the current study was to determine how meprin β modulation of the IL‐6 signaling pathway impacts downstream cellular proliferation in IR‐induced kidney injury. We used the unilateral IR as a model of renal inflammation in wild‐type (WT) and meprin β knockout (βKO) male mice, with the contralateral kidneys serving as controls. The mice were sacrificed at 96 h post‐IR, and kidney tissue processed for evaluation by RT‐PCR and immunohistochemistry (IHC). To determine staining intensity, the tissue sections were evaluated for IL‐6 and PCNA levels using light microscopy and imaged using Image J analysis Software. Statistical analysis of the optical density (OD) data utilized two‐way ANOVA. Our PCR data showed a significant increase in mRNA levels for IL‐6 and PCNA in WT and βKO mice (P ≤ 0.01) at 96 h‐post IR when compared to WT control kidneys. However, the baseline mRNA levels for PCNA were significantly higher in βKO (P ≤ 0.01) when compared to WT kidneys. Immunohistochemical data showed significant increases (P&lt;0.05) in IL‐6 and PCNA in select tubules in both genotypes at 96 h post‐IR when compared to control kidneys for each genotype. Data from immunofluorescence counterstaining of kidney tissues showed that the levels of IL‐6, and PCNA were higher in meprin β‐expressing proximal tubules (PTs), at 96 h post‐IR when compared to the distal kidney tubules (DTs), which lack meprins. High levels of IL‐6 were also present in the lumen of PTs and DTs from WT and βKO kidneys at 96 h post‐IR, suggesting increased release into filtrate and subsequently into urine. However, high levels of PCNA were present in the lumen of PTs only from both genotypes at 96 h post‐IR. In conclusion, our data shows that meprin β expression modulates cellular proliferation through the IL‐6 signaling pathway in IR‐induced kidney injury.

The Role of the Superior Cervical Sympathetic Ganglion in Ischemia Reperfusion-Induced Acute Kidney Injury in Rats.

Acute kidney injury (AKI) has been found to be a serious clinical problem with high morbidity and mortality, and is associated with acute inflammatory response and sympathetic activation that subsequently play an important role in the development of AKI. It is well known that the sympathetic nervous system (SNS) and immune system intensely interact and mutually control each other in order to maintain homeostasis in response to stress or injury. Evidence has shown that the superior cervical sympathetic ganglion (SCG) participates in the bidirectional network between the immune and the SNS, and that the superior cervical ganglionectomy has protective effect on myocardial infarction, however, the role of the SCG in the setting of renal ischemic reperfusion injury has not been studied. Here, we sought to determine whether or not the SCG modulates renal ischemic reperfusion (IR) injury in rats. Our results showed that bilateral superior cervical ganglionectomy (SCGx) 14 days before IR injury markedly reduced the norepinephrine (NE) in plasma, and down-regulated the increased expression of tyrosine hydroxylase (TH) in the kidney and hypothalamus. Sympathetic denervation by SCGx in the AKI group increased the level of blood urea nitrogen (BUN) and kidney injury molecule-1 (KIM-1), and exacerbated renal pathological damage. Sympathetic denervation by SCGx in the AKI group enhanced the expression of pro-inflammatory cytokines in plasma, kidney and hypothalamus, and increased levels of Bax in denervated rats with IR injury. In addition, the levels of purinergic receptors, P2X3R and P2X7R, in the spinal cord were up-regulated in the denervated rats of the IR group. In conclusion, these results demonstrate that the sympathetic denervation by SCGx aggravated IR-induced AKI in rats via enhancing the inflammatory response, thus, the activated purinergic signaling in the spinal cord might be the potential mechanism in the aggravated renal injury.

Maslinic Acid Attenuates Ischemia/Reperfusion-Induced Acute Kidney Injury by Suppressing Inflammation and Apoptosis Through Inhibiting NF-κB and MAPK Signaling Pathway.

Inflammation and apoptosis are the major contributors to the mechanisms of acute kidney injury (AKI) due to renal ischemia-reperfusion injury (IRI). Maslinic acid (MA), a pentacyclic triterpene acid mostly found in dietary plants, the current study was to demonstrate the renoprotective effect of MA on IRI-induced AKI, and to investigate the role of inflammation and apoptosis-related signaling pathways as a molecular mechanism. C57BL/6J mice were subjected to IRI for 72 h, and MA was daily administered by intraperitoneal injection during this period. In parallel, rat renal proximal tubule cells (NRK52E) were prophylactically treated with MA and then exposed to hydrogen peroxide (H2O2). MA treatment significantly inhibited the mRNA expression of interleukin (IL-1β), tumor necrosis factor-α (TGF-α), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1(ICAM-1). Also, MA reduced the expression of Bax/Bcl2 ratio and cleaved caspase-3. In NRK52 cells, MA inhibited the IκBα degradation, blocked NF-κB/p65 phosphorylation, and nuclear translocation. The phosphorylation of ERK, JNK, and p38 was attenuated by MA in IRI-induced kidney injury and H2O2-stimulated NRK52 cells. The expression levels of IL-1β, MCP-1, and ICAM-1 were upregulated in H2O2-stimulated NRK52E cells, which was attenuated by NF-κB inhibitor. H2O2 treatment increased the Bax/Bcl2 ratio and cleaved caspase-3 in NRK52E cells, which was counteracted by MAPK inhibitors. Together, our data demonstrate that MA suppresses IR-induced AKI injury through NF-κB and MAPK signaling pathways and that MA is a promising agent in the treatment of kidney diseases.

Roxadustat (FG-4592) protects against ischaemia/reperfusion-induced acute kidney injury through inhibiting the mitochondrial damage pathway in mice.

Ischaemia-reperfusion (I/R) is one of the main factors of acute kidney injury (AKI). mitochondrial damage pathway are important features of I/R induced-acute kidney injury (IRI-AKI). Hypoxia-inducible factor (HIF) expression in renal tubule segments is up-regulated during AKI. Herein, we investigated the role of FG-4592 in a mouse model of IRI-AKI to confirm whether FG-4592 is beneficial in AKI. We found that pretreatment with FG-4592significantly ameliorated renal function and renal histological damage in mice after IRI. Furthermore, these results suggest that pretreatment with FG-4592significantly reduced the tubular cells apoptosis (decreased TUNEL-positive cells, Bax, caspase12levels), attenuated mitochondrial damage (increased ATPβ, PPARγ, mitochondrial DNA copy number, and decreased cytoplasmic cytochrome C), and alleviated DNA damage after IRI. In conclusion, pretreatment with FG-4592may effectively prevent kidney from IRI possibly by via diminishing tubular cells injuries and protection of mitochondrial damage pathway. These results further validate that FG-4592may be an effective drug in the clinical treatment of IRI-AKI.

Immunomodulatory Effects of Mesenchymal Stem Cells on Drug-Induced Acute Kidney Injury.

Drug-induced nephrotoxicity is an important and increasing cause of acute kidney injury (AKI), which accounts for approximately 20% of hospitalized patients. Previous reviews studies on immunity and AKI focused mainly on ischemia-reperfusion (IR), whereas no systematic review addressing drug-induced AKI and its related immune mechanisms is available. Recent studies have provided a deeper understanding on the mechanisms of drug-induced AKI, among which acute tubular interstitial injury induced by the breakdown of innate immunity was reported to play an important role. Emerging research on mesenchymal stem cell (MSC) therapy has revealed its potential as treatment for drug-induced AKI. MSCs can inhibit kidney damage by regulating the innate immune balance, promoting kidney repair, and preventing kidney fibrosis. However, it is important to note that there are various sources of MSCs, which impacts on the immunomodulatory ability of the cells. This review aims to address the immune pathogenesis of drug-induced AKI versus that of IR-induced AKI, and to explore the immunomodulatory effects and therapeutic potential of MSCs for drug-induced AKI.

Meprin β Regulates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and associated with high morbidity and mortality rate. The underlying mechanisms involved in IR-induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically process several mediators of cell signaling pathways mediators involved in apoptosis and extracellular matrix (ECM) metabolism such as osteopontin (OPN). The objective of the current study was to determine how meprin β expression affects OPN and downstream mediators of the OPN-signaling pathway. Surgical procedures were used to induce unilateral IR with contralateral nephrectomy in wild-type (WT) and meprin β knockout (βKO) mice which are deficient in the meprin B (β-β) and the heteromeric isoform of meprin A (α-β). The mice were sacrificed at 24 h post-IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real-time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase-3, Bcl-2, and NFκB. Statistical analysis utilized 2-way ANOVA. Our data show NGAL level were elevated significantly in WT (P=0.0001) and βKO (P=0.01) at 24 h post-IR compare to their control counterparts. However, the fold change was higher in WT when compared to βKO mice indicating that meprin β enhances kidney injury. OPN mRNA increased in both genotypes at 24 h post-IR. In contrast, OPN protein levels only increased in WT kidneys. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules. To determine whether meprins expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in distal tubules (DTs), which do not express meprins. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti-apoptotic molecules in different diseases, therefore we evaluated its impact on caspase-3, Bcl-2, and NFκB. The mRNA levels for caspase-3, Bcl-2 and NFκB decreased in WT but increased in βKO mice at 24 h post-IR. Immunohistochemical staining for caspase-3, Bcl-2 and NFκB also showed high staining levels in tubules of WT kidney compared to βKO at 24 h post-IR. These findings suggest that meprin β expression has a direct impact on the levels of OPN in the kidney and affects apoptotic genes regulated by the OPN signaling pathway.