Interleukin-19 Mediates Tissue Damage in Murine Ischemic Acute Kidney Injury

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl₂-induced AKI animal models, real-time quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-β1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-α and IL-10 in cultured HepG2 cells, and it increased IL-1β and TNF-α expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl₂ treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.     

Renoprotective effect of paricalcitol via a modulation of the TLR4-NF-κB pathway in ischemia/reperfusion-induced acute kidney injury

Background

The pathophysiology of ischemic acute kidney injury (AKI) is thought to include a complex interplay between vascular endothelial cell dysfunction, inflammation, and tubular cell damage. Several lines of evidence suggest a potential anti-inflammatory effect of vitamin D in various kidney injury models. In this study, we investigated the effect of paricalcitol, a synthetic vitamin D analog, on renal inflammation in a mouse model of ischemia/reperfusion (I/R) induced acute kidney injury (AKI).

Methods

Paricalcitol was administered via intraperitoneal (IP) injection at 24 h before ischemia, and then I/R was performed through bilateral clamping of the renal pedicles. Twenty-four hours after I/R, mice were sacrificed for the evaluation of injury and inflammation. Additionally, an in vitro experiment using HK-2 cells was also performed to examine the direct effect of paricalcitol on tubular cells.

Results

Pre-treatment with paricalcitol attenuated functional deterioration and histological damage in I/R induced AKI, and significantly decreased tissue neutrophil and macrophage infiltration and the levels of chemokines, the pro-inflammatory cytokine interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). It also decreased IR-induced upregulation of Toll-like receptor 4 (TLR4), and nuclear translocation of p65 subunit of NF-κB. Results from the in vitro study showed pre-treatment with paricalcitol suppressed the TNF-α-induced depletion of cytosolic IκB in HK-2 cells.

Conclusion

These results demonstrate that pre-treatment with paricalcitol has a renoprotective effect in ischemic AKI, possibly by suppressing TLR4-NF-κB mediated inflammation.     

Deficiency for the Chemokine Monocyte Chemoattractant Protein-1 Aggravates Tubular Damage after Renal Ischemia/Reperfusion Injury

Temporal expression of chemokines is a crucial factor in the regulation of renal ischemia/reperfusion (I/R) injury and repair. Beside their role in the migration and activation of inflammatory cells to sites of injury, chemokines are also involved in other processes such as angiogenesis, development and migration of stem cells. In the present study we investigated the role of the chemokine MCP-1 (monocyte chemoattractant protein-1 or CCL2), the main chemoattractant for monocytes, during renal I/R injury. MCP-1 expression peaks several days after inducing renal I/R injury coinciding with macrophage accumulation. However, MCP-1 deficient mice had a significant decreased survival and increased renal damage within the first two days, i.e. the acute inflammatory response, after renal I/R injury with no evidence of altered macrophage accumulation. Kidneys and primary tubular epithelial cells from MCP-1 deficient mice showed increased apoptosis after ischemia. Taken together, MCP-1 protects the kidney during the acute inflammatory response following renal I/R injury.     

PTEN alleviates maladaptive repair of renal tubular epithelial cells by restoring CHMP2A-mediated phagosome closure

Phosphatase and Tensin Homolog on chromosome Ten (PTEN) has emerged as a key protein that governs the response to kidney injury. Notably, renal adaptive repair is important for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. To test the role of PTEN in renal repair after acute injury, we constructed a mouse model that overexpresses PTEN in renal proximal tubular cells (RPTC) by crossing PTEN^fl-stop-fl mice with Ggt1-Cre mice. Mass spectrometry-based proteomics was performed after subjecting these mice to ischemia/reperfusion (I/R). We found that PTEN was downregulated in renal tubular cells in mice and cultured HK-2 cells subjected to renal maladaptive repair induced by I/R. Renal expression of PTEN negatively correlated with NGAL and fibrotic markers. RPTC-specific PTEN overexpression relieved I/R-induced maladaptive repair, as indicated by alleviative tubular cell damage, apoptosis, and subsequent renal fibrosis. Mass spectrometry analysis revealed that differentially expressed proteins in RPTC-specific PTEN overexpression mice subjected to I/R were significantly enriched in phagosome, PI3K/Akt, and HIF-1 signaling pathway and found significant upregulation of CHMP2A, an autophagy-related protein. PTEN deficiency downregulated CHMP2A and inhibited phagosome closure and autolysosome formation, which aggravated cell injury and apoptosis after I/R. PTEN overexpression had the opposite effect. Notably, the beneficial effect of PTEN overexpression on autophagy flux and cell damage was abolished when CHMP2A was silenced. Collectively, our study suggests that PTEN relieved renal maladaptive repair in terms of cell damage, apoptosis, and renal fibrosis by upregulating CHMP2A-mediated phagosome closure, suggesting that PTEN/CHMP2A may serve as a novel therapeutic target for the AKI to CKD transition.Subject terms: Macroautophagy, Kidney     

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1^−/− mice, the cellular NADPH/NADP⁺ ratio was significantly higher and NOX activity was markedly higher than in those of NQO1^+/+ mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP⁺ ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP⁺ modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.     

Protective Role of AMP-Activated Protein Kinase-Evoked Autophagy on an In Vitro Model of Ischemia/Reperfusion-Induced Renal Tubular Cell Injury

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.     

Cell Atavistic Transition: Paired Box 2 Re-Expression Occurs in Mature Tubular Epithelial Cells during Acute Kidney Injury and Is Regulated by Angiotensin II

The regeneration of tubular epithelial cells (TECs) after acute kidney injury (AKI) is crucial for the recovery of renal structure and function. The mechanism by which quiescent TECs re-obtain a potential to regenerate remains unknown. In this study, we observed a transient re-expression of embryonic gene Paired box 2 (Pax2) in adult rat TECs in vivo during ischemia-reperfusion induced AKI and most Pax2 positive TECs co-expressed kidney injury molecule-1 (KIM-1), a tubular injury marker. The re-expression of Pax2 was accompanied by increased levels of intrarenal Angiotensin II, which is a crucial injury factor of AKI. Furthermore, we also found a temporary re-expression of Pax2 in NRK-52E cells under the stimulation of Angiotensin II. This stimulatory effect could be blocked by PD123319 (Angiotensin II type 2 receptor (AT2R) inhibitor) and AG490 (Janus Kinase 2 (JAK2) inhibitor). As Pax2 is essential for the phenotypic conversion from mesenchymal stem cells to TECs during kidney development, we proposed that the re-expression of Pax2 in mature TECs may be an indicator of “atavistic” transition which mimics but reverses the processes of development of TECs. This could be proved by that a progenitor marker, CD24, was also found to be transiently expressed shortly after the expression of Pax2 in NRK-52E cells stimulated with Angiotensin II. The expression of CD24 was also suppressed by PD123319 and AG490. Moreover, knockdown of Pax2 by RNA interference could significantly reduce the expression of CD24 in NRK-52E cells stimulated with Angiotension II. Those findings suggest that mature TECs can trans-differentiate into progenitor-like cells by “atavistic transition”, which may participate in the recovery of tissue structure and Pax2 may play a pivotal role in this process. That might have important implications for further understanding of tubular regeneration after injury.     

Autophagy activation attenuates renal ischemia-reperfusion injury in rats

Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future.     

TRAF1 improves cisplatin-induced acute kidney injury via inhibition of inflammation and metabolic disorders

BackgroundCisplatin-induced acute kidney injury (AKI) is a severe clinical complication with no satisfactory therapies in the clinic. Tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) plays a vital role in both inflammation and metabolism. However, the TRAF1 effect in cisplatin induced AKI needs to be evaluated.MethodsWe observed the role of TRAF1 in eight-week-old male mice and mouse proximal tubular cells both treated with cisplatin by examining the indicators associated with kidney injury, apoptosis, inflammation, and metabolism.ResultsTRAF1 expression was decreased in cisplatin-treated mice and mouse proximal tubular cells (mPTCs), suggesting a potential role of TRAF1 in cisplatin-associated kidney injury. TRAF1 overexpression significantly alleviated cisplatin-triggered AKI and renal tubular injury, as demonstrated by reduced serum creatinine (Scr) and urea nitrogen (BUN) levels, as well as the ameliorated histological damage and inhibited upregulation of NGAL and KIM-1. Moreover, the NF-κB activation and inflammatory cytokine production enhanced by cisplatin were significantly blunted by TRAF1. Meanwhile, the increased number of apoptotic cells and enhanced expression of BAX and cleaved Caspase-3 were markedly decreased by TRAF1 overexpression both in vivo and vitro. Additionally, a significant correction of the metabolic disturbance, including perturbations in energy generation and lipid and amino acid metabolism, was observed in the cisplatin-treated mice kidneys.ConclusionTRAF1 overexpression obviously attenuated cisplatin-induced nephrotoxicity, possibly by correcting the impaired metabolism, inhibiting inflammation, and blocking apoptosis in renal tubular cells.General significanceThese observations emphasize the novel mechanisms associated to metabolism and inflammation of TRAF1 in cisplatin-induced kidney injury.     

Deficiency of apoptosis‐stimulating protein two of p53 ameliorates acute kidney injury induced by ischemia reperfusion in mice through upregulation of autophagy

Acute kidney injury (AKI) has become a common disorder with a high risk of morbidity and mortality, which remains major medical problem without reliable and effective therapeutic intervention. Apoptosis‐stimulating protein two of p53 (ASPP2) is a proapoptotic member that belongs to p53 binding protein family, which plays a key role in regulating apoptosis and cell growth. However, the role of ASPP2 in AKI has not been reported. To explore the role of ASPP2 in the progression of AKI, we prepared an AKI mouse model induced by ischaemia reperfusion (I/R) in wild‐type (ASPP2^+/+) mice and ASPP2 haploinsufficient (ASPP2^+/?) mice. The expression profile of ASPP2 were examined in wild‐type mice. The renal injury, inflammation response, cellular apoptosis and autophagic pathway was assessed in ASPP2^+/+ and ASPP2^+/? mice. The renal injury, inflammation response and cellular apoptosis was analysed in ASPP2^+/+ and ASPP2^+/? mice treated with 3‐methyladenine or vehicle. The expression profile of ASPP2 showed an increase at the early stage while a decrease at the late stage during renal injury. Compared with ASPP2^+/+ mice, ASPP2 deficiency protected mice against renal injury induced by I/R, which mainly exhibited in slighter histologic changes, lower levels of blood urea nitrogen and serum creatinine, and less apoptosis as well as inflammatory response. Furthermore, ASPP2 deficiency enhanced autophagic activity reflecting in the light chain 3‐II conversion and p62 degradation, while the inhibition of autophagy reversed the protective effect of ASPP2 deficiency on AKI. These data suggest that downregulation of ASPP2 can ameliorate AKI induced by I/R through activating autophagy, which may provide a novel therapeutic strage for AKI.     

Aberrant macrophages mediate defective kidney repair that triggers nephritis in lupus-susceptible mice

CSF-1, required for macrophage (M?) survival, proliferation, and activation, is upregulated in the tubular epithelial cells (TECs) during kidney inflammation. CSF-1 mediates M?-dependent destruction in lupus-susceptible mice with nephritis and, paradoxically, M?-dependent renal repair in lupus-resistant mice after transient ischemia/reperfusion injury (I/R). We now report that I/R leads to defective renal repair, nonresolving inflammation, and, in turn, early-onset lupus nephritis in preclinical MRL/MpJ-Faslpr/Fas(lpr) mice (MRL-Fas(lpr) mice). Moreover, defective renal repair is not unique to MRL-Fas(lpr) mice, as flawed healing is a feature of other lupus-susceptible mice (Sle 123) and MRL mice without the Fas(lpr) mutation. Increasing CSF-1 hastens renal healing after I/R in lupus-resistant mice but hinders healing, exacerbates nonresolving inflammation, and triggers more severe early-onset lupus nephritis in MRL-Fas(lpr) mice. Probing further, the time-related balance of M1 "destroyer" M? shifts toward the M2 "healer" phenotype in lupus-resistant mice after I/R, but M1 M? continue to dominate in MRL-Fas(lpr) mice. Moreover, hypoxic TECs release mediators, including CSF-1, that are responsible for stimulating the expansion of M1 M? inherently poised to destroy the kidney in MRL-Fas(lpr) mice. In conclusion, I/R induces CSF-1 in injured TECs that expands aberrant M? (M1 phenotype), mediating defective renal repair and nonresolving inflammation, and thereby hastens the onset of lupus nephritis.     

A1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury

Activation of A(1) adenosine receptors (ARs) protects against renal ischemia-reperfusion (I/R) injury by reducing necrosis, apoptosis, and inflammation. However, extrarenal side effects (bradycardia, hypotension, and sedation) may limit A(1)AR agonist therapy for ischemic acute kidney injury. Here, we hypothesized that an allosteric enhancer for A(1)AR (PD-81723) protects against renal I/R injury without the undesirable side effects of systemic A(1)AR activation by potentiating the cytoprotective effects of renal adenosine generated locally by ischemia. Pretreatment with PD-81723 produced dose-dependent protection against renal I/R injury in A(1)AR wild-type mice but not in A(1)AR-deficient mice. Significant reductions in renal tubular necrosis, neutrophil infiltration, and inflammation as well as tubular apoptosis were observed in A(1)AR wild-type mice treated with PD-81723. Furthermore, PD-81723 decreased apoptotic cell death in human proximal tubule (HK-2) cells in culture, which was attenuated by a specific A(1)AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 induced sphingosine kinase (SK)1 mRNA and protein expression in HK-2 cells and in the mouse kidney. Supporting a critical role of SK1 in A(1)AR allosteric enhancer-mediated renal protection against renal I/R injury, PD-81723 failed to protect SK1-deficient mice against renal I/R injury. Finally, proximal tubule sphingosine-1-phosphate type 1 receptors (S1P(1)Rs) are critical for PD-81723-induced renal protection, as mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(flox/flox) PEPCK(Cre/-) mice) were not protected against renal I/R injury with PD-81723 treatment. Taken together, our experiments demonstrate potent renal protection with PD-81723 against I/R injury by reducing necrosis, inflammation, and apoptosis through the induction of renal tubular SK1 and activation of proximal tubule S1P(1)Rs. Our findings imply that selectively enhancing A(1)AR activation by locally produced renal adenosine may be a clinically useful therapeutic option to attenuate ischemic acute kidney injury without systemic side effects.     

SAP130 released by damaged tubule drives necroinflammation via miRNA-219c/Mincle signaling in acute kidney injury

Tubules injury and immune cell activation are the common pathogenic mechanisms in acute kidney injury (AKI). However, the exact modes of immune cell activation following tubule damage are not fully understood. Here we uncovered that the release of cytoplasmic spliceosome associated protein 130 (SAP130) from the damaged tubular cells mediated necroinflammation by triggering macrophage activation via miRNA-219c(miR-219c)/Mincle-dependent mechanism in unilateral ureteral obstruction (UUO) and cisplatin-induced AKI mouse models, and in patients with acute tubule necrosis (ATN). In the AKI kidneys, we found that Mincle expression was tightly correlated to the necrotic tubular epithelial cells (TECs) with higher expression of SAP130, a damaged associated molecule pattern (DAMP), suggesting that SAP130 released from damaged tubular cells may trigger macrophage activation and necroinflammation. This was confirmed in vivo in which administration of SAP130-rich supernatant from dead TECs or recombinant SAP130 promoted Mincle expression and macrophage accumulation which became worsen with profound tubulointerstitial inflammation in LPS-primed Mincle WT mice but not in Mincle deficient mice. Further studies identified that Mincle was negatively regulated via miR-219c-3p in macrophages as miR-219c-3p bound Mincle 3′-UTR to inhibit Mincle translation. Besides, lentivirus-mediated renal miR-219c-3p overexpression blunted Mincle and proinflammatory cytokine expression as well as macrophage infiltration in the inflamed kidney of UUO mice. In conclusion, SAP130 is released by damaged tubules which elicit Mincle activation on macrophages and renal necroinflammation via the miR-219c-3p-dependent mechanism. Results from this study suggest that targeting miR-219c-3p/Mincle signaling may represent a novel therapy for AKI.Subject terms: Cell death and immune response, Acute kidney injury     

The impact of Caspase-1 deletion on apoptosis and acute kidney injury in a murine transplant model

BackgroundCaspase-1 knockout mice (Casp1KO) are protected from Acute Kidney Injury (AKI) after warm ischemia/reperfusion injury in non-transplant models. Since Caspase-1 plays a central role as an inflammatory response initiator, we hypothesized that Casp1KO mice would be protected from AKI following transplant.MethodsRenal tubular cells (RTECs) were subjected to cold storage and rewarming (CS/REW). C57Bl/6 J wild type or Casp1KO kidneys were subjected to CI for 30 min and then transplanted into wild type recipients (CI + Txp). The recipients underwent bilateral native nephrectomy at the time of transplant. Serum creatinine (sCr) was measured 24 h after native nephrectomy to assess transplant function.ResultsWe found that RTECs subjected to CS/REW had significantly increased expression of the Caspase-1 and inflammasome protein NLRP1. Wild type kidneys subjected to CI + Txp into wild type recipients also demonstrated significantly increased Caspase-1 and NLRP1 protein expression compared to kidneys transplanted from Casp1KO donors into wild type recipients. Caspase-1 deletion results in significantly decreased RTEC apoptosis in transplanted Casp1KO vs WT kidneys. Surprisingly, however, renal function, ATN scores including brush border injury, cast formation and tubular simplification were similar in both groups and not significantly different.ConclusionsOur data suggest that other triggers of inflammation and programmed necrosis may need to be inhibited in addition to attenuating Caspase-1 to fully prevent AKI after kidney transplant. Importantly, requirements may be distinct for AKI induced by transplantation as opposed to other transient models such as the clamp model of AKI.     

Increased Cellular Senescence and Vascular Rarefaction Exacerbate the Progression of Kidney Fibrosis in Aged Mice Following Transient Ischemic Injury

Recent findings indicate that elderly patients with acute kidney injury (AKI) have an increased incidence of progression to chronic kidney disease (CKD) due to incomplete recovery from an acute insult. In the current study, a co-morbid model of AKI was developed to better mimic the patient population and to investigate whether age exacerbates the fibrosis and inflammation that develop in the sequelae of progressive kidney disease following acute injury. Young (8–10 weeks) and aged (46–49 weeks) C57BL/6 mice were subjected to 30 min bilateral renal ischemia-reperfusion (I/R) to induce AKI. The aged animals have greater mortality and prolonged elevation of plasma creatinine correlating with less tubular epithelial cell proliferation compared to the young. Six weeks post-reperfusion, interstitial fibrosis is greater in aged kidneys based on picrosirius red staining and immunolocalization of cellular fibronectin, collagen III and collagen IV. Aged kidneys 6 weeks post-reperfusion also express higher levels of p53 and p21 compared to the young, correlating with greater increases in senescence associated (SA) β-galactosidase, a known marker of cellular senescence. A higher influx of F4/80⁺ macrophages and CD4⁺ T lymphocytes is measured and is accompanied by increases in mRNA of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). Importantly, microvascular density is significantly less, correlating with an increase in nitro-tyrosine, a marker of oxidative stress. Collectively, these data demonstrate that prolonged acute injury in the aged animals results in an accelerated progression of kidney disease in a chronic state.