Intervention treatment reducing cellular senescence inhibits tubulointerstitial fibrosis in diabetic mice following acute kidney injury.

Senescence of kidney tubules leads to tubulointerstitial fibrosis (TIF). Proximal tubular epithelial cells undergo stress-induced senescence during diabetes and episodes of acute kidney injury (AKI), and combining these injuries promotes the progression of diabetic kidney disease (DKD). Since TIF is crucial to progression of DKD, we examined the therapeutic potential of targeting senescence with a senolytic drug (HSP90 inhibitor) and/or a senostatic drug (ASK1 inhibitor) in a model of TIF in which AKI is superimposed on diabetes. After 8 weeks of streptozotocin-induced diabetes, mice underwent bilateral clamping of renal pedicles to induce mild AKI, followed by 28 days of reperfusion. Groups of mice (n=10-12) received either vehicle, HSP90 inhibitor (alvespimycin), ASK1 inhibitor (GS-444217), or both treatments. Vehicle-treated mice displayed tubular injury at day 3 and extensive tubular cell senescence at day 10, which remained unresolved at day 28. Markers of senescence (Cdkn1a and Cdkn2a), inflammation (Cd68, Tnf, and Ccl2), and TIF (Col1a1, Col4a3, α-Sma/Acta2, and Tgfb1) were elevated at day 28, coinciding with renal function impairment. Treatment with alvespimycin alone reduced kidney senescence and levels of Col1a1, Acta2, Tgfb1, and Cd68; however, further treatment with GS-444217 also reduced Col4a3, Tnf, Ccl2, and renal function impairment. Senolytic therapy can inhibit TIF during DKD, but its effectiveness can be improved by follow-up treatment with a senostatic inhibitor, which has important implications for treating progressive DKD.

Mice with Established Diabetes Show Increased Susceptibility to Renal Ischemia/Reperfusion Injury: Protection by Blockade of Jnk or Syk Signaling Pathways.

Patients with diabetes are at an increased risk for acute kidney injury (AKI) after renal ischemia/reperfusion injury (IRI). However, there is a lack preclinical models of IRI in established diabetes. The current study characterized renal IRI in mice with established diabetes and investigated potential therapies. Diabetes was induced in C57BL/6J mice by low-dose streptozotocin injection. After 7 weeks of sustained diabetes, mice underwent 13 minutes of bilateral renal ischemia and were euthanized after 24 hours of reperfusion. Age-matched, nondiabetic controls underwent the same surgical procedure. Renal IRI induced two- and sevenfold increases in plasma creatinine level in nondiabetic and diabetic mice, respectively (P < 0.001). Kidney damage, as indicated by histologic damage, tubular cell death, tubular damage markers, and inflammation, was more severe in the diabetic IRI group. The diabetic IRI group showed greater accumulation of spleen tyrosine kinase (Syk)-expressing cells, and increased c-Jun N-terminal kinase (Jnk) signaling in tubules compared to nondiabetic IRI. Prophylactic treatment with a Jnk or Syk inhibitor substantially reduced the severity of AKI in the diabetic IRI model, with differential effects on neutrophil infiltration and Jnk activation. In conclusion, established diabetes predisposed mice to renal IRI-induced AKI. Two distinct proinflammatory pathways, JNK and SYK, were identified as potential therapeutic targets for anticipated AKI in patients with diabetes.

Corrigendum: c-Jun amino terminal kinase signaling promotes aristolochic acid-induced acute kidney injury.

[This corrects the article DOI: 10.3389/fphys.2021.599114.].

Cyclophilin D Promotes Acute, but Not Chronic, Kidney Injury in a Mouse Model of Aristolochic Acid Toxicity.

The plant-derived toxin, aristolochic acid (AA), is the cause of Chinese Herb Nephropathy and Balkan Nephropathy. Ingestion of high dose AA induces acute kidney injury, while chronic low dose ingestion leads to progressive kidney disease. Ingested AA is taken up by tubular epithelial cells of the kidney, leading to DNA damage and cell death. Cyclophilin D (CypD) participates in mitochondrial-dependent cell death, but whether this mechanism operates in acute or chronic AA-induced kidney injury is unknown. We addressed this question by exposing CypD-/- and wild type (WT) mice to acute high dose, or chronic low dose, AA. Administration of 5 mg/kg AA to WT mice induced acute kidney injury 3 days later, characterised by loss of kidney function, tubular cell damage and death, and neutrophil infiltration. All of these parameters were significantly reduced in CypD-/- mice. Chronic low dose (2 mg/kg AA) administration in WT mice resulted in chronic kidney disease with impaired renal function and renal fibrosis by day 28. However, CypD-/- mice were not protected from AA-induced chronic kidney disease. In conclusion, CypD facilitates AA-induced acute kidney damage, but CypD does not contribute to the transition of acute kidney injury to chronic kidney disease during ongoing AA exposure.

c-Jun Amino Terminal Kinase Signaling Promotes Aristolochic Acid-Induced Acute Kidney Injury.

Aristolochic acid (AA) is a toxin that induces DNA damage in tubular epithelial cells of the kidney and is the cause of Balkan Nephropathy and Chinese Herb Nephropathy. In cultured tubular epithelial cells, AA induces a pro-fibrotic response via the c-Jun amino terminal kinase (JNK) signaling pathway. This study investigated the in vivo role of JNK signaling with a JNK inhibitor (CC-930) in mouse models of acute high dose AA-induced kidney injury (day 3) and renal fibrosis induced by chronic low dose AA exposure (day 22). CC-930 treatment inhibited JNK signaling and protected from acute AA-induced renal function impairment and severe tubular cell damage on day 3, with reduced macrophage infiltration and expression of pro-inflammatory molecules. In the chronic model, CC-930 treatment inhibited JNK signaling but did not affect AA-induced renal function impairment, tubular cell damage including the DNA damage response and induction of senescence, or renal fibrosis; despite a reduction in the macrophage pro-inflammatory response. In conclusion, JNK signaling contributes to acute high dose AA-induced tubular cell damage, presumably via an oxidative stress-dependent mechanism, but is not involved in tubular atrophy and senescence that promote chronic kidney disease caused by ongoing DNA damage in chronic low dose AA exposure.

JUN Amino-Terminal Kinase 1 Signaling in the Proximal Tubule Causes Cell Death and Acute Renal Failure in Rat and Mouse Models of Renal Ischemia/Reperfusion Injury.

Activation of the JUN amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including acute renal ischemia/reperfusion injury (IRI). Two forms of JNK, JNK1 and JNK2, are expressed in the kidney. Systemic administration of pan-JNK inhibitors suppresses renal IRI; however, the contribution of JNK1 versus JNK2, and the specific role of JNK activation in the proximal tubule in IRI, remains unknown. These questions were addressed in rat and mouse models of acute bilateral renal IRI. Administration of the JNK inhibitor, CC-930, substantially reduced the severity of renal failure, tubular damage, and inflammation at 24 hours in a rat IRI model. Additionally, Jnk1-/- mice, but not Jnk2-/- mice, were shown to be significantly protected against acute renal failure, tubular damage, and inflammation in the IRI model. Furthermore, mice with conditional Jnk1 deletion in the proximal tubule also showed considerable protection from IRI-induced renal failure, tubular damage, and inflammation. Finally, primary cultures of Jnk1-/-, but not Jnk2-/-, tubular epithelial cells were protected from oxidant-induced cell death, in association with preventing phosphorylation of proteins (receptor interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase) in the necroptosis pathway. In conclusion, JNK1, but not JNK2, plays a specific role in IRI-induced cell death in the proximal tubule, leading to acute renal failure.

Cyclophilin A Promotes Inflammation in Acute Kidney Injury but Not in Renal Fibrosis.

Cyclophilin A (CypA) is a highly abundant protein in the cytoplasm of most mammalian cells. Beyond its homeostatic role in protein folding, CypA is a Damage-Associated Molecular Pattern which can promote inflammation during tissue injury. However, the role of CypA in kidney disease is largely unknown. This study investigates the contribution of CypA in two different types of kidney injury: acute tubular necrosis and progressive interstitial fibrosis. CypA (Ppia) gene deficient and wild type (WT) littermate controls underwent bilateral renal ischaemia/reperfusion injury (IRI) and were killed 24h later or underwent left unilateral ureteric obstruction (UUO) and were killed 7 days later. In the IRI model, CypA-/- mice showed substantial protection against the loss of renal function and from tubular cell damage and death. This was attributed to a significant reduction in neutrophil and macrophage infiltration since CypA-/- tubular cells were not protected from oxidant-induced cell death in vitro. In the UUO model, CypA-/- mice were not protected from leukocyte infiltration or renal interstitial fibrosis. In conclusion, CypA promotes inflammation and acute kidney injury in renal IRI, but does not contribute to inflammation or interstitial fibrosis in a model of progressive kidney fibrosis.

Targeting apoptosis signal-regulating kinase 1 in acute and chronic kidney disease.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase (MAP3K) family which acts as an upstream regulator for the activation of p38 MAPK and c-Jun N-terminal kinase (JNK). Experimental studies have demonstrated a pathogenic role for p38 MAPK and JNK activation in a number of kidney disease models; however, clinical studies targeting these kinases directly have been problematic due to their role in homeostatic functions. In comparison, ASK1 is activated in pathological states and is not essential for homeostatic functions, suggesting that ASK1 may be a safe and effective therapeutic target to inhibit p38 MAPK and JNK signaling in disease. Animal model studies using Ask1 gene deficient mice or a selective ASK1 inhibitor have demonstrated that ASK1 blockade is effective in a variety of acute and chronic kidney diseases; preventing cell injury, inflammation, fibrosis, albuminuria, and renal function impairment. Positive outcomes from these experimental studies have led to the current evaluation of an ASK1 inhibitor in patients with moderate to advanced diabetic kidney disease. This review summarizes the preclinical studies of ASK1 blockade in models of acute and chronic kidney injury and a clinical study examining ASK1 inhibitor treatment in diabetic kidney disease.

Cyclophilin Inhibition Protects Against Experimental Acute Kidney Injury and Renal Interstitial Fibrosis.

Cyclophilins have important homeostatic roles, but following tissue injury, cyclophilin A (CypA) can promote leukocyte recruitment and inflammation, while CypD can facilitate mitochondrial-dependent cell death. This study investigated the therapeutic potential of a selective cyclophilin inhibitor (GS-642362), which does not block calcineurin function, in mouse models of tubular cell necrosis and renal fibrosis. Mice underwent bilateral renal ischemia/reperfusion injury (IRI) and were killed 24 h later: treatment with 10 or 30 mg/kg/BID GS-642362 (or vehicle) began 1 h before surgery. In the second model, mice underwent unilateral ureteric obstruction (UUO) surgery and were killed 7 days later; treatment with 10 or 30 mg/kg/BID GS-642362 (or vehicle) began 1 h before surgery. GS-642362 treatment gave a profound and dose-dependent protection from acute renal failure in the IRI model. This protection was associated with reduced tubular cell death, including a dramatic reduction in neutrophil infiltration. In the UUO model, GS-642362 treatment significantly reduced tubular cell death, macrophage infiltration, and renal fibrosis. This protective effect was independent of the upregulation of IL-2 and activation of the stress-activated protein kinases (p38 and JNK). In conclusion, GS-642362 was effective in suppressing both acute kidney injury and renal fibrosis. These findings support further investigation of cyclophilin blockade in other types of acute and chronic kidney disease.

Protease-activated receptor 2 does not contribute to renal inflammation or fibrosis in the obstructed kidney.

AIM: Protease-activated receptor 2 (PAR2) has been implicated in the development of renal inflammation and fibrosis. In particular, activation of PAR2 in cultured tubular epithelial cells induces extracellular signal-regulated kinase signalling and secretion of fibronectin, C-C Motif Chemokine Ligand 2 (CCL2) and transforming growth factor-ß1 (TGF-ß1), suggesting a role in tubulointerstitial inflammation and fibrosis. We tested this hypothesis in unilateral ureteric obstruction (UUO) in which ongoing tubular epithelial cell damage drives tubulointerstitial inflammation and fibrosis. METHODS: Unilateral ureteric obstruction surgery was performed in groups (n = 9/10) of Par2-/- and wild type (WT) littermate mice which were killed 7 days later. Non-experimental mice were controls. RESULTS: Wild type mice exhibited a 5-fold increase in Par2 messenger RNA (mRNA) levels in the UUO kidney. In situ hybridization localized Par2 mRNA expression to tubular epithelial cells in normal kidney, with a marked increase in Par2 mRNA expression by tubular cells, including damaged tubular cells, in WT UUO kidney. Tubular damage (tubular dilation, increased KIM-1 and decreased α-Klotho expression) and tubular signalling (extracellular signal-regulated kinase phosphorylation) seen in WT UUO were not altered in Par2-/- UUO. In addition, macrophage infiltration, up-regulation of M1 (NOS2) and M2 (CD206) macrophage markers, and up-regulation of pro-inflammatory molecules (tumour necrosis factor, CCL2, interleukin-36α) in WT UUO kidney were unchanged in Par2-/- UUO. Finally, the accumulation of α-SMA+ myofibroblasts, deposition of collagen IV and expression of pro-fibrotic factors (CTGF, TGF-ß1) were not different between WT and Par2-/- UUO mice. CONCLUSION: Protease-activated receptor 2 expression is substantially up-regulated in tubular epithelial cells in the obstructed kidney, but this does not contribute to the development of tubular damage, renal inflammation or fibrosis.

ASK1 contributes to fibrosis and dysfunction in models of kidney disease.

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

ASK1 inhibitor treatment suppresses p38/JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis.

Activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun amino terminal kinase (JNK) is prominent in human crescentic glomerulonephritis. p38 and JNK inhibitors suppress crescentic disease in animal models; however, the upstream mechanisms inducing activation of these kinases in crescentic glomerulonephritis are unknown. We investigated the hypothesis that apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) promote p38/JNK activation and renal injury in models of nephrotoxic serum nephritis (NTN); acute glomerular injury in SD rats, and crescentic disease in WKY rats. Treatment with the selective ASK1 inhibitor, GS-444217 or vehicle began 1 hour before nephrotoxic serum injection and continued until animals were killed on day 1 (SD rats) or 14 (WKY rats). NTN resulted in phosphorylation (activation) of p38 and c-Jun in both models which was substantially reduced by ASK1 inhibitor treatment. In SD rats, GS-444217 prevented proteinuria and glomerular thrombosis with suppression of macrophage activation on day 1 NTN. In WKY rats, GS-444217 reduced crescent formation, prevented renal impairment and reduced proteinuria on day 14 NTN. Macrophage activation, T-cell infiltration and renal fibrosis were also reduced by GS-444217. In conclusion, GS-444217 treatment inhibited p38/JNK activation and development of renal injury in rat NTN. ASK1 inhibitors may have therapeutic potential in rapidly progressive glomerulonephritis.

An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis.

Non-selective inhibitors of spleen tyrosine kinase (SYK) efficiently suppress disease in T cell-dependent models of crescentic glomerulonephritis. However, the therapeutic potential of selective SYK inhibitors in this disease has not been established. In addition, we lack knowledge regarding SYK expression in non-myeloid cells in glomerulonephritis. We addressed these two issues in a rat model of nephrotoxic serum nephritis (NTN) using a SYK inhibitor, GS-492429. Disease was induced in Sprague-Dawley rats (Study 1) or Wistar-Kyoto (WKY) rats (Study 2) by immunization with sheep IgG and administration of sheep anti-rat nephrotoxic serum. Animals were untreated or received GS-492429 (30 mg/kg/bid) or vehicle treatment from 2 h before nephrotoxic serum injection until being killed 3 or 24 h later (Study 1) or 14 days later (Study 2). Two-colour confocal microscopy found that SYK expression in NTN kidney was restricted to myeloid cells and platelets, with no evidence of SYK expression by T cells, mesangial cells, podocytes or tubular epithelial cells. In Study 1, GS-492429 treatment significantly reduced glomerular neutrophil and macrophage infiltration, with protection from glomerular thrombosis and proteinuria. In Study 2, GS-492429 treatment reduced glomerular crescent formation by 70% on day 14 NTN in conjunction with reduced glomerular thrombosis, glomerulosclerosis and tubular damage. This was accompanied by a marked reduction in markers of inflammation (CCL2, TNF-α, NOS2, MMP-12). Importantly, the protective effects of GS-492429 were independent of T cell infiltration and activation and independent of JAK/STAT3 signalling. In conclusion, this study demonstrates that a SYK inhibitor can suppress the development of crescentic glomerulonephritis through effects upon myeloid cells and platelets.

Matrix metalloproteinase-12 deficiency attenuates experimental crescentic anti-glomerular basement membrane glomerulonephritis.

AIM: Matrix metalloproteinase-12 (MMP-12; macrophage elastase) is an enzyme that can cleave various extracellular matrix proteins and is required for macrophage infiltration and pulmonary fibrosis in experimental emphysema. We have shown previously that MMP-12 is highly up-regulated in experimental anti-glomerular basement membrane (GBM) disease. The aim of this study was to determine whether MMP-12 is required for glomerular macrophage infiltration and crescent formation in anti-GBM glomerulonephritis. METHODS: Accelerated anti-GBM disease was induced in groups of MMP-12 gene deficient mice (MMP-12-/-) and wild-type C57BL/6J controls, which were killed 12 days after injection of anti-GBM serum. RESULTS: Wild-type and MMP-12-/- mice developed glomerular damage and glomerular tuft adhesions to Bowman's capsule. Both groups developed severe proteinuria. Wild-type mice also developed significant loss of renal function and crescents in 22% of glomeruli, which were associated with macrophage infiltration and Bowman's capsule rupture. In contrast, MMP-12-/- mice were partially protected from renal function decline, crescent formation and Bowman's capsule rupture. This was associated with reduced macrophage infiltration in both glomeruli and the interstitium, and with reduced expression of CCL2, TNF-α and iNOS mRNA in MMP-12-/- kidneys. In addition, KIM-1 mRNA levels were reduced in MMP-12-/- mice indicating less tubular damage. CONCLUSION: These data demonstrate that endogenous MMP-12 facilitates macrophage accumulation and activation in anti-GBM glomerulonephritis which is required for glomerular crescent formation, Bowman's capsule rupture, tubular damage and renal function decline.

A Model of Acute Antibody-Mediated Renal Allograft Rejection in the Sensitized Rata.

OBJECTIVES: Antibody-mediated rejection in transplant recipients with preexisting donor-specific antibodies is a challenging clinical situation. However, we lack suitable animal models to study this scenario. The aim of this study was to develop an animal model of acute antibody-mediated rejection of renal allografts in sensitized recipients. MATERIALS AND METHODS: We used major histocompatibility complex class I and II incompatible rat strains (Dark Agouti RT1av1 and Lewis RT1l), which develop aggressive rejection. Recipient Lewis rats were immunized with donor strain spleen cells 5 days before surgery to induce donor-specific antibodies. Rats underwent bilateral nephrectomy and orthotopic transplant of the donor kidney. To minimize T-cell-mediated rejection while allowing the development of donor-specific antibodies, recipient animals were given tacrolimus starting the day before surgery. RESULTS: Hyperacute rejection was not seen, but acute graft dysfunction was evident on day 1 with a rapid deterioration of graft function by day 3. Histologic damage featured glomerulopathy, capillaritis, capillary thrombosis, and acute tubular injury. Recipients exhibited high serum levels of donor-specific antibodies and deposition of immunoglobulin G and C4d on graft endothelium. Immunostaining showed substantial endothelial damage, fibrin deposition in glomerular and peritubular capillaries, and infiltrates of macrophages, neutrophils, and natural killer cells. T-cell activation was efficiently suppressed by tacrolimus. CONCLUSIONS: We have developed a clinically relevant model of acute antibody-mediated rejection in recipients with preexisting donor-specific antibodies, which is suitable for testing novel therapies.

Cyclophilin D promotes tubular cell damage and the development of interstitial fibrosis in the obstructed kidney.

Cyclophilin D (CypD) is an important component in mitochondrial-dependent tubular cell death in acute kidney injury. However, it is not known whether CypD contributes to tubular cell damage in chronic interstitial fibrosis. We investigated this question in the unilateral ureter obstruction (UUO) model of renal interstitial fibrosis. Groups of CypD-/- and wild type (WT) mice were killed 7 or 12 days after UUO surgery. The significant tubular cell apoptosis seen in WT UUO was significantly reduced in CypD-/- UUO based on TUNEL and cleaved caspase 3 staining. Other markers of tubular cell damage; loss of E-cadherin and AQP1 expression, were also reduced in the CypD-/- UUO kidney. This reduced tubular damage was associated with less inflammation and a partial protection against loss of peritubular capillaries. The prominent accumulation of α-SMA+ myofibroblasts and interstitial collagen deposition seen in WT UUO was significantly reduced in CypD-/- UUO on day 12, but not day 7. Activation of several pro-fibrotic signalling pathways (p38 MAPK, JNK and Smad3) was unaltered in CypD-/- UUO, arguing that CypD acts independently to promote renal fibrosis. CypD deletion in cultured tubular cells attenuated oxidative stress-induced pro-inflammatory, pro-fibrotic and apoptotic responses; however, responses to angiotensin II and LPS were unaffected. In contrast, CypD deletion in cultured renal fibroblasts did not affect PDGF-induced proliferation or TGF-ß1-induced collagen I expression, suggesting no direct role of CypD in the fibroblast response. In conclusion, we have identified a role for CypD in chronic tubular cell damage and in the development of renal interstitial fibrosis.

The JNK Signaling Pathway in Renal Fibrosis.

Fibrosis of the glomerular and tubulointerstitial compartments is a common feature of chronic kidney disease leading to end-stage renal failure. This fibrotic process involves a number of pathologic mechanisms, including cell death and inflammation. This review focuses on the role of the c-Jun amino terminal kinase (JNK) signaling pathway in the development of renal fibrosis. The JNK pathway is activated in response to various cellular stresses and plays an important role in cell death and inflammation. Activation of JNK signaling is a common feature in most forms of human kidney injury, evident in both intrinsic glomerular and tubular cells as well as in infiltrating leukocytes. Similar patterns of JNK activation are evident in animal models of acute and chronic renal injury. Administration of JNK inhibitors can protect against acute kidney injury and suppress the development of glomerulosclerosis and tubulointerstitial fibrosis. In particular, JNK activation in tubular epithelial cells may be a pivotal mechanism in determining the outcome of both acute kidney injury and progression of chronic kidney disease. JNK signaling promotes tubular epithelial cell production of pro-inflammatory and pro-fibrotic molecules as well as tubular cell de-differentiation toward a mesenchymal phenotype. However, the role of JNK within renal fibroblasts is less well-characterized. The JNK pathway interacts with other pro-fibrotic pathways, most notable with the TGF-ß/SMAD pathway. JNK activation can augment TGF-ß gene transcription, induce expression of enzymes that activate the latent form of TGF-ß, and JNK directly phosphorylates SMAD3 to enhance transcription of pro-fibrotic molecules. In conclusion, JNK signaling plays an integral role in several key mechanisms operating in renal fibrosis. Targeting of JNK enzymes has therapeutic potential for the treatment of fibrotic kidney diseases.

Inhibition of Spleen Tyrosine Kinase Reduces Renal Allograft Injury in a Rat Model of Acute Antibody-Mediated Rejection in Sensitized Recipients.

BACKGROUND: Organ transplantation into sensitized patients with preexisting donor-specific antibodies (DSA) is very challenging. Spleen tyrosine kinase (Syk) promotes leukocyte recruitment and activation via signaling through various cell surface receptors. We investigated whether a selective Syk inhibitor (GS-492429) could suppress antibody-mediated rejection (AMR) in a rat model of AMR in sensitized recipients. METHODS: Recipient Lewis rats (RT1) were immunized with donor (Dark Agouti, RT1) spleen cells (day -5). Recipients underwent bilateral nephrectomy and orthotopic renal transplantation (day 0). Cellular rejection was minimized by tacrolimus treatment from day -1. Groups received GS-492429 (30 mg/kg, twice a day) (n = 11) or vehicle (n = 12) from 1 hour before transplantation until being killed on day 3. RESULTS: Vehicle-treated recipients developed graft dysfunction on day 1 which rapidly worsened by day 3. Histology showed severe damage (thrombosis, acute tubular injury, capillaritis) and infiltration of many Syk leukocytes. GS-492429 did not affect graft dysfunction on day 1, but treatment reduced allograft damage and prevented the rapid deterioration of graft function on day 3. GS-492429 reduced the prominent macrophage infiltrate and reduced the M1 proinflammatory response. Neutrophil and NK cell infiltration and capillary thrombosis were also significantly reduced by GS-492429 treatment. Serum DSA levels and the deposition of IgG and C4d in the allograft were equivalent in the 2 groups. CONCLUSIONS: Treatment with a Syk inhibitor significantly reduced renal allograft injury in a model of severe antibody-mediated damage in highly sensitized recipients. Further studies are warranted to determine whether Syk inhibition is a potential adjunctive treatment in clinical AMR.

Spleen Tyrosine Kinase Signaling Promotes Myeloid Cell Recruitment and Kidney Damage after Renal Ischemia/Reperfusion Injury.

Ischemia/reperfusion (I/R) injury is an important cause of acute and chronic renal failure. Neutrophils and macrophages, by integrin-based recruitment, play a key role in renal I/R injury. Integrin-based activation of spleen tyrosine kinase (Syk) contributes to myeloid cell adhesion to activated endothelial cells in vitro; however, whether Syk is required for myeloid cell recruitment and tubular damage in I/R injury is unknown. Therefore, we investigated the function of Syk in mouse I/R injury using two different approaches. C57Bl/6J mice underwent bilateral warm ischemia and were sacrificed after 30 minutes or 24 hours of reperfusion. Mice were treated with the Syk inhibitor CC0417, or vehicle, beginning 1 hour before surgery. Syk was expressed by infiltrating neutrophils, macrophages, and platelets in vehicle-treated I/R injury which exhibited severe renal failure and tubular damage at 24 hours. CC0417 treatment markedly reduced neutrophil, macrophage, and platelet accumulation with improved renal function and reduced tubular damage. Next, we compared mice with conditional Syk gene deletion in myeloid cells (Syk(My)) versus Syk(f/f) littermate controls in a 24-hour study. Syk(My) mice also showed a marked reduction in neutrophil and macrophage infiltration with significant protection from I/R-induced acute renal failure and tubular damage. These studies define a pathologic role for myeloid Syk signaling in renal I/R injury and identify Syk as a potential therapeutic target in this condition.

ASK1: a new therapeutic target for kidney disease.

Stress-induced activation of p38 MAPK and JNK signaling is a feature of both acute and chronic kidney disease and is associated with disease progression. Inhibitors of p38 MAPK or JNK activation provide protection against inflammation and fibrosis in animal models of kidney disease; however, clinical trials of p38 MAPK and JNK inhibitors in other diseases (rheumatoid arthritis and pulmonary fibrosis) have been disappointing. Apoptosis signal-regulating kinase 1 (ASK1) acts as an upstream regulator for the activation of p38 MAPK and JNK in kidney disease. Mice lacking the Ask1 gene are healthy with normal homeostatic functions and are protected from acute kidney injury induced by ischemia-reperfusion and from renal interstitial fibrosis induced by ureteric obstruction. Recent studies have shown that a selective ASK1 inhibitor substantially reduced renal p38 MAPK activation and halted the progression of nephropathy in diabetic mice, and this has led to a current clinical trial of an ASK1 inhibitor in patients with stage 3 or 4 diabetic kidney disease. This review explores the rationale for targeting ASK1 in kidney disease and the therapeutic potential of ASK1 inhibitors based on current experimental evidence.