Proximal tubular epithelial cells preferentially endocytose covalently-modified albumin compared to native albumin.

AIM: Albumin can be covalently modified at surface lysine residues and thus the circulation contains a mixture of native albumin (i.e. not modified) and albumin with varying degrees of modification. Uptake and lysosomal degradation of glomerular filtered albumin by proximal tubular cells via the megalin scavenger receptor is considered an important mechanism to limit albumin loss in the urine. However, whether this is a general mechanism of tubular uptake of albumin or if this is restricted to modified albumin is unknown. To address this question, we investigated the uptake of modified versus native albumin by proximal tubular cells. METHODS: A well-characterized proximal tubular cell model of albumin uptake was used to compare the uptake of modified albumin (covalent labelling of lysine residues with fluorescent probes) to that of native recombinant human albumin (rHA) labelled with 14 C during protein synthesis (14 C-rHA). RESULTS: Opossum kidney (OK) cells showed significant uptake of fluorescence-labelled albumin via an endocytosis mechanism. This uptake was inhibited by an equimolar ratio of different types of covalently modified albumin; however, purified bovine serum albumin and rHA failed to compete with the uptake of fluorescence-labelled albumin. In contrast, OK cells failed to endocytose native 14 C-rHA despite efficiently endocytosing covalently modified rHA. CONCLUSION: Our studies show that OK cells preferentially endocytose covalently-modified albumin compared to native albumin. This apparent selectivity of the megalin scavenger receptor complex suggests a specific role for this pathway in the removal of modified albumin from the circulation.

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.

Reduced tubular degradation of glomerular filtered plasma albumin is a common feature in acute and chronic kidney disease.

Tubular epithelial cells take up and degrade plasma albumin filtered by the glomerulus. Tubular damage resulting in reduced albumin uptake or degradation has been suggested as one mechanism contributing to albuminuria in kidney disease. This study investigated whether tubular albumin uptake or degradation is altered in acute and chronic glomerular disease. Mouse models of acute glomerular injury (anti-GBM disease and LPS-induced albuminuria) and chronic disease (streptozotocin-induced diabetes and db/db mice) were examined. Mice were injected intravenously with Alexa-albumin plus DQ-albumin and killed 20 minutes later. Tubular uptake of albumin (Alexa-albumin) and albumin degradation (Dye Quenched (DQ)-albumin) was assessed in tissue sections via confocal microscopy. Tubular uptake of Alexa-albumin in the models of diabetic nephropathy was not different to normal mice. However, the fluorescence signal resulting from degradation of DQ-albumin was significantly reduced in db/db mice, and the ratio of degraded to intact albumin was reduced in both models. The ratio of degraded to intact albumin in tubules was also reduced in the anti-GBM model. In the LPS model, both tubular uptake and degradation of albumin were significantly reduced, with a substantial reduction in the ratio of degraded to intact albumin in tubules. LPS stimulation of cultured tubular epithelial cells inhibited albumin uptake, indicating a direct role for LPS in modifying tubular handling of albumin. In conclusion, reduced degradation of filtered albumin in the proximal tubule is a common feature of glomerular diseases. This may be a general mechanism whereby tubular dysfunction contributes to the development of albuminuria.

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.

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.

Diabetic nephropathy - is this an immune disorder?

Chronic diabetes is associated with metabolic and haemodynamic stresses which can facilitate modifications to DNA, proteins and lipids, induce cellular dysfunction and damage, and stimulate inflammatory and fibrotic responses which lead to various types of renal injury. Approximately 30-40% of patients with diabetes develop nephropathy and this renal injury normally progresses in about a third of patients. Due to the growing incidence of diabetes, diabetic nephropathy is now the main cause of end-stage renal disease (ESRD) worldwide. Accumulating evidence from experimental and clinical studies has demonstrated that renal inflammation plays a critical role in determining whether renal injury progresses during diabetes. However, the immune response associated with diabetic nephropathy is considerably different to that seen in autoimmune kidney diseases or in acute kidney injury arising from episodes of ischaemia or infection. This review evaluates the role of the immune system in the development of diabetic nephropathy, including the specific contributions of leucocyte subsets (macrophages, neutrophils, mast cells, T and B lymphocytes), danger-associated molecular patterns (DAMPs), inflammasomes, immunoglobulin and complement. It also examines factors which may influence the development of the immune response, including genetic factors and exposure to other kidney insults. In addition, this review discusses therapies which are currently under development for targeting the immune system in diabetic nephropathy and indicates those which have proceeded into clinical trials.

miR-378 reduces mesangial hypertrophy and kidney tubular fibrosis via MAPK signalling.

The regulatory role of a novel miRNA, miR-378, was determined in the development of fibrosis through repression of the MAPK1 pathway, miR-378 and fibrotic gene expression was examined in streptozotocin (STZ)-induced diabetic mice at 18 weeks or in unilateral ureteral obstruction (UUO) mice at 7 days. miR-378 transfection of proximal tubular epithelial cells, NRK52E and mesangial cells was assessed with/without endogenous miR-378 knockdown using the locked nucleic acid (LNA) inhibitor. NRK52E cells were co-transfected with the mothers against decapentaplegic homolog 3 (SMAD3) CAGA reporter and miR-378 in the presence of transforming growth factor-ß (TGF-ß1) was assessed. Quantitative polymerase chain reaction (qPCR) showed a significant reduction in miR-378 (P<0.05) corresponding with up-regulated type I collagen, type IV collagen and α-smooth muscle actin (SMA) in kidneys of STZ or UUO mice, compared with controls. TGF-ß1 significantly increased mRNA expression of type I collagen (P<0.05), type IV collagen (P<0.05) and α-SMA (P<0.05) in NRK52E cells, which was significantly reduced (P<0.05) following miR-378 transfection and reversed following addition of the LNA inhibitor of endogenous miR-378 Overexpression of miR-378 inhibited mesangial cell expansion and proliferation in response to TGF-ß1, with LNA-miR-378 transfection reversing this protective effect, associated with cell morphological alterations. The protective function of MAPK1 on miR-378 was shown in kidney cells treated with the MAPK1 inhibitor, selumetinib, which inhibited mesangial cell hypertrophy in response to TGF-ß1. Taken together, these results suggest that miR-378 acts via regulation of the MAPK1 pathway. These studies demonstrate the protective function of MAPK1, regulated by miR-378, in the induction of kidney cell fibrosis and mesangial hypertrophy.

Myeloid cell-mediated renal injury in rapidly progressive glomerulonephritis depends upon spleen tyrosine kinase.

Antibody-dependent activation of myeloid cells within the glomerulus plays a central role in rapidly progressive forms of glomerulonephritis. The spleen tyrosine kinase (Syk) is expressed by all leukocytes, except mature T cells, and is required for signalling via the B-cell receptor, Fc receptors, and some integrins. Syk has been proposed as a therapeutic target in glomerulonephritis. However, little is known of Syk activation in human kidney disease, while studies in experimental glomerulonephritis using non-selective Syk inhibitors require validation via conditional gene deletion. The current study addressed both of these important points. Syk activation (Tyr(525/526) phosphorylation) was examined in a cohort of 96 patients with different glomerulonephritides. Syk activation was evident in infiltrating leukocytes, mainly neutrophils and macrophages, in 36/40 cases of rapidly progressive glomerulonephritis. In contrast, non-proliferative diseases showed little or no Syk activation. Glomerular and interstitial cells exhibiting Syk activation correlated with renal function and systemic inflammation. Next, we examined mice with conditional Syk gene deletion in myeloid cells (Syk(My) ) versus Syk(f/f) littermate controls in nephrotoxic serum nephritis - a model of rapidly progressive glomerulonephritis. Control Syk(f/f) mice featured a transient neutrophil influx at 3 h and severe disease on day 9 of nephrotoxic serum nephritis, with crescent formation, macrophage infiltration, inflammation, kidney fibrosis, and renal dysfunction. In contrast, Syk(My) mice had significantly reduced neutrophil and macrophage infiltration despite equivalent glomerular deposition of humoral reactants. Syk(My) mice exhibited reduced crescent formation, inflammation, and fibrosis, with improved renal function on day 9 of nephrotoxic serum nephritis. In conclusion, Syk activation is prominent in infiltrating myeloid cells in human rapidly progressive glomerulonephritis, and functional studies demonstrate that Syk deletion in myeloid cells is protective in mouse nephrotoxic serum nephritis.

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.

Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor.

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

Spleen tyrosine kinase contributes to acute renal allograft rejection in the rat.

Kidney allografts induce strong T-cell and antibody responses which mediate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leucocytes, except mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and some integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but little is known of Syk in acute renal allograft rejection. Sprague-Dawley rats underwent bilateral nephrectomy and received an orthotopic Wistar renal allograft. Recipient rats were treated with a Syk inhibitor (CC0482417, 30 mg/kg/bid), or vehicle, from 1 h before surgery until being killed 5 days later. Vehicle-treated recipients developed severe allograft failure with marked histologic damage in association with dense leucocyte infiltration (T cells, macrophages, neutrophils and NK cells) and deposition of IgM, IgG and C3. Immunostaining identified Syk expression by many infiltrating leucocytes. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries. In conclusion, this study identifies a role for Syk in acute renal allograft rejection. Syk inhibition may be a useful addition to T-cell-based immunotherapy in renal transplantation.

Myeloid mineralocorticoid receptor activation contributes to progressive kidney disease.

Clinical and experimental studies have shown that mineralocorticoid receptor (MR) antagonists substantially reduce kidney injury. However, the specific cellular targets and mechanisms by which MR antagonists protect against kidney injury must be identified. We used conditional gene deletion of MR signaling in myeloid cells (MR(flox/flox) LysM(Cre) mice; MyMRKO) or podocytes (MR(flox/flox) Pod(Cre) mice; PodMRKO) to establish the role of MR in these cell types in the development of mouse GN. Accelerated anti-glomerular basement membrane GN was examined in groups of mice: MyMRKO, PodMRKO, wild-type (WT) littermates, and WT mice receiving eplerenone (100 mg/kg twice a day; EPL-treated). At day 15 of disease, WT mice had glomerular crescents (37%±5%), severe proteinuria, and a 6-fold increase in serum cystatin-C. MyMRKO, PodMRKO, and EPL-treated mice with GN displayed proteinuria similar to that in these disease controls. However, MyMRKO and EPL-treated groups had a 35% reduction in serum cystatin-C levels and reduced crescent numbers compared with WT mice, whereas PodMRKO mice were not protected. The protection observed in MyMRKO mice appeared to result predominantly from reduced recruitment of macrophages and neutrophils into the inflamed kidney. Suppression of kidney leukocyte accumulation in MyMRKO mice correlated with reductions in gene expression of proinflammatory molecules (TNF-α, inducible nitric oxide synthase, chemokine (C-C motif) ligand 2, matrix metalloproteinase-12), tubular damage, and renal fibrosis and was similar in EPL-treated mice. In conclusion, MR signaling in myeloid cells, but not podocytes, contributes to the progression of renal injury in mouse GN, and myeloid deficiency of MR provides protection similar to eplerenone in this disease.

ASK1/p38 signaling in renal tubular epithelial cells promotes renal fibrosis in the mouse obstructed kidney.

Stress-activated kinases p38 MAPK and JNK promote renal fibrosis; however, how the pathways by which these kinases are activated in kidney disease remain poorly defined. Apoptosis signal-regulating kinase 1 (ASK1/MAPKKK5) is a member of the MAPKKK family that can induce activation of p38 and JNK. The present study examined whether ASK1 induces p38/JNK activation and renal fibrosis in unilateral ureteric obstruction (UUO) using wild-type (WT) and Ask1-deficient (Ask1(-/-)) mice. Basal p38 and JNK activation in WT kidneys was increased three- to fivefold in day 7 UUO mice in association with renal fibrosis. In contrast, there was no increase in p38 activation in Ask1(-/-) UUO mice, whereas JNK activation was only partially increased. The progressive increase in kidney collagen (hydroxyproline) content seen on days 7 and 12 of UUO in WT mice was significantly reduced in Ask1(-/-) UUO mice in association with reduced α-smooth muscle actin-positive myofibroblast accumulation. However, cultured WT and Ask1(-/-) renal fibroblasts showed equivalent proliferation and matrix production, indicating that ASK1 acts indirectly on fibroblasts. Tubular epithelial cells are the main site of p38 activation in the obstructed kidney. Angiotensin II and H2O2, but not IL-1 or lipopolysaccharide, induced p38 activation and upregulation of transforming growth factor-ß1, platelet-derived growth factor-B, and monocyte chemoattractant protein-1 production was suppressed in Ask1(-/-) tubular epithelial cells. In addition, macrophage accumulation was significantly inhibited in Ask1(-/-) UUO mice. In conclusion, ASK1 is an important upstream activator of p38 and JNK signaling in the obstructed kidney, and ASK1 is a potential therapeutic target in renal fibrosis.

Role of macrophages in the fibrotic phase of rat crescentic glomerulonephritis.

The ability of macrophages to cause acute inflammatory glomerular injury is well-established; however, the role of macrophages in the fibrotic phase of chronic kidney disease remains poorly understood. This study examined the role of macrophages in the fibrotic phase (days 14 to 35) of established crescentic glomerulonephritis. Nephrotoxic serum nephritis (NTN) was induced in groups of eight Wistar-Kyoto rats that were given a selective c-fms kinase inhibitor, fms-I, or vehicle alone from day 14 until being killed on day 35. Rats killed on day 14 NTN had pronounced macrophage infiltration with glomerular damage, fibrocellular crescents in 50% of glomeruli, tubulointerstitial damage, heavy proteinuria, and renal dysfunction. Glomerulosclerosis was more severe by day 35 in vehicle-treated rats, as was periglomerular and interstitial fibrosis, while proteinuria and renal dysfunction continued unabated and some parameters of tubular damage worsened. During the day 14-to-35 period, glomerular and interstitial macrophage infiltration decreased with an apparent change from a proinflammatory M1 phenotype to an alternatively activated M2 phenotype. Treatment with fms-I over days 14 to 35 selectively reduced blood monocyte numbers and abrogated glomerular and interstitial macrophage infiltration. This resulted in improved renal function, significantly reduced glomerular and interstitial fibrosis, and protection against further peritubular capillary loss. However, sustained proteinuria, tubular damage, and interstitial T cell infiltration and activation were unaffected. In conclusion, this study demonstrates that macrophages contribute to renal dysfunction and tissue damage in established crescentic glomerulonephritis as it progresses from the acute inflammatory to a chronic fibrotic phase.

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

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

TGF-ß1-activated kinase-1 regulates inflammation and fibrosis in the obstructed kidney.

Activation of c-Jun amino kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and the transcription factor nuclear factor-κB (NF-κB) drives renal inflammation and fibrosis. However, the upstream MAP kinase kinase kinase (MAP3K) enzyme(s) that activate these pathways in kidney disease are unknown. We determined the role of one candidate MAP3K enzyme, transforming growth factor-ß1-activated kinase-1 (TAK1/ MAP3K7), in activation of JNK, p38, and NF-κB in the obstructed kidney using conditional gene deletion in adult mice, and assessed the potential protective effect of TAK1 deletion on renal pathology. TAK1 deletion in cultured tubular epithelial cells substantially inhibited IL-1 and TNF-α-induced JNK, p38, and NF-κB signaling and the proinflammatory response. Map3k7(f/f)Cre-ER(TM) mice (in which tamoxifen induces global TAK1 deletion) and control Map3k7(f/f) mice were given tamoxifen at the time of unilateral ureteric obstruction (UUO) and then killed 2, 4, or 5 days later. Tamoxifen-treated control Map3k7(f/f) mice showed the expected activation of JNK, p38, and NF-κB signaling on days 2, 4, and 5, with macrophage infiltration and upregulation of mRNA levels of proinflammatory molecules (IL-1α, TNF-α, NOS2, and CCL2). Control Map3k7(f/f) mice also showed interstitial myofibroblast accumulation and collagen deposition in the obstructed kidney. Tamoxifen treatment of Map3k7(f/f)Cre-ER(TM) mice caused a 60% reduction in renal TAK1 expression on day 4 and >80% on day 5 UUO. Coincident with TAK1 deletion, activation of JNK, p38, and NF-κB signaling was markedly suppressed on days 4 to 5 UUO, which halted renal macrophage accumulation and expression of proinflammatory molecules. TAK1 deletion also halted the development of renal fibrosis in terms of myofibroblast accumulation, collagen deposition, and expression of profibrotic molecules. In conclusion, these studies establish TAK1 as a major upstream activator of JNK, p38, and NF-κB signaling in the obstructed kidney, and they define a pathologic role for TAK1 in renal inflammation and fibrosis.

c-fms blockade reverses glomerular macrophage infiltration and halts development of crescentic anti-GBM glomerulonephritis in the rat.

Depletion and adoptive transfer studies have demonstrated that macrophages induce glomerular lesions in experimental anti-glomerular basement membrane (anti-GBM) glomerulonephritis. However, there is no current therapeutic strategy that can rapidly and selectively remove these cells from the glomerulus in order to halt disease development. This study examined whether inhibition of the receptor for macrophage colony-stimulating factor (known as c-fms), which is selectively expressed by monocyte/macrophages, can eliminate the macrophage infiltrate in a rat model of crescentic anti-GBM glomerulonephritis. Wistar-Kyoto rats were treated with 10 or 30 mg/kg bid of fms-I (a selective c-fms kinase inhibitor) from the time of anti-GBM serum injection until being killed 1, 5 or 14 days later. fms-I treatment had only a minor effect upon the glomerular macrophage infiltrate on day 1 and did not prevent the subsequent induction of proteinuria. However, fms-I treatment reduced the glomerular macrophage infiltrate by 60% at day 5 and completely reversed the macrophage infiltrate by day 14. In addition, fms-I treatment downregulated the glomerular expression of pro-inflammatory molecules (TNF-α, NOS2, MMP-12, CCL2 and IL-12) on days 1 and 5, suggesting a suppression of the macrophage M1-type response. Despite a significant early loss of glomerular podocytes, ongoing proteinuria and glomerular tuft adhesions to Bowman's capsule, the reversal of the macrophage infiltrate prevented the development of glomerulosclerosis, crescent formation, tubulointerstitial damage and renal dysfunction. In conclusion, this study has identified c-fms kinase inhibition as a selective approach to target infiltrating macrophages in acute glomerular injury, which may have therapeutic potential in rapidly progressive crescentic glomerulonephritis.

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.

Lefty antagonises TGF-beta1 induced epithelial-mesenchymal transition in tubular epithelial cells.

Lefty is a novel member of the transforming growth factor (TGF) supergene family which has the potential to antagonise actions of TGF-beta1 - the main factor driving fibrotic disease in the kidney and in other organs. TGF-beta1 can induce fibrosis through several mechanisms, including epithelial-mesenchymal transition (EMT) which contributes to myofibroblast accumulation in the renal interstitium. This study examined whether Lefty can antagonise TGF-beta1 mediated EMT. A rat tubular epithelial cell line (NRK52E) was stably transfected with a Lefty expression plasmid (52E-Lefty) or control plasmid (52E-Control). 52E-Control cells underwent TGF-beta1 induced EMT with up-regulation of alpha-smooth muscle actin (alpha-SMA), down-regulation of E-cadherin, and transition to an elongated fibroblast-like morphology. In contrast, 52E-Lefty cells were substantially protected from TGF-beta1 induced EMT. Analysis of signalling pathways showed that 52E-Lefty cells had a marked reduction in TGF-beta1 induced Smad activity and suppression of the secondary phase of JNK (but not p38) signalling. Treatment of NRK52E cells with a JNK inhibitor was shown to suppress TGF-beta1 induced EMT. In conclusion, Lefty can antagonise TGF-beta1 mediated EMT in renal tubular epithelial cells. Lefty may have potential as an anti-fibrotic molecule in the treatment of renal fibrosis.

Review: Serum and urine biomarkers of kidney disease: A pathophysiological perspective.

The use of reliable biomarkers is becoming increasingly important for the improved management of patients with acute and chronic kidney diseases. Recent developments have identified a number of novel biomarkers in serum or urine that can determine the potential risk of kidney damage, distinguish different types of renal injury, predict the progression of disease and have the potential to assess the efficacy of therapeutic intervention. Some of these biomarkers can be used independently while others are more beneficial when used in combination with knowledge of other clinical risk factors. Advances in gene expression analysis, chromatography, mass spectrometry and the development of sensitive enzyme-linked immunosorbent assays have facilitated accurate quantification of many biomarkers. This review primarily focuses on describing new and established biomarkers, which identify and measure the various pathophysiological processes that promote kidney disease. It provides an overview of some of the different classes of renal biomarkers that can be assessed in serum/plasma and urine, including markers of renal function, oxidative stress, structural and cellular injury, immune responses and fibrosis. However, it does not explore the current status of these biomarkers in terms of their clinical validation.