Successful treatment of JAK1-associated inflammatory disease.

BACKGROUND: Gain-of-function variants of JAK1 drive a rare immune dysregulation syndrome associated with atopic dermatitis, allergy, and eosinophilia. OBJECTIVES: This study sought to describe the clinical and immunological characteristics associated with a new gain-of-function variant of JAK1 and report the therapeutic efficacy of Janus kinase (JAK) inhibition. METHODS: The investigators identified a family affected by JAK1-associated autoinflammatory disease and performed clinical assessment and immunological monitoring on 9 patients. JAK1 signaling was studied by flow and mass cytometry in patients' cells at basal state or after immune stimulation. A molecular disease signature in the blood was studied at the transcriptomic level. Patients were treated with 1 of 2 JAK inhibitors: either baricitinib or upadacitinib. Clinical, cellular, and molecular response were evaluated over a 2-year period. RESULTS: Affected individuals displayed a syndromic disease with prominent allergy including atopic dermatitis, ichthyosis, arthralgia, chronic diarrhea, disseminated calcifying fibrous tumors, and elevated whole blood histamine levels. A variant of JAK1 localized in the pseudokinase domain was identified in all 9 affected, tested patients. Hyper-phosphorylation of STAT3 was found in 5 of 6 patients tested. Treatment of patients' cells with baricitinib controlled most of the atypical hyper-phosphorylation of STAT3. Administration of baricitinib to patients led to rapid improvement of the disease in all adults and was associated with reduction of systemic inflammation. CONCLUSIONS: Patients with this new JAK1 gain-of-function pathogenic variant displayed very high levels of blood histamine and showed a variable combination of atopy with articular and gastrointestinal manifestations as well as calcifying fibrous tumors. The disease, which appears to be linked to STAT3 hyperactivation, was well controlled under treatment by JAK inhibitors in adult patients.

Neuropilin-1 cooperates with PD-1 in CD8+ T cells predicting outcomes in melanoma patients treated with anti-PD1.

Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by restoring antitumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development, also expressed in immune cells. We hypothesized that NRP1 could be an immune checkpoint co-receptor modulating CD8+ T cells activity in the context of the antitumor immune response. Here, we show that NRP1 is recruited in the cytolytic synapse of PD1+CD8+ T cells, cooperates and enhances PD-1 activity. In mice, CD8+ T cells specific deletion of Nrp1 improves anti-PD1 antibody antitumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8+ T cells predicts poor outcome of patients treated with anti-PD1. NRP1 is a promising target to overcome resistance to anti-PD1 therapies.

Erythrocytosis associated with IgA nephropathy.

BACKGROUND: Erythrocytosis is a hematological disorder usually related to hematopoietic stem cell somatic mutations. However, unexplained erythrocytosis remains frequent. In this study, we evaluated the involvement of IgA1, a regulator of erythropoiesis also implicated in IgA nephropathy (IgAN) pathophysiology, in unexplained polycythemia/erythrocytosis (PE) of IgAN patients. METHODS: IgAN-PE patients' serum was collected, analyzed and used to study IgA1 effect on proliferation and differentiation of erythroid progenitors. Hematological parameters of transgenic mice for human alpha1 heavy chain were studied. Multicentric observational cohorts of chronic kidney disease (CKD) patients, including both native kidney diseases and renal transplants, were studied to analyze patient hemoglobin levels. FINDINGS: We retrospectively identified 6 patients with IgAN and unexplained PE. In large CKD cohorts, IgAN was associated with PE in 3.5% of patients (p<0.001 compared to other nephropathies). IgAN was an independent factor associated with higher hemoglobin levels (13.1g/dL vs 12.2 g/dL, p=0.01). During post-transplant anemia, anemia recovery was faster in IgAN patients. Elevated polymeric/monomeric IgA1 ratio as well as high Gd-IgA1 rate were observed in circulating IgA1 of the 6 IgAN-PE patients as compared with control or IgAN patients without PE. IgA1 from these patients increased the sensitivity of erythroid progenitors to Epo. In mice, we also observed an elevation of hematocrit in alpha1 knock-in mice compared to wild type controls. INTERPRETATION: These data identify a new etiology of erythrocytosis and demonstrate the role of pIgA1 in human erythropoiesis. This syndrome of IgA-related erythrocytosis should be investigated in case of unexplained erythrocytosis and renal disease. FUNDING: This work was supported by INSERM (French national institute for health and medical research), Labex GRex and Imagine Institute (Paris, France).

Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors.

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoprotein cytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.

A novel, highly potent and selective phosphodiesterase-9 inhibitor for the treatment of sickle cell disease.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

Plasma histamine elevation in a large cohort of sickle cell disease patients.

The role of mast cells has been questioned in sickle cell disease (SCD). We performed a prospective study evaluating plasma histamine and tryptase levels in a cohort of paediatric and adult patients, in steady state (n = 132) and during vaso-occlusive crisis (VOC) (n = 121). Histamine level was elevated in 18% of patients in steady state and in 61% during VOC. Median histamine level was significantly higher during VOC than in steady state (24·1 [7·0-45·0] vs 9·6 [6·2-14·4] nmol/l, P < 0·0001). Tryptase level was slightly increased during VOC without reaching pathological values. These results suggest a role of mast cell activation in SCD pathophysiology.

Serum Iron Protects from Renal Postischemic Injury.

Renal transplants remain a medical challenge, because the parameters governing allograft outcome are incompletely identified. Here, we investigated the role of serum iron in the sterile inflammation that follows kidney ischemia-reperfusion injury. In a retrospective cohort study of renal allograft recipients (n=169), increased baseline levels of serum ferritin reliably predicted a positive outcome for allografts, particularly in elderly patients. In mice, systemic iron overload protected against renal ischemia-reperfusion injury-associated sterile inflammation. Furthermore, chronic iron injection in mice prevented macrophage recruitment after inflammatory stimuli. Macrophages cultured in high-iron conditions had reduced responses to Toll-like receptor-2, -3, and -4 agonists, which associated with decreased reactive oxygen species production, increased nuclear localization of the NRF2 transcription factor, increased expression of the NRF2-related antioxidant response genes, and limited NF-κB and proinflammatory signaling. In macrophage-depleted animals, the infusion of macrophages cultured in high-iron conditions did not reconstitute AKI after ischemia-reperfusion, whereas macrophages cultured in physiologic iron conditions did. These findings identify serum iron as a critical protective factor in renal allograft outcome. Increasing serum iron levels in patients may thus improve prognosis of renal transplants.

FLT3 inhibitors: clinical potential in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy that is cured in as few as 15%-40% of cases. Tremendous improvements in AML prognostication arose from a comprehensive analysis of leukemia cell genomes. Among normal karyotype AML cases, mutations in the FLT3 gene are the ones most commonly detected as having a deleterious prognostic impact. FLT3 is a transmembrane tyrosine kinase receptor, and alterations of the FLT3 gene such as internal tandem duplications (FLT3-ITD) deregulate FLT3 downstream signaling pathways in favor of increased cell proliferation and survival. FLT3 tyrosine kinase inhibitors (TKI) emerged as a new therapeutic option in FLT3-ITD AML, and clinical trials are ongoing with a variety of TKI either alone, combined with chemotherapy, or even as maintenance after allogenic stem cell transplantation. However, a wide range of molecular resistance mechanisms are activated upon TKI therapy, thus limiting their clinical impact. Massive research efforts are now ongoing to develop more efficient FLT3 TKI and/or new therapies targeting these resistance mechanisms to improve the prognosis of FLT3-ITD AML patients in the future.

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis.

BACKGROUND: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS. METHODS: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset. RESULTS: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %. CONCLUSIONS: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.

Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

The role of mast cells in cancers.

Mast cells are immune cells that accumulate in the tumors and their microenvironment during disease progression. Mast cells are armed with a wide array of receptors that sense environment modifications and, upon stimulation, they are able to secrete several biologically active factors involved in the modulation of tumor growth. For example, mast cells are able to secrete pro-angiogenic and growth factors but also pro- and anti-inflammatory mediators. Recent studies have allowed substantial progress in understanding the role of mast cells in tumorigenesis/disease progression but further studies are necessary to completely elucidate their impact in the pathophysiology of cancer. Here we review observations suggesting that mast cells could modulate tumor growth in humans. We also discuss the drawbacks related to observations from mast cell-deficient mouse models, which could have consequences in the determination of a potential causative relationship between mast cells and cancer. We believe that the understanding of the precise role of mast cells in tumor development and progression will be of critical importance for the development of new targeted therapies in human cancers.

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in ß-thalassemia.

The pathophysiology of ineffective erythropoiesis in ß-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of ß-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with ß-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in ß-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in ß-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Transglutaminase is essential for IgA nephropathy development acting through IgA receptors.

IgA nephropathy (IgAN) is a common cause of renal failure worldwide. Treatment is limited because of a complex pathogenesis, including unknown factors favoring IgA1 deposition in the glomerular mesangium. IgA receptor abnormalities are implicated, including circulating IgA-soluble CD89 (sCD89) complexes and overexpression of the mesangial IgA1 receptor, TfR1 (transferrin receptor 1). Herein, we show that although mice expressing both human IgA1 and CD89 displayed circulating and mesangial deposits of IgA1-sCD89 complexes resulting in kidney inflammation, hematuria, and proteinuria, mice expressing IgA1 only displayed endocapillary IgA1 deposition but neither mesangial injury nor kidney dysfunction. sCD89 injection into IgA1-expressing mouse recipients induced mesangial IgA1 deposits. sCD89 was also detected in patient and mouse mesangium. IgA1 deposition involved a direct binding of sCD89 to mesangial TfR1 resulting in TfR1 up-regulation. sCD89-TfR1 interaction induced mesangial surface expression of TGase2 (transglutaminase 2), which in turn up-regulated TfR1 expression. In the absence of TGase2, IgA1-sCD89 deposits were dramatically impaired. These data reveal a cooperation between IgA1, sCD89, TfR1, and TGase2 on mesangial cells needed for disease development. They demonstrate that TGase2 is responsible for a pathogenic amplification loop facilitating IgA1-sCD89 deposition and mesangial cell activation, thus identifying TGase2 as a target for therapeutic intervention in this disease.

Pathologic calcification of adult vascular smooth muscle cells differs on their crest or mesodermal embryonic origin.

Vascular calcifications can occur in the elderly and in patients suffering from various diseases. Interestingly, depending on the pathology, different regions of the arterial system can be affected. Embryonic observations have clearly indicated that vascular smooth muscle cell (VSMC) origin is notably heterogeneous. For instance, in the aorta, VSMCs colonizing the aortic arch region derive from cardiac neural crest cells, whereas those populating the descending aorta derive from the mesoderm. We examined here whether the embryonic origin of aortic VSMCs would correlate with their ability to mineralize. Under hyperphosphatemic conditions that induce vascular calcifications, we performed ex vivo aortic explant cultures as well as in vitro VSMC cultures from wild-type mice. Our data showed that VSMC embryonic origin affects their ability to mineralize. Indeed, the aortic arch media made up of VSMCs of neural crest origin calcifies significantly earlier than the descending aorta composed of VSMCs, which are mesoderm-derived. Similar results were obtained with cultured VSMCs harvested from both aortic regions. We also demonstrated that in a mouse model deficient in matrix Gla protein, a potent calcification inhibitor, developing extensive and spontaneous medial calcifications of the aorta, lesions initiate in the aortic arch. Subsequently, calcifications progress outside the aortic arch region and ultimately spread all over the entire arterial tree, including the descending aorta. Altogether, our results support an unsuspected correlation between VSMCs of embryonic origin and the timing of appearance of calcifications.

Targeting bone morphogenetic protein signaling on renal and vascular diseases.

PURPOSE OF REVIEW: Normal development and adult physiology of the kidney and vasculature rely heavily on bone morphogenetic proteins (BMPs). Here we compile evidence that favors the notion that BMPs are also critically involved in the process of generation and maintenance of renal and vascular diseases. RECENT FINDINGS: Molecular manipulation of BMP signaling in vivo and in vitro has been instrumental in showing the protective role of BMPs on renal fibrosis and diabetic nephropathy. Similarly, activation of those pathways produces phenotypic changes in vascular smooth muscle and endothelial cells, tightly linked to the pathogenesis of vascular calcification, hypertrophy and atherosclerosis. SUMMARY: Gain-of-function and loss-of-function experiments targeting BMP pathway agonists and inhibitors lead to significant progress in the comprehension of renal and vascular normal and altered behavior. The demonstration that BMP signaling plays an important part in pathological conditions of the vasculature and the kidney opens up possibilities for the development of diagnostic and therapeutic tools.

Amitriptyline attenuates interstitial inflammation and ameliorates the progression of renal fibrosis.

Amitriptyline is a pleiotropic tricyclic antidepressant, which has anti-oxidant and anti-inflammatory properties. We tested whether amitriptyline might be useful in the treatment of chronic renal disease using the mouse model of unilateral ureteral obstruction. Amitriptyline caused a significant reduction of interstitial fibrosis, determined by Masson's staining, with minimal myofibroblast formation and macrophage infiltration following ureteral obstruction. Using quantitative PCR we found that this treatment significantly reduced the expression of key molecular markers of progressive tubulointerstitial injury such as osteopontin, MCP-1, ICAM-1, and TGF-beta1 compared to their level in a saline-treated control group. Sublethal X-irradiation or mycophenolate mofetil, treatments that reduce inflammation, were comparable to amitriptyline in the reduction of interstitial fibrosis and macrophage infiltration. These studies in animals suggest that amitriptyline is worth testing as a therapeutic agent that might preserve renal function by blocking inflammation and renal fibrosis.

Endostatin, an antiangiogenic protein, is expressed in the unilateral ureteral obstruction mice model.

BACKGROUND: Extracellular matrix accumulation, epithelial-to-mesenchymal transition, tubular atrophy and loss of peritubular capillary network are hallmarks of tubulointerstitial injury in progressive renal diseases. In this study, we analyzed endostatin expression in kidneys subjected to unilateral ureteral obstruction (UUO). METHODS: Collagen XVIII mRNA expression was evaluated by real-time polymerase chain reaction (PCR). Endostatin and CD31 protein levels were analyzed by Western blot and immunohistochemistry. In vitro quantification of collagen XVIII and fibrosis-related genes in HK2 cells was performed by real-time PCR. RESULTS: UUO significantly increased collagen XVIII mRNA expression and released a 30-kDa endostatin fragment. Immunohistochemistry revealed endostatin expression increased in injured tissue, mainly on tubular cells. Of interest, expression of CD31 was significantly reduced by UUO. Endostatin administration in vitro did not modify the expression of genes related to fibrosis development. However, in vitro TGF-beta1 administration induced expression of collagen XVIII/endostatin mRNA in human tubular cells. CONCLUSION: Endostatin is expressed during the progression of renal fibrosis in vitro and in vivo, suggesting a role for endostatin in development of tubulointerstitial injury.

Gremlin promotes vascular smooth muscle cell proliferation and migration.

Recent reports highlight the importance of BMP in the vasculature. We investigated the expression pattern and role of the BMP antagonist gremlin in VSMC. We detected gremlin mRNA constitutive expression in adult and embryonic rat aortic VSMC, and in rat carotids. In vitro analysis demonstrated that angiotensin II, TGF-beta1 and PDGF induced significant changes in gremlin mRNA expression. Gremlin stable overexpression in A7r5 cells blocked BMP signaling. BMP-induced reduction in VSMC DNA synthesis was markedly inhibited by gremlin overexpression. In fact, gremlin overexpression increased DNA synthesis and cell counts, and accelerated cell cycle progression of VSMC, through mechanisms that include p27(kip1) down-regulation. Gremlin also led to marked increments in VSMC migration. In addition, gremlin gene silencing promoted a significant blockade on cell proliferation and migration. In vivo studies disclosed increased gremlin protein expression in the neointima of balloon-injured carotid arteries. In summary, the BMP antagonist gremlin is constitutively expressed in the normal vasculature. Gremlin induces VSMC proliferation and migration and is significantly regulated by growth factors and injury. We postulate that gremlin plays a part in the development of pathological phenotypic changes of adult VSMC.

Endostatin expression in the murine model of ischaemia/reperfusion-induced acute renal failure.

BACKGROUND: Renal ischaemia-hypoxia is a leading cause of acute renal failure, a clinical condition associated with rapid loss of renal function and high rates of mortality. Renal proximal tubular cells are the most severely injured during renal ischaemia, caused by the breakdown of the extracellular matrix of the tubular basement membrane. Endostatin is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage and it is well-known as being an inhibitor of angiogenesis. In vitro, endostatin inhibits endothelial cell proliferation and migration, as well as tubule formation. In vivo, it has a potent inhibitory effect on tumour growth. In this study, we analysed endostatin gene expression in C57BL/6 mouse kidneys subjected to ischaemia/reperfusion. METHODS: Ischaemic renal failure was induced via 45 min of bilateral occlusion of the renal artery and vein, followed by 12 h or 24 h of reperfusion. Whole-kidney homogenate and total RNA were extracted for examination by western blot analysis and quantitative polymerase chain reaction. The immunohistological examination revealed increased endostatin expression in injured kidney, mainly in the proximal tubule and collecting ducts. RESULTS: Endostatin/collagen XVIII mRNA and protein expression increased during ischaemia and within 12 h of reperfusion. In the western blot assay, we identified increased expression of the 30 kDa endostatin-related fragment and of matrix metalloproteinase-9. CD31 was significantly expressed during reperfusion (P < 0.05). Immunohistological examination revealed glomerular and tubulointerstitial expression of endostatin. CONCLUSION: These data suggest the local synthesis of a 30 kDa endostatin-related fragment following acute renal failure and suggest its role in the modulation of renal capillary density.