Pieter Klok

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation

BACKGROUND/AIMS To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.

Specificity of Antibodies to Nitric Oxide Synthase Isoforms in Human, Guinea Pig, Rat, and Mouse Tissues

Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several antibodies did not react with the expected cell populations in a certain species, or reacted in previously unknown patterns. In addition, different antibodies to the same isoform rarely detected identical cell populations, even within one species. Most of these unexpected immunoreactivities were observed in bronchial epithelial, glomerular epithelial, and vascular smooth muscle cells. These unexpected results usually occurred when the antibodies were tested in other organs or species than that to which they were originally raised. We therefore strongly recommend the use of anti-NOS antibodies only after careful immunohistological and biochemical analysis of their reactivity in the organ and species to be studied.

Inhibition of Renal Rho Kinase Attenuates Ischemia/Reperfusion-Induced Injury

The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen Ialpha1, TGF-beta1, tissue inhibitor of metalloproteinase 1, and alpha-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of alpha-smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.

Pregnancy and preeclampsia affect monocyte subsets in humans and rats.

Introduction Both nonclassical and intermediate monocytes have been implicated in different inflammatory conditions. We hypothesized that these monocytes would increase during pregnancy, a condition associated with generalized activation of inflammatory responses and that they would increase even more during preeclampsia, in which inflammatory responses are further stimulated. In the present study we investigated changes in monocyte subsets during healthy pregnancy and preeclampsia in humans and rats. Methods Blood monocyte subsets of nonpregnant, preeclamptic and healthy pregnant women were identified with CD14 and CD16. In nonpregnant and pregnant rats, blood monocytes were identified with CD172a and CD43, as well as in rats infused with adenosine triphosphate (ATP), a pro-inflammatory stimulus known to induce preeclampsia-like symptoms. Total and CD206-positive macrophages were quantified in placentas of these animals. Results Lower percentages of classical monocytes were found in pregnant women (91%–[83–98%]) compared to nonpregnant women (94%–[90–98%]) and even less in preeclamptic patients (90%–[61–92%]). In contrast, the percentage of combined nonclassical/intermediate monocytes was higher in pregnant women (8.5%–[2.3–16.6%] vs. 5.6%–[1.9–9.5%]) and even higher in preeclamptic patients (9.9%–[7.8–38.7%]), which was caused by a selective increase of intermediate monocytes. In rats, we also found lower percentages of classical monocytes and higher percentages of nonclassical monocytes in pregnant versus nonpregnant rats. ATP infusion increased the percentage of nonclassical monocytes in pregnant rats even further but not in nonpregnant rats. These nonclassical monocytes showed a more activated phenotype in pregnant ATP-infused rats only. Mesometrial triangles of ATP-infused rats had less CD206-positive macrophages as compared to those of saline-infused rats. Conclusion The higher percentage of nonclassical/intermediate monocytes found in pregnancy and preeclampsia confirms their association with inflammatory responses. The observation that ATP stimulated numbers/activation of nonclassical monocytes in pregnant rats only, suggests that nonclassical monocytes are specifically altered in pregnancy and may play a role in the pathophysiology of preeclampsia.

c-Jun NH2-terminal kinase is crucially involved in renal tubulo-interstitial inflammation.

Chronic inflammation is a major outcome determinant in several renal disorders. Induction of monocyte chemoattractant protein (MCP)-1 expression in tubular epithelial cells contributes importantly to the recruitment of inflammatory cells from the circulation toward the damaged tubulo-interstitium. Because the MCP-1 gene contains several c-Jun binding sites, we hypothesized that the c-Jun NH2-terminal kinase (JNK) pathway regulates MCP-1 expression and subsequently tubulo-interstitial inflammation. This was investigated in cultured rat tubular epithelial cells (NRK-52E) and in the rat unilateral ischemia/reperfusion (I/R) model. In NRK-52E cells, the JNK inhibitor anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125) reduced interleukin-1β-, transforming growth factor-β-, or bovine serum albumin-induced MCP-1 expression in a potent manner (up to 150-fold). In the rat I/R model, JNK activation was low in controls but induced in tubular cells from 30 min after I/R. The extent of JNK activation correlated with interstitial macrophage accumulation. Treatment with SP600125 (30 mg/kg/day i.p. for 4 days) reduced renal c-Jun activation; MCP-1, osteopontin, and vimentin expression; and interstitial macrophage and T-cell accumulation (all p < 0.05). In human renal disease, we also found induction of JNK activation, which correlated strongly with interstitial macrophage accumulation, tubulointerstitial fibrosis, and renal function loss. In conclusion, these data indicate that the JNK pathway plays an important role in renal inflammation, at least in part through induction of MCP-1 gene expression in tubular epithelial cells.

Pneumococcal Conjugate Vaccines Overcome Splenic Dependency of Antibody Response to Pneumococcal Polysaccharides

ABSTRACT Protection against infections with Streptococcus pneumoniae depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasible. We show that in rats, vaccination with a pneumococcal conjugate vaccine can induce good antibody responses even after splenectomy, particularly after a second dose. The spleen remains necessary for a fast, primary response to (blood-borne) polysaccharides, even when they are presented in a conjugated form. Coadministration of a conjugate vaccine with additional nonconjugated polysaccharides of other serotypes did not improve the response to the nonconjugated polysaccharides. We conclude that pneumococcal conjugate vaccines can be of value in protecting asplenic or hyposplenic patients against pneumococcal infections.

Chronic blockade of angiotensin II action prevents glomerulosclerosis, but induces graft vasculopathy in experimental kidney transplantation

Long‐term renin–angiotensin system blockade is beneficial in a variety of renal diseases. This study examines the long‐term (34 weeks) effects of the angiotensin‐converting enzyme inhibitor lisinopril and the angiotensin II receptor type I blocker L158,809 in the Fisher to Lewis rat model of chronic renal transplant failure. Treatment in allografted rats with lisinopril or L158,809 was initiated 10 days after transplantation, or at the time when proteinuria exceeded 50 mg/24 h. Untreated allografts and syngrafts served as controls. In contrast to syngrafts, untreated allografts developed proteinuria, hypercholesterolaemia, interstitial damage, and glomerulosclerosis. Lisinopril or L158,809 treatment in allografts starting at day 10 after transplantation completely prevented this, with the exception of interstitial damage, but this treatment also caused a reduction in blood pressure and renal function. Moreover, the intimal surface area of the renal arteries was dramatically increased in allografts treated with either lisinopril or L158,809 compared with untreated allografted rats. Treatment once proteinuria had developed was less effective in preventing glomerulosclerosis, but also caused less intimal expansion. Thus, chronic renin–angiotensin system blockade preserves glomerular morphology in the absence of proteinuria, but enhances intimal hyperplasia and reduces renal function in experimental transplantation. In view of these results, it should be questioned whether such treatment benefits renal transplant patients in the long term. Copyright

Induction of glomerular alkaline phosphatase after challenge with lipopolysaccharide

Summary.  Alkaline phosphatase (AP) can be considered as a host defence molecule since this enzyme is able to detoxify bacterial endotoxin at physiological pH. The question emerged whether this anti‐endotoxin principle is inducible in the glomerulus and if so, which glomerular cells might be involved in the expression of ectoAP after stimulation with pro‐inflammatory agents. Therefore kidneys of rats treated with either lipopolysaccharide (LPS), E. coli bacteria or non‐toxic monophosphoryl lipid A (MPLA) were examined for AP activity 6 or 24 h after challenge. In addition cultures of endothelial cells or mesangial cells were evaluated for AP activity after stimulation with either LPS, TNFα or IL‐6, and mRNA for AP was studied in TNFα‐stimulated and control mesangial cells. The results show significant up‐regulation of glomerular AP in LPS‐ or E. coli‐injected rats compared to rats injected with MPLA. Endothelial and mesangial cells in vitro showed significant up‐regulation of AP activity following stimulation with LPS, TNFα or IL‐6, whereas increased mRNA for AP was observed in mesangial cells after TNFα stimulation compared to non‐stimulated control cells. Since it appeared that hydrolysis occurred when endotoxin was used as a substrate in the histochemical staining, we concluded that inducible glomerular ectoAP may reflect a local endotoxin detoxifying principle of the kidney.

Minimal change-like glomerular alterations induced by a human plasma factor

Circulating factors, including the plasma protease (100 KF) described previously, have been suspected to play a role in the pathogenesis of minimal change disease (MCD) for several decades. This factor was able to induce MCD-like alterations in kidney tissue in vitro, i.e. impairment of glomerular polyanion (GPA), as well as glomerular ecto-ATPase. We conducted permeability studies using alternate perfusion of the rat kidney ex vivo according to standard techniques. Either native 100 KF (n = 7) or control factor (n = 7) perfusion, followed by perfusion with diluted rat serum was carried out, while urine samples were collected by ureter cannulation. Total urinary protein (by spectrophotometry) as well as IgG (by ELISA) and albumin (by rocket electrophoresis) were measured. Sections of perfused kidneys were stained (immuno-) histochemically for GPA and glomerular ecto-ATPase, and the stainability was quantified using image analysis and expressed as arbitrary units. The results show significantly increased protein leakage after perfusion of 100 KF versus control factor (150.0 +/- 48.9 vs. 33.2 +/- 7.7 micrograms/min, p < or = 0.01), while the IgG/albumin ratio has decreased (12.0 +/- 9.4 vs 26.9 +/- 14.4%, p < or = 0.01). Plasma protein leakage after 100KF perfusion is associated with a significant loss of GPA (57.3 +/- 27.5 vs. 98.4 +/-12.0, p < or = 0.01) and significant decrease of glomerular ecto-ATPase expression (28.7 +/- 11.5 vs. 79.5 +/- 15.0, p < or = 0.001). The capability of 100KF to induce MCD-like glomerular lesions, in association with selectively increased permeability for plasma proteins, suggests that this human plasma constituent may be important in the pathogenesis of MCD.

Selective delivery of IFN-γ to renal interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis

Renal fibrosis leads to end‐stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN‐γ is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN‐γ causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell‐specific delivery of IFN‐γ to platelet‐derived growth factor receptor β (PDGFRβ)‐expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN‐γ conjugated to PDGFRβ‐recognizing peptide [(PPB)‐polyethylene glycol (PEG)‐IFN‐γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN‐γ. PDGFRβ expression was >3‐fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with α‐smooth muscle actin‐positive (SMA+) myofibroblasts. In vitro, PPB‐PEG‐IFN‐γ significantly inhibited col1a1, col1a2, and α‐SMA mRNA expression in TGF‐β‐activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB‐PEG‐IFN‐γ specifically accumulated in PDGFRβ‐positive myofibroblasts. PPB‐PEG‐IFN‐γ treatment significantly reduced renal collagen I, fibronectin, and α‐SMA mRNA and protein expression. Compared with vehicle treatment, PPB‐PEG‐IFN‐γ preserved tubular morphology, reduced interstitial T‐cell infiltration, and attenuated lymphangiogenesis (all P < 0.05) without affecting peritubular capillary density. PPB‐PEGIFN‐γ reduced IFN‐γ‐related side effects as manifested by reduced major histocompatibility complex class II expression in brain tissue (P < 0.05 vs. free IFN‐γ). Our findings demonstrate that specific targeting of IFN‐γ to PDGFRβ‐expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects.—Poosti, F., Bansal, R., Yazdani, S., Prakash, J., Post, E., Klok, P., vanden Born J. deBorst M.H. vanGoor H. Poelstra K. Hillebrands, J.‐L. Selective delivery of IFN‐γ to renal interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis. FASEB J. 29, 1029–1042 (2015). www.fasebj.org