Saleh Yazdani

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

Vascular endothelial growth factor C levels are modulated by dietary salt intake in proteinuric chronic kidney disease patients and in healthy subjects

BACKGROUND Recent experimental findings demonstrate vascular endothelial growth factor C (VEGF-C)-mediated water-free storage of salt in the interstitium, which prevents a salt-sensitive blood pressure state. It is unknown whether this mechanism plays a role in salt homeostasis and regulation of blood pressure in humans as well. Therefore, we investigated circulating VEGF-C levels and blood pressure during different well-controlled salt intake in chronic kidney disease (CKD) patients and in healthy subjects. METHODS In two crossover studies, non-diabetic proteinuric CKD patients (n = 32) and healthy subjects (n = 31) were treated with consecutively a low-sodium diet (LS, aim 50 mmol Na(+)/day) and a high-sodium diet (HS, aim 200 mmol Na(+)/day) in random order, during two 6-week (CKD patients) and two 1-week periods (healthy subjects). RESULTS We found that VEGF-C levels are higher during HS than during LS in CKD patients (P = 0.034) with a trend towards higher VEGF-C in healthy subjects as well (P = 0.070). In CKD patients, HS was associated with higher NT-proBNP levels (P = 0.005) and body weight (P = 0.013), consistent with extracellular volume (ECV) expansion and with higher blood pressure (P < 0.001), indicating salt sensitivity. In healthy subjects, blood pressure was not affected by dietary salt (P = 0.14), despite a rise in ECV (P = 0.023). DISCUSSION Our findings support a role for VEGF-C-mediated salt homeostasis in humans. Considering the salt sensitivity of blood pressure, this buffering mechanism appears to be insufficient in proteinuric CKD patients. Future studies are needed to provide causality and to substantiate the clinical and therapeutic relevance of this VEGF-C regulatory mechanism in humans.

Proteinuria Triggers Renal Lymphangiogenesis Prior to the Development of Interstitial Fibrosis

Proteinuria is an important cause of progressive tubulo-interstitial damage. Whether proteinuria could trigger a renal lymphangiogenic response has not been established. Moreover, the temporal relationship between development of fibrosis, inflammation and lymphangiogenesis in chronic progressive kidney disease is not clear yet. Therefore, we evaluated the time course of lymph vessel (LV) formation in relation to proteinuria and interstitial damage in a rat model of chronic unilateral adriamycin nephrosis. Proteinuria and kidneys were evaluated up to 30 weeks after induction of nephrosis. LVs were identified by podoplanin/VEGFR3 double staining. After 6 weeks proteinuria was well-established, without influx of interstitial macrophages and myofibroblasts, collagen deposition, osteopontin expression (tubular activation) or LV formation. At 12 weeks, a ∼3-fold increase in cortical LV density was found (p<0.001), gradually increasing over time. This corresponded with a significant increase in tubular osteopontin expression (p<0.01) and interstitial myofibroblast numbers (p<0.05), whereas collagen deposition and macrophage numbers were not yet increased. VEGF-C was mostly expressed by tubular cells rather than interstitial cells. Cultured tubular cells stimulated with FCS showed a dose-dependent increase in mRNA and protein expression of VEGF-C which was not observed by human albumin stimulation. We conclude that chronic proteinuria provoked lymphangiogenesis in temporal conjunction with tubular osteopontin expression and influx of myofibroblasts, that preceded interstitial fibrosis.

Lymphangiogenesis in renal diseases: passive bystander or active participant?

Lymphatic vessels (LVs) are involved in a number of physiological and pathophysiological processes such as fluid homoeostasis, immune surveillance, and resolution of inflammation and wound healing. Lymphangiogenesis, the outgrowth of existing LVs and the formation of new ones, has received increasing attention over the past decade on account of its prominence in organ physiology and pathology, which has been enabled by the development of specific tools to study lymph vessel functions. Several studies have been devoted to renal lymphatic vasculature and lymphangiogenesis in kidney diseases, such as chronic renal transplant dysfunction, primary renal fibrotic disorders, proteinuria, diabetic nephropathy and renal inflammation. This review describes the most recent findings on lymphangiogenesis, with a specific focus on renal lymphangiogenesis and its impact on renal diseases. We suggest renal lymphatics as a possible target for therapeutic interventions in renal medicine to dampen tubulointerstitial tissue remodelling and improve renal functioning.

Basement membrane zone collagens XV and XVIII/proteoglycans mediate leukocyte influx in renal ischemia/reperfusion.

Collagen type XV and XVIII are proteoglycans found in the basement membrane zones of endothelial and epithelial cells, and known for their cryptic anti-angiogenic domains named restin and endostatin, respectively. Mutations or deletions of these collagens are associated with eye, muscle and microvessel phenotypes. We now describe a novel role for these collagens, namely a supportive role in leukocyte recruitment. We subjected mice deficient in collagen XV or collagen XVIII, and their compound mutant, as well as the wild-type control mice to bilateral renal ischemia/reperfusion, and evaluated renal function, tubular injury, and neutrophil and macrophage influx at different time points after ischemia/reperfusion. Five days after ischemia/reperfusion, the collagen XV, collagen XVIII and the compound mutant mice showed diminished serum urea levels compared to wild-type mice (all p<0.05). Histology showed reduced tubular damage, and decreased inflammatory cell influx in all mutant mice, which were more pronounced in the compound mutant despite increased expression of MCP-1 and TNF-α in double mutant mice compared to wildtype mice. Both type XV and type XVIII collagen bear glycosaminoglycan side chains and an in vitro approach with recombinant collagen XVIII fragments with variable glycanation indicated a role for these side chains in leukocyte migration. Thus, basement membrane zone collagen/proteoglycan hybrids facilitate leukocyte influx and tubular damage after renal ischemia/reperfusion and might be potential intervention targets for the reduction of inflammation in this condition.

Targeted inhibition of renal Rho kinase reduces macrophage infiltration and lymphangiogenesis in acute renal allograft rejection

The Rho kinase pathway plays an important role in epithelial dedifferentiation and inflammatory cell infiltration. Recent studies suggest that inflammation promotes lymphangiogenesis, which has been associated with renal allograft rejection. We investigated whether targeted inhibition of the Rho kinase pathway in proximal tubular cells reduces inflammation and lymphangiogenesis in acute renal allograft rejection. The Rho kinase inhibitor Y27632 was coupled to lysozyme (Y27632-lysozyme), providing a kidney-specific conjugate that can release its drug in proximal tubular cells. Isogenic (Fisher-Fisher, n=18), or allogenic (Fisher-Lewis, n=24) kidney transplantations were performed, with the contralateral kidney remaining in situ. To elicit acute rejection, no immunosuppressive treatment was given. Animals were treated daily with Y27632-lysozyme (10 mg/kg/day i.v.) or vehicle (saline i.v.) until sacrifice (1 or 4 days post-transplantation). After allogenic transplantation, interstitial macrophage accumulation was strongly reduced by Y27632-lysozyme at day 4 after transplantation. Interstitial lymphangiogenesis, which was induced in allografts as compared to control kidney, was also reduced by renal Rho kinase inhibition at day 4 after transplantation. The increase of vimentin and procollagen-1alpha1 gene expression in renal allografts from day 1 to day 4 after transplantation was significantly reduced by Y27632-lysozyme. Y27632-lysozyme did not affect systolic blood pressure in isogenic or allogenic transplantation groups. In cultured tubular epithelial cells (NRK-52E), Rho kinase inhibition dose-dependently reduced IL-1β-induced MCP-1 gene expression. Renal inhibition of Rho kinase causes a marked reduction in renal inflammation and renal lymphangiogenesis during acute transplant rejection, suggesting that this treatment regimen is a valuable future treatment in renal transplantation.

Identification of novel genes associated with renal tertiary lymphoid organ formation in aging mice.

A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10−137) in male mice; Ctnnbip1 (P = 6.42×10−267) and Tnfrsf8 (P = 5.42×10−245) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation.

dl-propargylglycine reduces blood pressure and renal injury but increases kidney weight in angiotensin-II infused rats

Hydrogen sulfide (H2S), carbon monoxide (CO) and nitric oxide (NO) share signaling and vasorelaxant properties and are involved in proliferation and apoptosis. Inhibiting NO production or availability induces hypertension and proteinuria, which is prevented by concomitant blockade of the H2S producing enzyme cystathionine γ-lyase (CSE) by d,l-propargylglycine (PAG). We hypothesized that blocking H2S production ameliorates Angiotensin II (AngII)-induced hypertension and renal injury in a rodent model. Effects of concomitant administration of PAG or saline were therefore studied in healthy (CON) and AngII hypertensive rats. In CON rats, PAG did not affect systolic blood pressure (SBP), but slightly increased proteinuria. In AngII rats PAG reduced SBP, proteinuria and plasma creatinine (180 ± 12 vs. 211 ± 19 mmHg; 66 ± 35 vs. 346 ± 92 mg/24 h; 24 ± 6 vs. 47 ± 15 μmol/L, respectively; p < 0.01). Unexpectedly, kidney to body weight ratio was increased in all groups by PAG (p < 0.05). Renal injury induced by AngII was reduced by PAG (p < 0.001). HO-1 gene expression was increased by PAG alone (p < 0.05). PAG increased inner cortical tubular cell proliferation after 1 week and decreased outer cortical tubular nucleus number/field after 4 weeks. In vitro proximal tubular cell size increased after exposure to PAG. In summary, blocking H2S production with PAG reduced SBP and renal injury in AngII infused rats. Independent of the cardiovascular and renal effects, PAG increased HO-1 gene expression and kidney weight. PAG alone increased tubular cell size and proliferation in-vivo and in-vitro. Our results are indicative of a complex interplay of gasotransmitter signaling/action of mutually compensatory nature in the kidney.

Drug targeting to myofibroblasts : Implications for fibrosis and cancer

ABSTRACT Myofibroblasts are the key players in extracellular matrix remodeling, a core phenomenon in numerous devastating fibrotic diseases. Not only in organ fibrosis, but also the pivotal role of myofibroblasts in tumor progression, invasion and metastasis has recently been highlighted. Myofibroblast targeting has gained tremendous attention in order to inhibit the progression of incurable fibrotic diseases, or to limit the myofibroblast‐induced tumor progression and metastasis. In this review, we outline the origin of myofibroblasts, their general characteristics and functions during fibrosis progression in three major organs: liver, kidneys and lungs as well as in cancer. We will then discuss the state‐of‐the art drug targeting technologies to myofibroblasts in context of the above‐mentioned organs and tumor microenvironment. The overall objective of this review is therefore to advance our understanding in drug targeting to myofibroblasts, and concurrently identify opportunities and challenges for designing new strategies to develop novel diagnostics and therapeutics against fibrosis and cancer. Graphical abstract Figure. No Caption available.

Interferon gamma peptidomimetic targeted to interstitial myofibroblasts attenuates renal fibrosis after unilateral ureteral obstruction in mice

Renal fibrosis cannot be adequately treated since anti-fibrotic treatment is lacking. Interferon-γ is a pro-inflammatory cytokine with anti-fibrotic properties. Clinical use of interferon-γ is hampered due to inflammation-mediated systemic side effects. We used an interferon-γ peptidomimetic (mimγ) lacking the extracellular IFNγReceptor recognition domain, and coupled it to the PDGFβR-recognizing peptide BiPPB. Here we tested the efficacy of mimγ-BiPPB (referred to as “Fibroferon”) targeted to PDGFβR-overexpressing interstitial myofibroblasts to attenuate renal fibrosis without inducing inflammation-mediated side effects in the mouse unilateral ureter obstruction model. Unilateral ureter obstruction induced renal fibrosis characterized by significantly increased α-SMA, TGFβ1, fibronectin, and collagens I and III protein and/or mRNA expression. Fibroferon treatment significantly reduced expression of these fibrotic markers. Compared to full-length IFNγ, anti-fibrotic effects of Fibroferon were more pronounced. Unilateral ureter obstruction-induced lymphangiogenesis was significantly reduced by Fibroferon but not full-length IFNγ. In contrast to full-length IFNγ, Fibroferon did not induce IFNγ-related side-effects as evidenced by preserved low-level brain MHC II expression (similar to vehicle), lowered plasma triglyceride levels, and improved weight gain after unilateral ureter obstruction. In conclusion, compared to full-length IFNγ, the IFNγ-peptidomimetic Fibroferon targeted to PDGFβR-overexpressing myofibroblasts attenuates renal fibrosis in the absence of IFNγ-mediated adverse effects.