Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and alpha-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling. Kidney International (2011) 79, 1217-1227; doi:10.1038/ki.2011.14; published online 16 March 2011

Invariant Natural Killer T Cell Agonist Modulates Experimental Focal and Segmental Glomerulosclerosis

A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis ( FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T ( iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin( ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1 alpha, IL-1 beta, IL-17, TNF-alpha, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-beta analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-beta could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-beta, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-beta through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management.

The polysaccharide fraction of Propionibacterium acnes modulates the development of experimental focal segmental glomerulosclerosis

The pathogenesis of focal segmental glomerulosclerosis (FSGS) appears to be associated with type-2 cytokines and podocyte dysfunction. In this study, we tested the hypothesis that immunization with the polysaccharide fraction of Propionibacterium acnes (PS), a pro-Th1 agonist, may subvert the type-2 profile and protect podocytes from adriamycin-induced glomerulosclerosis. Adriamycin injection resulted in albuminuria and increased serum creatinine in association with loss of glomerular podocin and podoplanin expression, which is consistent with podocyte dysfunction. Renal tissue analysis revealed the expression of transcripts for GATA3 and fibrogenic-related proteins, such as TGF-beta, tissue inhibitor of metalloproteinase-1 (TIMP-1) and metalloproteinase 9 (MMP9). In association with the expression of fibrogenic transcripts, we observed peri-glomerular expression of a-smooth muscle actin (alpha-SMA), indicating epithelial-to-mesenchymal transition, and increased expression of proliferating cell nuclear antigen (PCNA) in tubular cells, suggesting intense proliferative activity. Previous immunization with PS inhibited albuminuria and serum creatinine in association with the preservation of podocyte proteins and inhibition of fibrogenic transcripts and the expression of alpha-SMA and PCNA proteins. Tissue analysis also revealed that PS treatment induced expression of mRNA for GD3 synthase, which is a glycosiltransferase related to the synthesis of GD3, a ganglioside associated with podocyte physiology. In addition, PS treatment inhibited the influx of inflammatory CD8(pos) and CD11b(pos) cells to kidney tissue. Finally, PS treatment on day 4 post-ADM, a period when proteinuria was already established, was able to improve renal function. Thus, we demonstrate that the PS fraction of P. acnes can inhibit FSGS pathogenesis, suggesting that immunomodulation can represent an alternative approach for disease management. (C) 2011 Elsevier GmbH. All rights reserved.

PRESSER MECHANISMS IN ADRIAMYCIN-INDUCED NEPHROPATHY WITH HYPERTENSION IN RATS

We explored the role of angiotensin II and vasopressin in the maintenance of blood pressure during the nephrotic syndrome of adriamycin-induced nephropathy in rats. All 91 rats treated with adriamycin developed chronic renal failure with nephrotic syndrome, which was more pronounced in the normotensive rats than the 35% who became hypertensive. Angiotensin II blockade with DuP 753 produced a significantly greater hypotensive response in both the adriamycin-hypertensive (-16+/-3 mmHg) and adriamycin-normotensive (-14+/-5 mmHg) groups than the saline-treated controls (-5+/-1 mm Hg, P<.05). Vasopressin blockade with either a V1V2 inhibitor or a selective V-1 inhibitor produced a hypotensive response in adriamycin-hypertensive rats only (by -16+/-4 and -17+/-2 mm Hg, respectively, P<.01), although the nonselective vasopressin inhibitor produced similar fluid loss and body weight reduction in all three groups. The data suggest that in adriamycin-induced nephropathy with nephrotic syndrome, angiotensin II contributes to blood pressure maintenance in both hypertensive and normotensive animals, whereas the presser action of vasopressin contributes to elevated blood pressure in hypertensive animals only.

The hemolytic uremic syndrome as a complication of adriamycin nephropathy

Rats treated with two injections of adriamycin (week 0 and week 12) developed glomerusclerosis and severe tubulointerstitial lesions as described in the literature. In addition, a number of glomerular alterations were present. These included capillary loop dilation, insudation of eosinophilic material, necrosis, duplication of the glomerular basement membrane, severe mesangiolysis with disruption of the mesangial matrix and segmental double- contours. The renal arterioles and interlobular arteries showed endothelial cell swelling. The subendothelial space was infiltrated by fibrinoid material and there was intensive fibrinoid necrosis of the wall of both arteries and arterioles extending into the glomerular tuft. These alterations were very similar to those observed in the hemolytic uremic syndrome. This observation suggests that the two injections of adriamycin, with a long interval in between them, might induce renal lesions similar to those observed in the hemolytic uremic syndrome.

Effects of cyclosporine on adriamycin induced nephropathy

The effect of cyclosporine on adriamycin nephropathy was studied over both short (6 weeks) and long (26 weeks) periods. In the short-term study, cyclosporine was introduced 2 weeks after nephritis induction and in the long-term study, 14 weeks after the first adriamycin injection. The animals with adriamycin nephropathy treated with cyclosporine, studied for 6 weeks, developed proteinuria with increased renal size and glomerular area. The nephrotic animals treated with cyclosporine showed less proteinuria than the nephrotic control animals. There were no differences in glomerular area, creatinine clearance or serum creatinine and kidney weight between the treated nephrotic group and the health control group. The nephrotic animals, studied for the longer period, developed intense proteinuria, decreased creatinine clearance, glomerular necrosis and sclerosis, severe tubulointerstitial nephritis, increased hydroxyproline concentration and tubulointerstitial area. No difference was observed between the nephrotic animals treated with cyclosporine and those not treated. In conclusion, Cyclosporine A reduced proteinuria, glomerular hypertrophy and kidney weight in rats with short-term nephropathy but had no effect on the established nephropathy.

Effect of allopurinol in the course of adriamycin induced nephropathy

The role of superoxide in adriamycin-induced nephropathy (single dose; i.v. 3 mg/kg) has been studied by blocking superoxide synthesis through the administration of allopurinol (500 mg/L in drinking water). In Experiment I (EI), allopurinol administration was started 3 days prior to nephropathy induction and continued until day 14. In Experiment II (EII) allopurinol administration was started 2 weeks after nephropathy induction and was maintained until the end of the experiment (26 weeks). Affected glomeruli frequency and tubulointerstitial lesion index (TILI) were determined at Weeks 2 and 4 (EI) and Week 26 (EII). In EI, and 24 h mean proteinuria in the nephrotic control group (NCG-I) differed from that of the treated nephrotic group (TNG-I) at Week 1 (TNG = 33.3 ± 6.39 mg/24 h; NCG = 59.8 ± 6.3 mg/24 h; p < 0.05) and 2 (NCG-I = 80.0 ± 17.5 mg/24h; TNG-I = 49.1 ± 8.4 mg/24 h; p < 0.05). No glomerular alterations were observed and TILI medians were not different in both nephrotic groups at week 2 (NCG-I = 1+: TNG = 1+) and 4 (NCG = 4+; TNG = 4+). In EII, NCG-II and TNG-II presented different 24 h proteinuria values only at Week 6, (136.91 ± 22.23 mg/24 h ad 72.66 ± 10.72 mg/24 h, respectively; p < 0.05). Between nephrotic groups, there was no statistical difference in the median of affected glomeruli (CNG-II = 56%; TNG-II = 48% and TILI (NCG-II = 8+; TNG-II = 9+). Thus, allopurinol was associated with a transient reduction in proteinuria and it did not alter the progression of the nephropathy.

Role of ticlopidine on adriamycin-induced nephropathy

The effect of ticlopidine on rats with adriamycin nephropathy was observed during 26 weeks. In the ticlopidine-treated nephrotic animals (TNG), proteinuria was less than in the untreated nephrotic animals (NG), but this difference was significant only at week 6 (TNG = 47.27 ± 16.52 versus NG = 100.08 ± 13.83 mg/24h, p < 0.01) and week 26 (TNG = 157.00 ± 28.73 versus NG = 217.00 ± 21.73 mg/24h, p< 0.01) after ADR injection. NG presented severe tubulointerstitial abnormalities with a tubulointerstitial lesion index of 3+. No difference in glomerular lesions was observed among the groups (NG median = 6%, TNG median = 4% and TCG median = 2%). The tubulointerstitial lesion index of TNG was less intense (median = 2+) but not different from those of the control groups (CG median = 1+; TCG median = 0+) nor NG (median = 3+). We concluded that the treatment with ticlopidine produced some partially beneficial effects but did not prevent the development of adriamycin-induced nephropathy.

Importance of early and continuous use of protein restriction on the progression of adriamycin nephropathy

Three experimental protocols were carried out with the aim of evaluating the role of protein restriction on the progression of the established adriamycin-induced nephropathy, and whether the protective effect of the diet persists after the diet is discontinued. The effect of a low protein diet (LPD) was studied for 6 weeks in protocol 1, 16 weeks in protocol 2 and for 28 weeks in protocol 3. In protocol 3, one group (LL) received LPD and another (NN) was given a normal protein diet (NPD). A third group (LN) received LPD for 16 weeks and then NPD for 12 weeks and a fourth group (NL) was fed NPD for 16 weeks and then LPD for 12 weeks. In protocol I the tubulo- interstitial index (TILl) of rats on LPD (Md = 2, P25 = 0.0; P75 = 3.5) after six weeks, was smaller than that of the animals on NPD (Md = 6.0; P25 = 3.0; P75 = 8.0; p < 0.05). In protocol 2, the group taking LPD presented an area of interstitial fibrosis (IF) (Md= 0.5%, P25 0.2%; P75 = 1.9%) smaller than that of the NPD group (Md = 6.8%; P25 = 5.2%; P75 = 7.1%; P < 0.05). No significant difference in the area of glomerulosclerosis (GSA) was observed between the animals on LPD (Md = 0.0%; P25 = 0.0%, P75 = 0.0%) and NPD (Md = 0.37%; P25 = 02% P75 = 1.25%; p > 0.05). In protocol 3, the group LL showed GSA (Md = 1.3%; P25 0.6%, P75 = 2.5%) and IF (Md = 3.60/0; P25 = 1.6%; P75 = 5.9%) smaller that those of LN (GSA Md = 10.1%; P25 = 6.6%; P75 = 14.8%; IF; Md = 17.3%; P25 = 14.1%; P75 = 24,5%), NL (GSA: Md = 9.1%; P25 = 5,8%; P75 = 11.7%; IF; Md = 25.0%; P25 = 20.4%; P75 = 30%), and NN (GSA: Md = 6. 75%; P25 = 4.9%; P75 = 11.7%; IF: Md = 20.9%; P25 = 16.2%; P75 = 32.4%). In conclusion, in order to be effective, LPD must be introduced early and maintained for a long period of tune.

Treatment of adriamycin-induced nephropathy with erythropoietin and G-CSF

Background: Granulocyte colony-stimulating factor (G-CSF) and Erythropoietin (EPO) are known to stimulate the growth and differentiation of progenitor cells to prevent acute renal injury. This study aimed to assess the use of growth factors to mobilize stem cell in a mouse model of adriamycin-induced chronic kidney disease. Methods: All animals were injected with adriamycin for kidney injury and allocated into three treatment groups (G-CSF, EPO and G-CSF + EPO), and a control group (adriamycin alone). Results: Number of atrophic sites, glomerulosclerosis rate and interstitial fibrosis severity score were assessed in all groups. In all treatment groups, histologic parameters did not significantly differ, but were lower than in the control group (P<.001). Scal and CD34 expressions among treatment groups showed no statistically significant difference, but were higher than in the control group (P<.0001). CD105 expression was higher in EPO and G+EPO as compared to G-CSF and the control group (P<.0001), with no statistically significant difference between the latter two groups (P = NS). Conclusion: G-CSF and EPO had a histologic protective effect, while treatment with EPO + G-CSF had no additive effects in a model of adriamycin-induced chronic kidney disease. © 2013 Societá Italiana di Nefrologia.

Fibroblast growth factor receptor inhibition synergizes with paclitaxel and doxorubicin in endometrial cancer cells

OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

A phase II study of Caelyx, liposomal doxorubicin : lack of activity in patients with advanced gastric cancer

Purpose A phase II study was designed to assess the efficacy and safety of Caelyx (liposomal doxorubicin) in patients with advanced or metastatic gastric cancer. Methods A total of 25 patients with gastric adenocarcinoma were treated with Caelyx 45 mg/m2 every 28 days as first-line therapy for advanced disease. Patients were treated until tumour progression or unacceptable toxicity. Results One patient was withdrawn from the study after experiencing a severe infusion reaction. Of the 24 evaluable patients, 1 had a partial response, 7 had stable disease and the others progressed. Side effects, in particular palmar-plantar erythrodysaesthesia and haematological toxicity, were minor. Conclusions We conclude that while this dose and schedule of Caelyx in this patient group is acceptable, further studies with this regimen cannot be recommended due to the lack of antitumour activity seen.