February 18, 2025

These data also present that TGF- from infiltrating cells might play a role in interstitial fibrosis after renal reperfusion in the later phase, but it appeared to contribute mainly, if not exclusively, to CX3CR1-independent fibrosis in the model

These data also present that TGF- from infiltrating cells might play a role in interstitial fibrosis after renal reperfusion in the later phase, but it appeared to contribute mainly, if not exclusively, to CX3CR1-independent fibrosis in the model. Open in a separate window Figure 6-6944 TGF- is induced during ischemia-reperfusion Prochloraz manganese injury of the kidney in a CX3CR1-independent manner. acute kidney allograft rejection, they have not had an impact on late graft failure, which may result in part from early ischemic injury.2,3 Late allograft failure afflicts a growing number of patients, fed by the large increase in patients living with end-stage renal disease since the advent of hemodialysis.4C6 Recently, chronic allograft nephropathy, characterized by progressive renal dysfunction, interstitial inflammation and fibrosis, and vascular occlusion, has been identified as the chief cause of late graft failure.7 All other causes of ischemic renal disease are also characterized by inflammation and fibrosis. Rodent models of renal ischemia-reperfusion injury have been developed in an effort to develop insights into pathogenesis at the molecular level. Recent studies Prochloraz manganese using such models have succeeded in Prochloraz manganese delineating many factors that are involved in inflammation8; however, osteopontin is the only molecular determinant of fibrosis identified to date.9 Transforming growth factor (TGF)- and platelet-derived growth factor (PDGF) are well-characterized factors that promote fibrosis in many diseases and organs, including the kidney.10,11 PDGF, which stimulates fibroblast proliferation and production of extracellular matrix, is actually a family of four molecules, PDGF-A and -B and the newly discovered PDGF-C and -D.12 PDGF-B has been implicated in renal fibrosis based on the effects of direct injection of the factor into rat kidney for 3 minutes, and platelet-rich plasma was then collected. Centrifugation of the platelet-rich plasma at 1300 for 10 minutes produced a platelet pellet. Platelets were labeled with PKH26 red fluorescent cell linker mini kit (Sigma, St. Louis, MO) using the method of Michelson and colleagues24 with minor modifications. Platelets were resuspended in Diluent C at 4 109/ml to which 10 mol/L prostaglandin I2 (PGI2) was added. An equal volume of Diluent C containing freshly prepared 4 mol/L PKH26 was added, and the suspension was mixed and incubated for 8 minutes at room temperature with occasional inversion. An equal volume of citrate-albumin PGE1 buffer (11 mmol/L dextrose, 128 mmol/L NaCl, 4.3 mmol/L NaH2PO4, Prochloraz manganese 7.5 mmol/L Na2HPO4, 4.8 mmol/L trisodium citrate, 2.4 mmol/L citric acid, 0.35% bovine serum albumin, 0.33 mol/L PGE1, pH 6.5) was added. The mixture was incubated for 1 minute and centrifuged. The pellet was resuspended in 5 ml of citrate-albumin-PGE1 buffer, incubated for 10 minutes, centrifuged, and resuspended in Tyrodes solution (Sigma) with 0.35% albumin and 3 U/ml apyrase at a platelet count of 2.0 109/ml. To evaluate the role of CX3CR1 in the accumulation of platelets in the injured kidney, 2 108 PKH26-labeled platelets from wild-type mice or CX3CR1-deficient mice in a total volume of 100 l were injected in the tail vein of wild-type mice just before ischemia-reperfusion injury. Immunohistochemistry One portion of the renal tissue was fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with periodic acid-Schiff reagent, naphthol AS-D chloroacetate esterase, Gomoris trichrome, or indicated antibodies. Another portion of fresh renal tissue was embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap-frozen on dry ice. Frozen sections were used to detect PKH26-labeled platelets and for immunohistochemistry using antibodies directed against CX3CR1 and F4/80. Deparaffinized sections were treated with Target river solution (DAKO, Carpinteria, PITX2 CA) before staining of fractalkine and -smooth.