Gpnmb Expression In The Kidney With Acute Ischemia/Reperfusion Injury And Antigen Presenting Cells

Acute kidney injury (AKI) is a common medical condition with high risk of death. The pathogenesis of AKI is complex and inflammation and immune response are confirmed to contribute to this pathophysiology. Macrophages (Mφ) infiltrate into the outer medulla of the postischemic kidneys and play key roles in inflammation and immunity. Although Mφare suspected to play a role in renal repair after acute kidney injury, their precise role is yet to be elucidated. In clinical medicine, early diagnosis is important to guide the treatment of AKI. The most frequently used serologic indicators for the diagnosis of renal disease are serum creatinine, serum urea nitrogen, and creatinine clearance, all of which are insensitive and nonspecific for detection of renal injury. We previously reported kidney injury molecule-1 (Kim-1) as a noninvasive and specific biomarker to detect AKI. Many other urinary proteins have been evaluated as indicators of renal injury, including neutrophil gelatinase-associated lipoclain (NGAL), interleukin-18 (IL-18) and fatty-acid binding protein (FABP), However, better individual biomarkers and/or biomarker panels will be still needed to enable earlier diagnosis and therapies, improved patient outcomes.Glyocoprotein non-metastatic melanoma b (Gpnmb), also known as hematopoietic growth factor-inducible neurokinin (HGFIN), dendritic cell-associated heparin sulfate proteoglycan integrin ligand (DC-HIL), and osteoactivin, is a type I transmembrane glycoprotein. Its extracellular domains include a heparin-binding domain, an integrin-binding arginine-glycme-aspartic acid motif, and a polycystic kidney disease sequence. Gpnmb is expressed in numerous cells and tissue, including embryonic nervous system, developing nephrons, basal layer of skin, germinal cells of hair follicles, osteoblasts, osteoclasts, Mφ, retinal pigment epithelium, and dendritic cells (DC). Depending on recent research, Gpnmb may serve as an osteoblast differentiation factor, and functions as a negative regulator of inflammation in macrophages and activation of T lymphocytes. Gpnmb may play a role in the inflammation response and immune regulation.Objective:To detect the expression of Gpnmb in the kidney and urine of normal mice or rats and I/R mice or rats. To observe Gpnmb expression in different types of primary cultured mouse and rat bone marrow derived macrophages (BMMφ) and bone marrow derived dendritic cells (BMDC). To study the Gpnmb expression in various renal cells.Methods:1,C57BL/6J mice or SD rats were subjected to 30 min of bilateral renal ischemia or sham-surgery on day 0. Ischemia was induced by clamping both renal pedicles with nontraumatic microaneurysm clamps. After the clamps were removed reperfusion of the kidneys was visually confirmed. At day 1,2,3,4,5,7,14, the animals were sacrificed after anticoagulant serum and urine were collected. The kidneys were harvested which was prepared for light microscopy, immunofluorescence staining and northern blot analysis and mRNA extraction.2,Gpnmb mRNA expression in renal tissue was detected by Northern blot analysis, qPCR and in situ hybridization; Gpnmb protein expression was detected by double immunofluorscence staining, flow cytometry analysis; Gpnmb expression in the urine was detected by Western blot analysis.3,Generate primary cultured mouse and rat BMMφ. Western blot analysis, flow cytometry analysis and double immunofluorscence staining of Gpnmb and Mφbiomarkers were used to study Gpnmb expression in the BMMφ. Differentiate MO BMMcp to M1 BMMφand M2 BMMφ, using the expression of biomarkers and cytokines to confirm the successful differentiation of these two types of BMMφ. Then to detect the expression of Gpnmb in M1 BMMφand M2 BMMφby immunofluorescence staining, flow cytometry analysis, Western blot analysis and ELISA.4,Treat the RAW264.7 cells with lipopolysaccharide (LPS) (50ng/ml) for 24 hours and paralleled unstimulated RAW264.7 cells were used as control. The Gpnmb expression in RAW264.7 cells with LPS or without LPS were detected by immunofluorescence staining, Western blot analysis and EL ISA.5,Generate primary cultured mouse BMDC, using the testing of CD11c expression to confirm there are really BMDC. Then detect Gpnmb expression in BMDC using immunofluorescence staining and cytometry analysis.6,Generate primary cultured mouse and rat proximal tubular epithelial cells (PTC) and cultured various kinds of renal tubular epithelial cell lines, including LLC-PK1, NRK52e, MDCK, MIMCD3, and a human renal carcinoma cell line (769-P) and a human renal embryonic cell line (293). qPCR and immunofluorescence staining were used to study the expression of Gpnmb in these cells.7,Feed primary cultured mouse and rat PTC with red fluorescence labled Gpnmb (Gpnmb-Fc-Cy3). Paralleled cells without feeding or fed with Ox-LDL were used as negative control and positive control, respectively. Feed the primary cultured mouse BMMcφwith Gpnmb-Fc-Cy3. Paralleled BMMφfed with BSA-Cy3 or goat IgG-Cy3 was used as negative control. Observed if the primary cultured mouse and rat PTC or mouse BMMcφ can bind or uptake exogenous Gpnmb-Fc-Cy3.Results:K I/R mouse and rat model were generated:(1) Compared with control groups, Serum creatinine (SCr) in I/R mice or rats increased the day 1 after 1/R injury and then decreased gradually, and returned to normal level until day 7. (2) In the control mouse or rat kidneys, the histology was normal; I/R kidneys showed that swelling, necrotic or exfoliated renal tubular epithelial cells was detected, tubular lumen enlarged, and many inflammatory cells in renal interstitium. (3)There was low Kim-1 expression in control mouse and rat kidneys, Kim-1 expression increased in I/R mouse and rat kidneys. (4)There was low vimentin expression in control rat kidneys. Vimentin expression increased in the rat kidneys after 4 days after I/R injury.2,Gpnmb expression was highly upregulated in the kidney and urine from mice or rats with I/R injury. (1) Results of Northern blot analysis, qPCR and In situ hybridization showed that there was no detectable Gpnmb mRNA in control rat kidneys. However, the Gpnmb mRNA level in rat 1/R kidneys was highly up-regulated and was predominantly located into the infiltrating cells in the outer stripe of the outer medulla (OSOM). (2) Using immunofluorescence staining, we detected that there was no detectable Gpnmb protein expression in control rat kidneys. After I/R injury, Gpnmb protein expression was up-regulated and mainly located into the infiltrating cells in the OSOM. After 2 days of I/R injury, the Gpnmbcells were mostly located to the interstium area between renal tubule, while after 4 days of I/R injury, the Gpnmb+cells predominantly existed in the lumen of renal tubules. Double immunofluorescence staining and flow cytometry results showed that Gpnmb was co-localized with macrophage marker, such as F4/80, CD68, MHC 11 and CD86.(3) Western blot anaylsis showed that Gpnmb protein level in the control mouse urine was low. In the urine of mice with 1 day or 2 days of I/R injury, the Gpnmb level is highly up-regulated and returned to normal level at day 3 after I/R injury.3,There was Gpnmb expression in the primary cultured mouse and rat BMMcp and Gpnmb could be secreted into the conditioned media:(1) Double immunofluorescence staining showed that there was co-localization of Gpnmb and F4/80 in mouse BMMcp and CD68 in rat BMMcp, respectively. (2) Flow cytometry showed that in our primary cultured mouse BMMcp, there were 94.7±6.1% cells positive for CD llb, a marker for mouse Mφ; and 75.1±4.8% CD11b+BMMcps cells were positive for Gpnmb. (3) There was no significant difference of the total Gpnmb protein expression between MO BMMφand M2 BMMcp. But after stimulation with IFN-y and LPS, MO BMMcp were differentiated into M1 BMMcp and expressed more Gpnmb protein. Compared to the M2 BMMcp, the level of Gpnmb protein in the conditioned media of M1 BMMcp was higher, but in the cell lysates of M1 BMMcp was lower. By immunofluorescence staining, we found that according to the Gpnmb expression, BMMcp could be divided into three different groups:small mononucleated Gpnmb+BMMcp; large multinucleated Gpnmb+BMMφ; Gpnmb-BMMφ.4,There was Gpnmb expression in a mouse Mcp cell line (RAW264.7) and Gpnmb could be secreted into the conditioned media: (1) qPCR and Western blot analysis results showed that compared to unstimulated RAW 264.7 cells, Gpnmb were upregulated in RAW 264.7 cells with LPS stimulation. (2) Immunofluorescence staining showed that there were three groups of RAW264.7 cells:small mononucleated Gpnmb+RAW264.7 cells; large multinucleated Gpnmb+RAW264,7 cells; Gpnmb-RAW264.7 cells. After LPS stimulation, the Gpnmb expressed by small mononucleated Gpnmb+RAW264.7 cells went into small vesicles and anchored onto the cell membrane and then were secreted to the conditioned media, while the Gpnmb expressed by large multinucleated Gpnmb+RAW264.7 cells were still located to the perinuclear area,5^ There was Gpnmb expression in the primary cultured mouse BMDC:(1) Double immunofluorescence staining showed that there was colocalization of Gpnmb and CDllc in mouse BMDC. (2). Flow cytometry showed that in our primary cultured mouse BMDC, there were 70.28±8.25% cells positive for CDllc, a marker for mouse DC, and 33.51±2.32% CD1Ic+BMDC were positive for Gpnmb.6,Gpnmb expression was low in the primary cultured mouse and rat PTC and various renal cells. (1) qPCR showed that compared to RAW 264.7 cells, the Gpnmb mRNA expression in the primary cultured mouse PTC and various kinds of renal tubular epithelial cell lines, including LLC-PK1, NRK52e, MDCK, MIMCD3, and a human renal carcinoma cell line (769-P) and a human renal embryonic cell line (293) was low. (2) Immunofluorescence staining showed the Gpnmb protein expression in the primary cultured mouse and rat PTC was low. (3) Primary cultured mouse or rat PTC could not bind or uptake exogenous red fluorescence labled Gpnmb (Gpnmb-Fc-Cy3). (4) Primary cultured mouse BMMφcould bind and uptake Gpnmb-Fc-Cy3 to the lysosome.Conclusion:1,Gpnmb expression is low in the kidney from control mice and rats, while Gpnmb expression is up-regulated in the kidney from mice and rats with I/R injury and mainly located in APC. Gpnmb may be involved in the inflammatory response and immune response and play a role in the pathophysiological process in the AKI.(2) Gpnmb level in the urine from the control mice is low and is increased in the urine from mice with I/R injury. Gpnmb may be used as a potential marker of AKI.