The Role Of Macrophage-ARG1-Polyamine Axis To Promote Cyst Growth Of Pkd1^-/- Mice

Background: Autosomal dominant polycystic kidney disease（ADPKD）is a single gene inheritance disease characterized by bilateral,multiple and gradual expanding fluid-filled cysts.ADPKD is derived from Pkd1 inactivating tubules oppresses and damages surrounding parenchyma.Patients will finally suffer irreversible loss of renal function and undergo end stage renal disease（ESRD）.ADPKD has already been regarded as the forth pathogen for ESRD and 75% of ADPKD patients are caused by the mutation of PKD1.Inflammation and immunitcal factors play an important role in the origination and development of ADPKD.As a most important member of immune cells,macrophages might have a specific contribution to the deterioration of ADPKD.Abundant macrophages have been detected in the kidney samples of ADPKD patients and its orthologous animal models.Besides,most of them showed M2 type.Macrophage depletion could obviously postpone cysts growth in vivo and more than that,macrophage could stimulate cyst-lining epithelial cells（CLEC）proliferation via some undefined soluble factors.Methods: Pkd1^-/- mice would manifest classic cystic kidneys phenotype by conditional inactivation of Pkd1 using tamoxifen.It is an orthologous model for human ADPKD.We have found faster kidneys proliferative rate after kidney development paralleling to higher cystic index caculation and impairment of renal function.By immunohistochemistry（IHC）,macrophages infiltrating to the cystic kidneys were counted and compared among different postnatal days in rapid-onset ADPKD mice.Further we investigated the relationship between macrophage counts and kidney proliferative rate.The kidney development was normally ended at approximatly 22 th postnatal day（P22）in mice;however,compared with Pkd1+/+ mice,the kidneys of Pkd1^-/- mice would still maintain a significantly higher proliferative rate even after P22.Then the m RNA expression of kidneys between Pkd1^-/- mice and their age-matched Pkd1+/+ controls at P22 and P30 were respectively analyzed by GO analysis.Totally 204 genes that meets the following criria were screened out:（1）significantly higher expression in the kidneys of Pkd1^-/- mice compared with their Pkd1+/+ controls both at P22 and P30;（2）significantly higher expression in the kidneys of P30 Pkd1^-/- mice than that of P22 Pkd1^-/- mice;and（3）the expression of candidate genes should be independent of the postnatral ages in Pkd1+/+ mouse kidneys.Finally,only one gene,Arg1,which classically expressed in macrophage,was locked among these candidate genes.The mRNA expression of the fourteen genes which are associated with macrophage polarization were compared between P22 Pkd1-/-and Pkd1+/+ mouse kidneys and between P30 Pkd1-/-and Pkd1+/+ mouse kidneys using the Q-PCR mehtod.Arg1 were regarded as the target gene that significantly higher expressed in P30 Pkd1^-/- mice kidneys compared with P22 even after considering physiological expression.Using laser confocal microscopy we investigated the expression of macrophage polarization markers.Futher the correlation between ARG1 expression in macrophages and the proliferation rate of neiphbour cyts was analyzed.Based on above results,we have depleted the macrophages in Pkd1^-/- mice duing three stages using liposomal clodronate（LC）and compared the renal function,cystic index and kidney Ki-67 nuclei staining among different groups.Finally we used nor-NOHA which is a specific inhibitor for ARG1 to disturb L-ornithine production in vivo and investigate whether it could suspend cysts growth of Pkd1^-/- mice.Results:（1）Macrophages are obviously recruited into the kidneys during P24-P26.（2）The number of ARG1 positive macrophages infltring into the kidneys is significantly associated with cysts growth rate.（3）Macrophage depletion during Stage1（P12-P22）neither improved the renal function and kidney gross appearance nor lowered kidney proliferation rate.However,depletion during Stage2（P22-P32）remarkably improved the overall prognosis of Pkd1^-/- mice.Macrophage depletion during Stage2 and Stage3（P16-P32）leaded to the approximate prognosis including renal function and cysts growth.（4）Specific ARG1 inhibitor,nor-NOHA,failed to improve overall survival and kidney gross appearance in rapid-onset ADPKD models,although lowered kidney proliferation and protected renal function to a certain degree.（5）chronic-onset Pkd1^-/- mice presents a similar PKD phenotype as the rapid-onset model espically in end stage of the disease.（6）Urea circle and polyamine metabolism was activated in cystic kidneys.The expression of key enzymes incluiding ARG1 and ODC1 were signifncalty higher in Pkd1-/-mouse kidneys than age-mathced Pkd1+/+ controls;while NOS2 expression and NO systhesis were inhibited on the contrary.（7）ARG1 could not express in other cells except the macrophage in cystic kidney and it was capable to pump L-arginine from the circulation and then catalyze and transform it into L-ornithine which is a substrate for polyamine systhesis.（8）nor-NOHA could disturb the production of L-ornithine and inhibit polyamine metabolism.In this way,it delayed cysts growth and improve the prognosis of ADPKD in the mouse models.（9）L-lactic acid could induce macrophage expressing ARG1.Conclusions:（1）macrophage expressing ARG1 is the real contributor to promote the cysts growth in ADPKD mice;（2）macrophage-ARG1-polyamine axis and the loop of L-lactic acid-macrophage-ARG1-cysts both contribute the progress of ADPKD;（3）nor-NOHA could significantly improve the prognosis of ADPKD model and ARG1 is probably a promising therapeutic target for ADPKD.