The second primary antibody was added to the slides, and incubated at 4☌ overnight. Slides were again washed twice with PBS and then blocked with Gentex block for 45 minutes at room temperature. Slides were then washed twice with PBS, and secondary antibodies were added at a concentration of 1:200, diluted in antibody diluent, and incubated at room temperature for 30 minutes. Primary antibodies were incubated at the concentrations in Supplemental Table 1 in antibody diluent (Abcam) and incubated overnight at 4☌. Slides were allowed to cool at room temperature, mounted in Sequenza (Thermo Fisher) racks, rinsed twice in PBS, and blocked for 45 minutes in Gentex block (120 µl) at room temperature. Slides were de-waxed in xylene (2×5 minutes), rehydrated, and antigen retrieved (7 minutes at 60% microwave power). The percentage of DAB staining per section ( n=6–8 per group) was determined by ImageJ.
The stained section was scanned with a Zeiss Axio Scan.Z1 Slide Scanner (Carl Zeiss Microscopy). Vectastain RTU ABC Reagent (PK7100 Vector Laboratories) was then applied, followed by incubation with the DAB+ Substrate Chromogen System (K3468 Dako), and then counterstaining with hematoxylin before dehydration and mounting with Pertex mounting medium (3808707E Histolab Products AB). Tissue sections were incubated with primary antibody ( Supplemental Table 1) diluted in antibody diluent (S202230 Dako UK Ltd.), overnight at 4☌, before incubation with biotinylated secondary antibody ( Supplemental Table 1) for 30 minutes at room temperature. Sections were incubated with the avidin/biotin blocking kit (SP2001 Vector Laboratories) and blocked with serum-free protein block (X0909 Dako). Sections were rehydrated and staining was performed using the Sequenza system (Thermo Scientific, Waltham, MA). Kidney tissue was fixed and formalin-fixed, paraffin-embedded, 4- μm tissue sections were prepared. Similar myeloid cell phenotypes are observed in the human kidney, suggesting they may represent specific targets to slow progression of CKD or promote renal repair. Pseudotime analysis and paired blood exchange (PBE) support dynamic changes in monocyte and macrophage phenotype in response to induction and removal of injury.
We identified myeloid cell subsets that were indistinguishable using standard flow cytometry markers, with the relative proportions of the subsets changing dynamically during injury and repair. Hence, in this study, we employ scRNA-seq to characterize myeloid cell subsets in the reversible, unilateral-ureteric-obstruction (R-UUO) model, in which we 32 and others 33 have demonstrated regression of established tubulointerstitial fibrosis after reversal of obstruction. 27 ⇓ ⇓ ⇓– 31 However, macrophage heterogeneity during regression of fibrosis in the kidney remains uncertain. Recent advances in transcriptomics, including single-cell RNA sequencing (scRNA-seq), have facilitated detailed analysis of myeloid cells in the healthy kidney, and after AKI, 24 ⇓– 26 and in other organs.
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Most studies have used panels of cell surface markers to characterize myeloid cell subsets by flow cytometry however, this approach is inherently biased and is unlikely to capture the full phenotypic spectrum. Hence, they may be injurious, but, in addition, they may mediate repair by scavenging cell debris, degrading excess extracellular matrix (ECM), and by secreting factors that may promote regeneration of injured tissue. Tissue macrophages are heterogeneous and inherently plastic, and may adopt different phenotypes in response to environmental cues. 11 ⇓ ⇓ ⇓– 15 Recruitment of proinflammatory monocytes 16, 17 to the injured kidney via CCL1-CCR2 signaling 18, 19 may exacerbate tissue damage through the release of proinflammatory factors and by activating myofibroblasts.
The innate immune system has been implicated in both progression and regression of fibrosis in multiple organs, including the kidney. 9, 10 However, the cellular and molecular pathways mediating injury regression are poorly understood, partly because renal biopsies are rarely performed in patients who are clinically improving. 5 ⇓– 7 Furthermore, regression of established fibrosis, the best histologic predictor of outcome, 8 has been observed after prolonged normalization of blood glucose levels after successful pancreas transplantation.
2 ⇓– 4 It is now recognized that CKD is not always progressive, but that regression of albuminuria and improvement in renal function can occur if the injurious stimulus is removed. Conclusions Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.ĬKD affects approximately 10% of the global population 1 and is a major risk factor for ESKD and cardiovascular disease.
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