Figure 1.
Outline of experimental protocol with dates of procedures indicated and splinted excisional wound model.
A) RF = Renal Failure, Hd = Harvest day. B) The splinted excisional wound model minimizes the role of wound contraction during rodent wound healing, allowing for a finer analysis of parameters such as epithelial gap closure and granulation tissue deposition.
Figure 2.
Mice exhibited different degrees of chronic kidney disease.
A) By controlling the amount of cautery to the injured kidney, moderate and severe degrees of chronic kidney disease were produced. Only mice with a severe degree of chronic kidney disease were wounded for experimental purposes. (n = 6–8 mice/group) B) These animals exhibited metabolic derangements analogous to those in human chronic kidney disease and a notable growth retardation.
Figure 3.
Chronic kidney disease (CKD) mice exhibited a significant increase in glomerular tuft area.
Using hematoxylin and eosin to stain the paraffin embedded kidneys glomerular tuft area was measured from glomeruli in which the vascular pole was evident to compare the control kidneys (A) to the CKD kidneys (B). CKD kidneys exhibited indirect signs of mesangial expansion with a significant increase in glomerular tuft area when compared to control kidneys. (Magnification ×400) (** p<0.01) (n = 6–8 mice/group).
Figure 4.
Hematoxylin and eosin (H&E) sections of representative wounds.
Wounds were obtained at different harvesting time points, processed, and stained with H&E. Red arrows indicate epithelial keratinocyte migration across the wounds. (Magnification ×20).
Figure 5.
Epithelial gap closure and granulation tissue deposition measurements.
A) Epithelial gap closure was significantly delayed in the chronic kidney disease model at days 7 and 14. B) Granulation tissue deposition exhibited a trend towards being decreased in the chronic kidney disease group. (* p<0.05) (n = 6–8 mice/group).
Figure 6.
Chronic kidney disease demonstrated significantly less cellular proliferation in the early stages of wound healing.
Representative immunofluorescent sections of wound edges stained with BrdU and DAPI in A) normal mice and B) chronic kidney disease mice at day 7. C) Utilizing Tissue Gnostics quantification technology, chronic kidney disease wounds demonstrated significantly less cellular proliferation in the early stages of wound healing. (Arrows indicate wound edges, white dots indicate epidermis, and brackets indicate dermis) (* p<0.05, ** p<0.01) (Magnification ×200) (n = 6–8 mice/group).
Figure 7.
Chronic kidney disease demonstrated a significant reduction in angiogenesis in wound healing tissue.
Representative immunofluorescent sections of wound edges stained with CD31 and DAPI in A) normal mice and B) chronic kidney disease mice at day 7. C) Utilizing Tissue Gnostics quantification technology, chronic kidney disease wounds demonstrated significantly less angiogenesis in the early stages of wound healing. (Arrows indicate wound edges, white dots indicate epidermis, and brackets indicate dermis) (* p<0.05, ** p<0.01) (Magnification ×200) (n = 6–8 mice/group).
Figure 8.
Chronic kidney disease demonstrated significantly increased and maintained inflammation in the early stages of wound healing.
Representative immunofluorescent sections of wound edges stained with CD45 and DAPI in A) normal mice and B) chronic kidney disease mice at day 7. C) Utilizing Tissue Gnostics quantification technology, chronic kidney disease wounds demonstrated a significantly increased and maintained inflammation in the early stages of wound healing. (Arrows indicate wound edges, white dots indicate epidermis, and brackets indicate dermis) (* p<0.05, ** p<0.01) (Magnification ×200) (n = 6–8 mice/group).
Figure 9.
Quantitative reverse-transcription PCR (qRT-PCR) of growth factor, nitric oxide, and inflammatory genese.
Genetic analysis of wounds by qRT-PCR exhibited differences in expression of important wound healing related genes due chronic kidney disease. At post-operative day (POD) 3, CKD wounds demonstrate significantly reduced expression of VEGF and eNOS, with an increase in IL-1β. These trends were maintained at POD7, except for a significant decrease in iNOS instead of eNOS. Rows show POD when the tissue was collected, POD0, 3, and 7 at top, middle and bottom, respectively. Genes analyzed are represented by columns: (A) growth factor genes, (B) nitric oxide genes and (C) cytokine genes. (* p<0.05) (n = 4 wounds/group).