Table 1.
Summary of donor information and disease status.
Fig 1.
Histology of donor human kidney tissue derived from normal kidneys (NK) and chronic kidney disease-affected kidneys (CKD).
H&E staining,(A & D); Periodic acid-Schiff (PAS) staining, (B & E); Masson’s-Trichrome staining (C & F). No major histological abnormalities were seen in the NK tissues. H&E staining of the CKD kidneys revealed a fibrotic cortex with sclerotic glomeruli and scattered chronic infiltration of inflammatory cells. The arterial walls appeared thickened, and the tubules were dilated and filled with pink casts, illustrating renal thyroidization (D). The PAS staining of CKD tissues showed sclerotic glomeruli with collagen deposition and interstitial fibrosis (E). Tubular atrophy and hyalinosis were also observed (D-E). MT staining of the CKD tissues showed collagen deposition in glomeruli (glomerulosclerosis) and interstitium, as well as a thickening of arterial walls (F). Original magnification x40.
Fig 2.
Photomicrograph of primary renal cell cultures derived from NK and CKD kidney at passage 3 (P3) and passage 9 (P9) (A-D). There were no differences in gross cell morphology between NK and CKD kidney cells at passages three (P3) and nine (P9). Original magnification x20; Cell growth curves of NK and CKD kidney derived primary renal cells. Cell growth curve of human NK and CKD cells (2E) from different age donors were counted after achieving confluency, had the same behavior in culture.
Fig 3.
Characterization of isolated primary renal cells from NK and CKD kidneys using cell-specific markers.
Florescent antibody staining was carried out on passage 3 (P3) cells. Staining with proximal tubular marker Aquaporin-1 (A-B); Quantitation of proximal tubular cells (Aquaporin-1) among the total isolated primary renal cells at different passages from P3 to P12; (C) Distal tubular marker E-cadherin1 staining of primary renal cells from NK and CKD kidneys. N = 3; (D-E) Quantitation of distal tubular cell (E-cadherin) among the total isolated primary renal cells at different passages from P3 to P12 (F). The overall amounts (percentage) of proximal tubular cells and distal tubular cell were similar in the renal cell population derived from NK and CKD kidneys. Original magnification x20.
Fig 4.
Identification of Podocytes among the primary renal cells from NK and CKD using antibody-based staining of cell surface markers.
(A-B) Podocytes and endothelial cells identified by Podocalyxin (PDX) staining; (C-D) Podocytes stained using Wilms’ tumor (WT-1) antibody; (E-F) Staining of Podocytes using another cell-specific marker Nephrin; Quantitation of Podocytes among the primary renal cells isolated from NK and CKD kidneys at P3. Note that use of WT-1 and Nephrin antibodies resulted in slightly different levels of Podocytes detection amount the renal cell population (G). However, the relative amounts of Podocytes in both NK and CKD kidney derived cells were similar.
Fig 5.
Characterization of specific renal cells among the total renal cells isolated from NK and CKD kidneys.
Cell-type specific antibodies and FACS was used to isolate proximal tubular cells (A-B), distal tubular cells (D-E) and podocytes (G-H) in different passages (P3 and P9) of cultured renal cells from NK and CKD kidneys. FACS-based quantification percentage of proximal tubular cells in passage 3 and 9 cells (C), percentage of distal tubular cells (F), percentage of podocytes (I). The result highlights that there is no significant difference between the groups.
Fig 6.
Photomicrograph of proximal tubular cells (PTC) purified from primary cell cultures that were originally derived from NK kidneys (A) and CKD kidneys (B) at passage 1 (P1) original magnification x10. Consolidated growth curve of proximal tubular cells isolated from NK and CKD human renal cells (C). Proximal tubular cells from different age donors were counted after achieving confluency, had the same behavior in culture.
Fig 7.
Ultrastructural analysis of renal proximal tubular cells using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM).
SEM analysis showing long microvilli on the apical surface membrane of a Proximal tubular cells derived from a NK kidney at passage 3 (A) and passage 6 (C) and the same cell type from a CKD kidney at passage 3 (B) and passage 6 (D); Upper panel shows the cellular morphology magnified x630. TEM analysis showing the integrity of tight junction (arrow) in proximal tubular cells isolated from primary renal NK kidneys (E) and CKD kidney cells (F) at passage 3 (P3) magnified x4780; TEM micrograph showing the ultrastructure of nucleus “N” and other intracellular components in proximal tubular cells of NK (G) and CKD (H) kidneys are similar morphology, magnified x11000.
Fig 8.
Oxidative Stress in NK and CKD kidney derived tissues.
Immunohistochemical staining to detect Superoxide dismutase 1 (SOD1) expression in human renal tissues from NK (A) and CKD (B) kidneys. Quantitation of oxidative stress in primary renal cells derived from NK and CKD kidneys using a Glutathione (GSH) assay that utilizes a fluorescent dye Monochlorobimane (MCB), (C). GSH was assayed in cells of passage 3 (P3) to (P9) and 12. The Glutathione levels in renal cells derived from both NK and CKD kidneys were almost similar during the entire cell culture
Fig 9.
Quantitative uptake analysis of primary proximal tubular cells (PTC) derived from NK and CKD kidneys confirms specific uptake of Na+ by the cells with no significant difference.
Ouabain treatment increases sodium uptake by inhibiting Na/K ATPase. FACS analysis of Intracellular Na+ uptake using the cell permeant Sodium Green Tetra-acetate in (PTC) cells from NK and CKD kidneys (B-C) were similar.
Fig 10.
Measurement of protein transport in primary renal cells from NK (A) and CKD (B) kidneys, as measured by uptake of rhodamine-conjugated albumin (ALB-RHO).Enhancement of ALB-RHO in the presence of Angiotensin II (ANGII) in the cells derived from NK (C) and CKD (D) kidneys. Reduction of ALB-RHO uptake by the NK (E) and CKD (F) kidneys derived cells in the presence of receptor-associated protein (RAP). NK and CKD kidneys derived cells Showing the same behavior of protein transport in culture.