Multiplex tissue profiling - Kidney

The kidneys are part of the urinary system and their main function is maintainance of the electrolyte homeostasis and acid-base balance of the body. These processes involve several different cell types that form distinct histological structures. As part of the Tissue resource, we used multiplex immunohistochemistry-based fluorescence (mIHC/IF) for in-depth profiling of protein expression in cells in the glomeruli and the different tubules/ducts. In the current version, 162 proteins have been analyzed in 5 cell types using 1 antibody panel.

  • 5 different cell types
  • 162 profiled proteins
  • 1 antibody panel


The kidney panel

The antibody panel for kidney was generated to profile the different renal tubules (collecting ducts, distal and proximal tubules) as well as cells within the glomerular compartment (podocytes and endothelial cells). The mIHC/IF technique is based on the overlap between the location of the candidate protein and location of the marker proteins targeted by the panel antibodies. Figure 1 shows the different cell types/states/structures, and the corresponding marker proteins included in each panel. For more information about the multiplex panels, please visit Assays & annotations.


Figure 1. A schematic depiction of the kidney panel on the left with a list of cell types, representing colors and marker proteins on the right.


Protein expression in kidney

Candidate proteins for in-depth profiling were selected from the Tissue resource based on cell type specific staining pattern using conventional immunohistochemistry. Multiplex immunohistochemistry has allowed us to analyze and visualize protein expression in kidney at a higher resolution than ever before. For kidney, the protein expression was analyzed in podocytes, endothelial cells, proximal tubules, distal tubules, and collecting ducts.


Table 1. The number of proteins mainly located to different cell types/structures in kidney based on manual annotation.

Antibody panel
Main location
# proteins
Kidney collecting ducts 53
Kidney distal tubules 54
Kidney Endothelia 26
Kidney Podocytes 29
Kidney Proximal tubules 60


Protein expression in podocytes

As shown in Table 1, 29 genes have main protein location in podocytes. Podocytes are a cell type present in the glomeruli, which are complex vascular structures responsible for filtering the blood, a process that ultimately results in the formation of urine. Podocytes, which are wrapped around the capillaries, facilitate the selective passage of cells and specific proteins from the blood. An example of a protein with main location in podocytes is podocin (NPHS2), suggested to be involved in the regulation of glomerular permeability by linking the cytoskeleton and plasma membrane. Another example is DYNC1I1, which is essential for the movement of vesicles and organelles along microtubules. TPPP3 is another cytoskeleton associated protein expressed in podocytes. It regulates the dynamic polymerization of microtubules which is crucial for cell division.



NPHS2

DYNC1I1

TPPP3

NPHS2

DYNC1I1

TPPP3


Protein expression in endothelial cells

As shown in Table 1, 26 genes have main protein location in endothelial cells. Endothelial cells are found in the capillaries of the glomeruli and, similarly to podocytes, are involved in the filtering of blood. An example of a protein with main location in endothelial cells is platelet and endothelial cell adhesion molecule 1 (PECAM1), which constitutes a large proportion of the intercellular junctions of endothelial cells. Another example is collagen type XV alpha 1 chain (COL15A1), suggested to stabilize microvessels such as capillaries by attaching the cells that produce it to the underlying connective tissue. A less known protein expressed in endothelial cells is adhesion G protein-coupled receptor L4 (ADGRL4), a receptor with predicted function in regulation of angiogenesis.



PECAM1

COL15A1

ADGRL4

PECAM1

COL15A1

ADGRL4


Protein expression in proximal tubules

As shown in Table 1, 60 genes have main protein location in proximal tubules. The proximal tubules are composed of cells with a brush border (microvilli). The primary function of proximal tubules is reabsorption of a substantial portion of various ions, glucose and amino acids that have been filtered out from the blood. Two examples of proteins with main location in proximal tubules are solute carrier family 4 member 4 (SLC4A4), a sodium bicarbonate cotransporter, and solute carrier family 3 member 1 (SLC3A1), an aminoacid transporter located in the microvilli. Another example is agmatinase (AGMAT), an enzyme involved in the processing of urea and amino acids.



SLC4A4

SLC3A1

AGMAT

SLC4A4

SLC3A1

AGMAT


Protein expression in distal tubules

As shown in Table 1, 54 genes have main protein location in distal tubules. Both distal tubules and collecting ducts are the sites where the ion concentrations and pH of urine are regulated by hormones such as aldosterone and vasopressin. An example of a protein with main location in distal tubules is BSND, an essential component of chloride channels that facilitate chloride reabsorption and potassium secretion in the renal tubules. Another example is ATPase Na+/K+ transporting subunit beta 1 (ATP1B1), a component of an enzyme involved in potassium and sodium transport across the plasma membrane.



BSND

ATP1B1


BSND

ATP1B1


Protein expression in collecting ducts

As shown in Table 1, 53 genes have main protein location in collecting ducts. Collecting ducts cells are the main site of salt and water transport, as well as acid-base regulation. An example of a protein with main location in collecting ducts is aquaporin 3 (AQP3), a water channel that is required for water transport across the cell membrane and, to a less extend, faciliates the transport of urea and glycerol. Another example is FXYD4, a member of a family of FXYD-domain containing ion transport regulators that is thought to be involved in the exchange of sodium and potassium across plasma membranes.



AQP3

FXYD4


AQP3

FXYD4



Relevant links and publications

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Karlsson M et al., A single-cell type transcriptomics map of human tissues. Sci Adv. (2021)
PubMed: 34321199 DOI: 10.1126/sciadv.abh2169

Lewis SM et al., Spatial omics and multiplexed imaging to explore cancer biology. Nat Methods. (2021)
PubMed: 34341583 DOI: 10.1038/s41592-021-01203-6

Tan WCC et al., Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). (2020)
PubMed: 32301585 DOI: 10.1002/cac2.12023