Background/Purpose: Tissue clearing approaches such as CLARITY renders tissue transparent, and in combination with two-photon microscopy, enables microscopic visualization deep internal structures within unaltered organs. These cutting edge approaches have drastically improved the understanding of cellular circuits in the brain. However, application of this approach has only recently been described for the kidney. Here, we clear mouse kidneys to better understand the immune cell architecture following induction of nephrotoxic nephritis (NTN) using a modified lipid removal approach that also worked for human kidney fragments.

Methods: 129 mice were injected intravenously with nephrotoxic serum to induce NTN. Mice were perfused with an acrylamide monomer solution to form the basis of the hydrogel. Lipid removal was accomplished using 8% sodium dodecyl sulfate (SDS). An aminoalcohol solution was used to quench light absorbing heme in red blood cells trapped within the tissue. Cleared mouse kidneys were stained with antibodies specific for B cells, T cells, macrophages, and dendritic cells (DCs). Human kidney fragments were incubated in acrylamide monomer solution, then cleared as mentioned above. Cleared tissue was imaged using two-photon microscopy.

Results: Compared to previously published protocols, perfusion of acrylamide monomers into mice significantly accelerated the tissue clearing process. Enhanced tissue clearing was observed when we incubated kidneys in aminoalcohols. We observed vast networks of lymphocytes, macrophages, and DCs in cleared NTN kidneys compared to control kidneys. Human kidneys also were cleared using this approach, and we noted DC networks in healthy donor controls.

Conclusion: We identified a new protocol that enhanced and accelerated tissue clearing in mouse and human kidneys. Using this approach, we found elaborate networks of lymphocytes and monocyte-derived cells in NTN mouse kidneys. We also observed DC networks in healthy human kidney donor fragments. These data demonstrate the utility of tissue clearing in evaluating cellular architecture in mouse and human kidneys.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/improved-tissue-clearing-and-2-photon-imaging-of-mouse-kidneys-reveals-immune-cell-architecture-in-nephrotoxic-nephritis/