Ultrastructural studies of the blood-urine barrier in proteinuric states

The kidneys filter enormous amounts of blood every day, removing excess fluid and waste products while retaining cells and large proteins in the circulation. The podocytes with their foot processes and slit diaphragms (SD) are important components of the renal filter and the barrier function. Proteinuria is a leading sign of kidney disease, irrespective of the cause, and can in itself be harmful, accelerating the disease towards renal failure and the need for dialysis or transplantation.

In this project, we used semiquantitative immunoelectron microscopy (iEM) to describe the distribution of novel podocyte proteins in diseases with foot process effacement (FPE), the most common ultrastructural finding in proteinuria.

Study I: Expression of nephrin in minimal change nephrotic syndrome (MCNS). Under the light microscope, nephrin immunofluorescence (IFL) turned from a linear pattern along the glomerular capillary loops, into a course granular one. iEM showed a decreased nephrin labeling in MCNS, even in ultrastructurally normal podocytes. This implies a role for nephrin and the SD in the pathogenesis of MCNS.

Study II: Dendrin is a novel intracellular protein in the SD region. We analyzed its expression in MCNS compared to ZO-1, a protein known to be stable in proteinuria. In areas with FPE, dendrin was redistributed from the SD to the podocyte cytoplasm, but in contrast to nephrin, there was no overall decrease. Thus, when compared to nephrin, dendrin seems less involved in the pathogenesis of FPE.

Study III: To enable studies on novel podocyte proteins before and around the onset of proteinuria, an experimental model was used; puromycin aminoneucleoside nephrosis in rat (PAN). Nephrin, dendrin, plekhh2 and alpha-actinin-4 were studied. Thorough ultrastuctural analyses at different time-points in PAN were performed. alpha-actinin-4, a cytoskeleton cross-linker, did not change. Nephrin started to decrease on day two after induction, i.e. before the appearance of proteinuria. Dendrin and plekhh2 decreased on day four when the animals had massive proteinuria. We concluded that a disturbed expression of nephrin in PAN rats, similar to our findings in MCNS, seems to be related to the appearance of proteinuria. Changes in plekhh2 and dendrin on the other hand, could be secondary to FPE and loss of SDs.

Study IV: Pdlim2 is a cytoskeleton-associated protein, not previously shown in the kidney. Here, its association with the actin cross-linker alpha-actinin-4 in the podocytes is demonstrated by several techniques. Under the electron microscope, it is found centrally in the foot processes, in association with the actin cytoskeleton. In patients with MCNS and membranous nephropathy (MN), the expression was significantly reduced, while it was preserved in FSGS patients. This implies that pdlim2 may have a specific role in the pathogenesis of MN and MCNS.

In conclusion, we have developed a semiquantitative immuno-EM technique to study the expression of novel glomerular proteins in proteinuric rats, in normal human kidney, and in renal biopsies from patients with acquired renal diseases. Our studies have brought new information about the subcellular localization of these proteins. Furthermore, the expression patterns differ between diseases, indicating different roles in the pathogenesis of proteinuria.

List of scientific papers

I. Wernerson A, Dunér F, Pettersson E, Widholm SM, Berg U, Ruotsalainen V, Tryggvason K, Hultenby K, Söderberg M (2003). "Altered ultrastructural distribution of nephrin in minimal change nephrotic syndrome." Nephrol Dial Transplant 18(1): 70-6.
https://doi.org/10.1093/ndt/18.1.70

II. Dunér F, Patrakka J, Xiao Z, Larsson J, Vlamis-Gardikas A, Pettersson E, Tryggvason K, Hultenby K, Wernerson A (2008). "Dendrin expression in glomerulogenesis and in human minimal change nephrotic syndrome." Nephrol Dial Transplant 23(8): 2504-11.
https://pubmed.ncbi.nlm.nih.gov/18356187

III. Dunér F, Lindström K, Hultenby K, Hulkko J, Patrakka J, Tryggvason K, Haraldsson B, Wernerson A, Pettersson E (2010). "Permeability, ultrastructural changes, and distribution of novel proteins in the glomerular barrier in early puromycin aminonucleoside nephrosis." Nephron Exp Nephron. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/20588063

IV. Sistani L, Dunér F, Udumala S, Xiao Z, Hultenby K, Uhlén M, Tryggvason K, Wernerson A, Patrakka J (2010). "Novel podocyte protein pdlim2 is differently expressed in proteinuric diseases and stabilizes stress fibers through interaction with alpha-actinin-4." [Manuscript]