Towards Individual Glomerular Phenotyping: Advent of Precision Medicine in Kidney Biopsies

Abstract

The road to precision medicine for nephrology is approaching quickly. In the present volume, the glomerular proteome has now been characterized at a single glomerulus level in mouse and human kidneys. Using the Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) approach the authors demonstrated that LAMP1 is a key lysosomal protein that is increased in glomerular diseases and may play a pathogenic role.

Every nephrologist, renal pathologist and trainee have noted the marked variability of glomerular structure in a human kidney biopsy. Despite a similar clinical picture of glomerular disease marked by proteinuria and reduced eGFR, a human kidney biopsy will often reveal some glomeruli with marked sclerosis and pathology and other, often nearby glomeruli that appear almost normal in appearance at the light microscopic level and sometimes even at the EM level (Figure 1). The reason behind the appearance is elusive as the biopsies are performed as a cross-sectional approach and the exact physiologic or pathophysiologic sequelae to arrive at that particular destination will almost never be fully known. Theories relating to regional hemodynamics and localization within the cortical regions may explain some of the heterogeneity however this will remain speculative until specific methods have been developed to quantitatively assess and visualize specific intra-nephron hemodynamics in real time and longitudindally.

Figure 1.

Adjacent glomeruli with a seemingly normal glomerulus next to a glomerulus with focal sclerosis (arrows)

Another theory is that regional cell alterations are occurring due to unclear etiologies and will dictate the structural appearance of one glomerulus vs another in the same kidney and same region of the cortex. A molecular characterization of an individual glomerulus may soon be developed, but translating the role of genes or transcripts to functional protein activity remains a limitation with purely sequencing approaches. Integrating transcriptomic to proteomic and ultimately metabolomics data will give a much better comprehensive analysis of the major pathways that are engaged or suppressed in a specific glomerulus. Namely, a systems approach based on quantitative multi-omics data will provide a molecular signatures that will enhance our understanding and provide new theories to determine why one glomerulus may be failing and another could survive in the same environmental milieu.

In the present KI issue, a group from Cologne, Germany has accomplished a major step towards this goal by applying a mass spectrometry based method to measure proteome at a single glomerulus level (1). The so-called Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) approach, introduced originally in 2014 by Hughes et al.(2), capitalizes on the affinity of proteins and peptides to bind tightly to carboxylated magnetic beads during sample processing to minimize sample losses and enable analysis of limited biomass. The current report purports to be able to quantitatively measure proteins with as few as 200 cells and monitor podocytes with as few as 80 podocytes from mouse and human kidneys using this protocol. After microdissecting individual glomeruli from mouse (or human kidneys), the team was able to perform LC-MS/MS analysis to detect podocyte specific proteins (nephrin, ACTN4, podocin, CD2AP), markers of mesangial cells (desmin) and type 4 collagen. Individual tubules were also microdissected and S1 segments, thick ascending limbs and cortical collecting ducts were characterized at the single tubule level via proteomics. Interestingly, the proximal tubule identified in excess of 1500 proteins and spanned 4 orders of magnitude. This feat of itself is a remarkable technological breakthrough, however the team went forward to determine how the technology could be applied to glomerular disorders in mice and human samples (Figure 2).

Figure 2 –

A) Schematic modeling the use of Single-Pot Solid-Phase-enhanced Sample Preparation analysis of individual glomeruli by LC/MS proteomics to identify intra- and intermouse heterogeneity in glomerular protein expression. B) The authors found that increased LAMP1 expression is a common feature of damaged glomeruli and tubules in several patients and mouse disease models.

Using Wilms tumor-het (Wt-het) mice that develop proteinuria and an FSGS like lesion, they developed a podocyte function sentinel assay comprising 20 proteins which were measured with parallel reaction monitoring. While a large regional heterogeneity was found within the same kidney, laminin 5 and LAMP1 (a lysosomal protein) abundances were strongly correlated with each other. There was also a negative correlation between LAMP1 and ACTN1/4. Similarly, proximal tubular heterogeneity was found in the same kidneys and Wt1het mice generally had reduced levels of the amino acid transport proteins (SLC3A1, SLC13A3 and SLC6A20a/b). In a separate model of FSGS with doxorubicin, a similar increase in albumin, laminin, collagen and LAMP1 with a reduction of ACTN1/4 and CD2AP was observed. There was a common correlation between LAMP1 and glomerular albumin and extracellular matrix proteins, suggesting that LAMP1 is a characteristic of the glomerular proteinuric signature and potentially linked to pathogenesis.

LAMP1 is a structural protein of the lysosome. Lysosomes also contain cathepsins which may be of greater interest in terms of a target for therapeutic intervention. Therefore, the team then developed a targeted proteomic assay to monitor cathepsin B, L and Z along with LAMP1. Cathepsin B correlated positively with LAMP1 in glomeruli from Wt1het mice and the doxorubicin-challenged mice glomeruli. There was a less consistent statistical relationship with cathepsin L and Z. To assess functional significance, KO mice for Cathepsin B, L and Z were evaluated and found not to be proteinuric at baseline and have less damage with nephrotoxic serum suggesting that cathepsin upregulation may be contributing to disease progression.

To translate these findings to humans, the team compared the proteome of single glomeruli in patients with nephrotic syndrome. In the kidney of a patient with primary idiopathic FSGS, there was an abundance of the lysosomal proteins LAMP1 and SCARB2. There was again an increase in LAMP1 in other causes of nephrotic syndrome, such as nephrin deficiency. Of interest, mitochondrial proteins were reduced in all three samples in comparison to control cells. Similar data was also found using laser dissection microscopy from 10 μm thick cryosections. Although the full extent of the technology was not exploited by this study, the authors have made a major contribution to the field of precision medicine in nephrology. In future studies, correlation with distinct glomerular pathologic features can be made with proteomic signatures. The technology may be of even more value at the tubular level and can be linked to biomarkers found in the urine and blood. Subtle changes of tubular structure may be observed to link with protein markers and explain pathobiology. There remain significant limitations such as adequate QC of various parameters and inability to define post-translational modifications of proteins.

The series of technical breakthroughs in the field of mass spectrometry and proteomics are remarkable and will dramatically enhance the understanding of glomerular and tubular diseases in animal models and humans. Recent proteomic studies have identified key proteins that have been putative or validated novel biomarkers for glomerular diseases.(3-6) However, the role of individual glomerular proteomes may provide even greater insight in diseases where there is glomerular heterogeneity in disease manifestation. At present, LCMS sensitivity has advanced to the level that even a single cell sized samples (~0.1 ng of protein for human) can be effectively measured. However, the amount of starting material required is typically orders of magnitude larger due to the losses incurred during sample preparation for LCMS analysis. The protocol described herein addresses this challenge by performing all sample processing (protein extraction, digestion, cleanup) in a single tube to minimize sample losses, and thus enhance sensitivity. Yet, additional improvements are needed to push the sensitivity to ever smaller samples and match demonstrated analytical sensitivity with the practical needs (i.e. single cell measurements). Microfluidic technology is an attractive venue to address the sample preparation bottleneck, and, when coupled with robust nanoLC platform and advanced mass analyzer, holds promise to close the gap that still exists in single cell proteomics. One could argue that sequencing technologies for analysis of small samples are well developed and sufficient, but proteome wide measurements would provide more direct information on the cellular state. It is also possible to envision similar approaches providing information about posttranslational modifications and their dynamics to better inform our understanding of cellular functions and regulatory networks. Furthermore, the full extent of a systems analysis will soon be possible as metabolomics can be performed with mass spectrometry based imaging of specific sub-structures (glomeruli and tubules) with spatial resolution of 20 μm (or better) and the same glomerulus or tubule could potentially be interrogated.

In coordination with proteomic and transcriptomic analysis, the full extent of a systems analysis will soon be possible as metabolomics can also be performed with mass spectrometry imaging of specific sub-structures (glomeruli and tubules) with spatial resolution of 20 μm and the same glomerulus or tubule could potentially be interrogated. Using a multi-omic approach will provide distinct definition of individual glomeruli and tubules and further the ability to perform precision medicine with defined tissue compartments within kidney biopsies. There is hope that nephrologists will soon be able to distinguish at a molecular level why one glomerulus appears normal while an adjacent glomerulus is markedly affected by a disease process.