From the Beginning to the End: Effects of Proteinuria along the Renal Tubule

Kidneys are essential regulators of fluid and electrolyte homeostasis and clear the body of waste products by processing about 900 L of blood plasma every day to produce 180 L of primary urinary filtrate in healthy adults. These are concentrated into 1–2 L of excreted urine. Although 1 L of human plasma contains approximately 60–80 g protein, only a minimal amount of protein is excreted in the urine. This highly restrictive capacity of the kidneys results from the glomerular filtration barrier composed of endothelial cells, glomerular basement membrane, and podocytes. The glomerular filtration barrier limits the passage of proteins from the blood into the primary filtrate on the basis of size, structure, and charge. Only low–molecular-weight proteins and a tiny fraction of larger proteins, such as albumin, leak through the filtration barrier and are then reabsorbed in the proximal tubule. Consequently, nephron segments downstream of the proximal tubule, including the loop of Henle, the distal tubule, and the collecting duct, do not come in contact with significant amounts of proteinuria under healthy physiologic conditions.

In kidney diseases, such as FSGS or diabetic nephropathy, alteration and disruption of the glomerular filtration barrier increase protein leakage into the primary filtrate. When the amount of protein in the primary filtrate exceeds the resorptive capacities of the proximal tubule, protein leakage results in proteinuria. Irrespective of its underlying cause, proteinuria is associated with a higher risk of progressive kidney disease and cardiovascular disease. A recent meta-analysis comprising over 9 million individuals from 114 global cohorts indicated that a higher urinary albumin-to-creatinine ratio is associated with higher all-cause and cardiovascular mortality as well as higher risk of AKI and kidney failure.¹ In addition, the risk of any hospitalization, stroke, and several other cardiac conditions, such as coronary heart disease and heart failure, was heightened in patients with higher urinary albumin-to-creatinine ratios.¹ Even levels of albuminuria below the canonical pathologic range were shown to be associated with adverse cardiovascular outcomes.²

How does proteinuria exert such detrimental effects? Several lines of evidence from in vitro and in vivo studies indicate that filtered proteins have a toxic effect on proximal tubule epithelial cells, suggesting that proteinuria not only represents a biomarker of progressive kidney disease but also directly contributes to disease progression.³ Proximal tubule cells mainly take up filtered proteins, including albumin and albumin-bound free fatty acids, through endocytosis, mediated by the megalin/cubilin receptor complex.³ Genetic mutations in CUBN, the gene encoding the cubilin receptor (involved in albumin uptake), lead to impaired albumin reabsorption in the proximal tubule, resulting in chronic albuminuria. Interestingly, no development of kidney dysfunction was observed in patients with chronic albuminuria due to CUBN mutations.⁴ This suggests that luminal protein exposure alone without protein uptake by tubule cells does not necessarily induce kidney disease progression.⁴ Conversely, receptor-mediated endocytosis of filtered proteins by proximal tubule cells was shown to trigger various pathophysiologic processes, including production of reactive oxygen species, autophagy dysfunction, lysosomal membrane permeabilization, and endoplasmic reticulum stress.³ Collectively, these processes lead to proinflammatory and profibrotic signaling and can eventually cause apoptosis of proximal tubule cells and development of tubulointerstitial inflammation and fibrosis.³ How the complex molecular and cellular mechanisms triggered by proteinuria affect different segments of the nephron and how exactly they link to kidney disease progression, however, remains unknown.

In this issue of JASN, Faivre et al. apply an elegant combination of intravital imaging and single-nuclei RNA-sequencing to shed new light on tubular protein handling and the effects of glomerular proteinuria on transcriptional state and function of different cell types along the consecutive segments of nephrons in mice.⁵

The authors present three key findings: (1) The kidney adapts to proteinuria by extending the protein reabsorption capacity from segment 1 to segment 2 of the proximal tubule. (2) Proteinuria causes tubular remodeling and injury. (3) Proteinuria affects transcriptional state and function of all nephron segments in a segment-specific way.

The authors start their analysis by spatially mapping protein reabsorption and organic anion secretion in different segments of the proximal tubule in healthy mice using intravital imaging and labeled ligands. They confirm previous findings by showing that protein reabsorption mainly takes place in segment 1, while organic anion transport processes are primarily located in segment 2.^6,7 The latter are, for example, responsible for excretion of uremic toxins.

In a mouse model of FSGS characterized by heavy proteinuria, this functional distinction between segment 1 and 2 of the proximal tubule is partially abolished. Under proteinuric conditions, the authors find a reduction in cells from proximal tubule segments 2 and 3, while a hybrid S1/S2 population with characteristics of both segment 1 and segment 2 appears. The hybrid S1/S2 population shows reduced expression of organic anion transport-related genes compared with segment 2 cells but increased expression of segment 1 markers. This suggests a potential remodeling of segment 2 cells to protein-reabsorbing S1/S2 cells with reduced organic anion secretion capacity. While the functional consequences of the observed transcriptomic changes require further investigation, proteinuria-triggered loss of organic anion secretion capacity could explain why excretion of certain solutes is reduced more than the GFR in advanced CKD.⁸ This finding could furthermore relate to the reduced effect of loop diuretics in end stage kidney disease, since loop diuretics have to be secreted through organic anion transport processes to exert their function.⁵

Using single-nuclei RNA-sequencing, the authors show that proteinuria results in appearance of dedifferentiated, injured proximal tubule cells. They describe a failed-repair proximal tubule cell population, which has striking similarities to injured proximal tubule cells observed in mouse models of ischemic AKI, although the spatiotemporal dynamics of proximal tubule injury differ between the diseases.^5,9 These failed-repair proximal tubule cells express the injury marker Vcam1 (encoding vascular cell adhesion molecule 1). Vcam1 has also been shown to be upregulated in proximal tubule cells in other kidney diseases, such as diabetic kidney disease and autosomal dominant polycystic kidney disease.¹⁰ Could these failed-repair proximal tubule cell populations represent essentially similar cell states to which proximal tubule cells converge across the distinct kidney diseases? This study certainly adds an interesting piece to the puzzle of Vcam1-positive proximal tubule cells.

Beyond the proximal tubule, the authors also observe proteinuria-induced gene expression changes in the subsequent segments of the nephron. Using single-nuclei RNA-sequencing, they detect the emergence of new cell populations in the thick ascending limb of Henle, the distal convoluted tubule, and the connecting tubule in response to luminal protein exposure. These novel cell populations are characterized by reduced expression of solute transport–related genes accompanied by increased expression of genes related to developmental processes and TGFβ signaling, and altered metabolic responses. Elucidating the mechanisms underlying the observed transcriptional reprogramming and its functional implications is of great interest in the search for novel therapeutic strategies to halt kidney disease progression.

Taken together, this study highlights functional tubular cell plasticity under proteinuric conditions, points to a role of injured proximal tubule cells in disease progression, and reveals novel aspects of tubular responses to proteinuria along the entire nephron, bringing not only proximal tubule cells but all other nephron segments into the spotlight.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/E688.

Funding

L.M.S. Gerhardt is supported by a clinician-scientist grant from the German Society of Internal Medicine (Deutsche Gesellschaft für Innere Medizin—DGIM).

Author Contributions

Conceptualization: Louisa M.S. Gerhardt.

Writing – original draft: Louisa M.S. Gerhardt.