Obesity-related proximal tubulopathy: an emerging threat to kidney health

ABSTRACT

Our previous studies have demonstrated that lipid overload leads to lysosomal dysfunction and autophagic stagnation in kidney proximal tubular epithelial cells (PTECs), which contributes to the renal lipotoxicity and eventually leading to the development of an obesity-related kidney disease. Here we identified that TFEB (transcription factor EB) is a modulator of PTECs lipotoxicity. Exposure to saturated fatty acid enhanced TFEB dephosphorylation and nuclear translocation in PTECs. In a mouse model fed with a high-fat diet (HFD), activated TFEB counteracted phospholipid accumulation in lysosomes by promoting lysosomal exocytosis in PTECs. Conversely, HFD-fed, PTECs-specific tfeb^−/− deficient mice exhibited increased phospholipid accumulation and autophagic stagnation, which made kidney vulnerable to injury following ischemia-reperfusion. Moreover, a higher body mass index was correlated to reductions in TFEB nuclear translocation in PTECs of chronic kidney disease patients. These data suggest that PTECs are involved in the pathogenesis of obesity-related kidney disease, which is called obesity-related proximal tubulopathy.

Abbreviations: EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase activating protein; HFD: high-fat diet; I/R: ischemia-reperfusion; LMP: lysosomal membrane permeabilization; LRP2: low density lipoprotein receptor-related protein 2; MLBs: multilamellar bodies; MTORC1: mechanistic target of rapamycin kinase complex 1; ORT: obesity-related proximal tubulopathy; PA: palmitic acid; PTEC: proximal tubular epithelial cell; RRAG: Ras related GTP binding; RPS6KB1, ribosomal protein S6 kinase B1; TFEB: transcription factor EB.

The prevalence of obesity has increased dramatically worldwide over the past four decades, making it an independent risk factor for kidney function decline and mortality. Previous studies have demonstrated that lysosomal dysfunction and impaired autophagic flux in kidney proximal tubular epithelial cells (PTECs) contribute to lipotoxicity in obesity-related kidney disease. However, the regulatory factors involved in fighting renal lipotoxicity have not been well understood. In our recently published research article [1], we identified a novel mechanism of renal lipotoxicity connected with the transcription factor EB (TFEB) and autophagy: First, lipid overload induces TFEB nuclear translocation via a Ras related GTP binding (RRAG) C/D-dependent inhibition of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway in PTECs, which helps to prevent multilamellar bodies (MLBs) accumulation by promoting lysosomal exocytosis. Second, TFEB deficiency results in autophagic stagnation and increased vulnerability to ischemia-reperfusion (I/R) injury in obese mice. Third, insufficient TFEB activity and impaired autophagic flux may be involved in obesity-related vacuolar lesions in chronic kidney disease (CKD) patients. Collectively, these findings provide fundamental insights into the mechanism through which TFEB transcriptionally controls MLBs accumulation in PTECs. We have proposed the novel disease concept “obesity-related proximal tubulopathy (ORT)” (Figure 1).

Figure 1.

TFEB-mediated lysosomal exocytosis of MLBs is involved in ORT. PTECs retrieve albumin-bound PA from the glomerular filtrate via LRP2-mediated albumin (Alb) endocytosis. PA induces autophagy, which mobilizes phospholipids from cellular membranes in lysosomes, resulting in MLBs accumulation. In parallel, PA promotes TFEB nuclear translocation via RRAG GTPase inactivation by sequestration of FLCN onto the lysosomal membrane, which in turn mediates lysosomal exocytosis to prevent MLBs accumulation and counteracts lipotoxicity.

In general, obesity-related kidney disease is characterized by glomerular hypertrophy and segmental sclerosis, known as “obesity-related glomerulopathy”. While much attention has been given to the glomerular lesions, recent evidence indicates that lipid overload-induced tubular lesions, such as MLBs accumulation, contribute to kidney dysfunction, inflammation, and fibrosis. The β-oxidation of free fatty acids is a major source of renal ATP synthesis, particularly in PTECs, which have a high-energy demand. However, obesity is associated with adipocyte hypertrophy, leading to the secretion of pro-inflammatory saturated fatty acids, including palmitic acid (PA). It has been suggested that PTECs are highly susceptible to lipid overload, as PTECs consistently uptake fatty acids not only from circulation, but also from glomerular filtrate through receptors such as LRP2 (low-density lipoprotein receptor–related protein/MEGALIN). The LRP2-mediated endocytosis of glomerular-filtered albumin-bound fatty acids is involved in MLBs formation, since this formation is blocked by lrp2^−/− deletion in HFD-fed obese mice. Moreover, we previously reported that lipid overload stimulates autophagy, which redistributes phospholipids from cellular membranes into lysosomes for renovation. However, prolonged lipid overload inevitably places a burden on the lysosomal system, resulting in phospholipid accumulation. Autophagy is involved in MLBs formation, since deletion of Atg5 prevents MLBs formation, although autophagy deficiency severely exaggerates HFD-induced mitochondrial dysfunction, inflammation, and fibrosis. Thus, MLBs formation in PTECs can be viewed as a process to sequester potentially cytotoxic metabolites and render them less toxic.

Despite recent advances in our understanding of MLBs formation, the regulatory mechanisms by which PTECs counteract obesity-related tubular lesions, have remained largely unknown. To address this, we conducted RNA sequencing transcriptomic analysis and identified TFEB as a crucial regulatory factor involved in PA-induced lipotoxicity in PTECs. PA strongly induces dephosphorylation and nuclear translocation of TFEB, although these effects gradually diminish with an extended treatment. What does TFEB activation trigger in response to lipid overload? We found that PA-induced TFEB activation depends on a substrate-specific inactivation of MTORC1. Unlike the canonical MTORC1 pathway in which components such as RPS6KB1/S6K1 (ribosomal protein S6 kinase B1) and EIF4EBP1/4E-BP1 (eukaryotic translation initiation factor 4E binding protein 1) are involved, the phosphorylation of TFEB depends on the amino acid–mediated activation of RRAGC/D GTPases. Focusing on FLCN (folliculin), which is an amino acid–dependent GTPase-activating protein (GAP) for RRAGC/D, we revealed that PA promotes TFEB nuclear translocation via RRAG GTPase inactivation by sequestration of FLCN onto the lysosomal membrane.

In addition, we demonstrated that lipid overload induces TFEB-mediated lysosomal exocytosis of phospholipids at the apical membrane. This serves to prevent excessive MLBs accumulation in PTECs, while PTEC-specific deletion of Tfeb exacerbates phospholipid accumulation and autophagic stagnation. Moreover, our study explains a multi-hit mechanism of renal lipotoxicity, in which lipid overload contributes to kidney injury in combination with other factors. Although lipid overload alone is not sufficient to lead to kidney injury, HFD-fed obese mice with massive MLBs accumulation not only have limited ability to augment autophagy, but giant MLBs are also more sensitive to lysosomal membrane permeabilization, making the obese mice more susceptible to additional stressors such as I/R injury. These findings demonstrate the critical role of TFEB-mediated lysosomal exocytosis in countering renal lipotoxicity. Furthermore, we discovered that trehalose, a natural disaccharide α,α-1,1-glucoside, as well as resveratrol, a plant-derived polyphenolic compound, which are known to enhance TFEB nuclear translocation, reduce HFD-induced formation of cytosolic vacuoles in PTECs. Thus, the activation of TFEB to promote lysosomal exocytosis and thereby alleviate autophagic stagnation, may be an attractive treatment strategy for ORT. Additionally, enhancing lysosomal biogenesis and function by TFEB could also be a strategy to reduce MLBs accumulation.

Finally, we examined whether findings in murine models can be translated to human obesity-related kidney disease. We found that higher body mass index is associated with higher vacuolation, i.e., phospholipid accumulation in the enlarged lysosomes, and lower TFEB nuclear localization in PTECs from patients with CKD. These findings indicate that insufficient TFEB activity and increased tubular vacuolar lesions may be the principal determinant for the kidney function decline in obese patients. Opposite to TFEB activation upon lipid overload in both in vitro and in vivo studies, the percentage of PTECs exhibiting TFEB nuclear translocation decreased in obese patients. One possible reason of this phenomenon is that longer PA exposure may somehow decrease the sequestration of FLCN onto lysosomes, which in turn prevents nuclear retention of TFEB by altering RRAGC/D activity.

In conclusion, TFEB-mediated lysosomal exocytosis is critical for alleviating autophagic stagnation and maintaining PTECs integrity, and protect against lipotoxicity. As a result, insufficient TFEB activity and MLBs accumulation may play a critical role in the pathogenesis of human ORT. Altogether, these data shed new light on the mechanisms underlying increased tubular vacuolar lesions, and we propose ORT as an emerging threat to kidney health.

Funding Statement

T.Y. is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (21K16163), G-7 Scholarship Foundation, and the TANITA Healthy Weight Community Trust.