Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome
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eLife assessment
The article is of importance to the field of glomerular diseases and rare diseases. The authors propose a link between the inhibition of SGLT2 and lipotoxicity-mediated renal injury in experimental Alport syndrome (AS) by modulation pathways linked to CKD progression, possibly through metabolic adaption in podocytes. Although there is scientific merit in the work presented, the functional analyses are incomplete to support the claim that effects pharmacological effects are mediated through podocytes in Alport Syndrome.
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Abstract
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are anti-hyperglycemic agents that prevent glucose reabsorption in proximal tubular cells. SGLT2i improves renal outcomes in both diabetic and non-diabetic patients, indicating it may have beneficial effects beyond glycemic control. Here, we demonstrate that SGLT2i affects energy metabolism and podocyte lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that the SGLT2 protein was expressed in human and mouse podocytes to a similar extent in tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wild-type podocytes. Treatment with SGLT2i empagliflozin reduces lipid droplet accumulation and apoptosis in AS podocytes. Empagliflozin inhibits the utilization of glucose/pyruvate as a metabolic substrate in AS podocytes but not in AS tubular cells. In vivo, we demonstrate that empagliflozin reduces albuminuria and prolongs the survival of AS mice. Empagliflozin-treated AS mice show decreased serum blood urea nitrogen and creatinine levels in association with reduced triglyceride and cholesterol ester content in kidney cortices when compared to AS mice. Lipid accumulation in kidney cortices correlates with a decline in renal function. In summary, empagliflozin reduces podocyte lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.
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Author Response
Reviewer #1 (Public Review):
Ge et. al., examined sodium-glucose cotransporter-2 inhibitors (SGLT2i) in Alport syndrome (AS), and demonstrate that it was beneficial in AS through reduced lipotoxicity in podocytes as a key mechanism of action. The SGLT2i empagliflozin has been previously shown to have positive effects on hyperglycemia control, as well as on cardiovascular and renal outcomes of type II diabetes mellitus through tubuloglomerular feedback, but its effect on glomerular diseases such as AS are unknown to date. The authors have previously identified that cholesterol efflux in podocytes plays a critical pathogenic role in a diabetic kidney disease setting. The evidence that authors provide in favor of their hypothesis in a disease of non-metabolic origin such as AS, was supported as the SGLT2i was effective …
Author Response
Reviewer #1 (Public Review):
Ge et. al., examined sodium-glucose cotransporter-2 inhibitors (SGLT2i) in Alport syndrome (AS), and demonstrate that it was beneficial in AS through reduced lipotoxicity in podocytes as a key mechanism of action. The SGLT2i empagliflozin has been previously shown to have positive effects on hyperglycemia control, as well as on cardiovascular and renal outcomes of type II diabetes mellitus through tubuloglomerular feedback, but its effect on glomerular diseases such as AS are unknown to date. The authors have previously identified that cholesterol efflux in podocytes plays a critical pathogenic role in a diabetic kidney disease setting. The evidence that authors provide in favor of their hypothesis in a disease of non-metabolic origin such as AS, was supported as the SGLT2i was effective in reducing the deleterious effects of lipotoxicity in podocytes, ameliorated glomerular injury and proteinuria, and extending the life span of Col4a3 knockout mice. They further show that empagliflozin treatment mitigated AS podocytes from cell death through apoptosis, but did not impact the cell's cytotoxicity. These results support the notion that empagliflozin affects the regulation of important metabolic switch in mouse kidneys, perhaps through decreasing lipid accumulation in podocytes.
However, the authors solely rely on one IHC staining image of a human biopsy to demonstrate SGLT2 expression in podocytes in vivo. Although the authors have done several experiments which greatly increase the confidence in their findings that empagliflozin is beneficial in AS and would have clinical significance, their data does not rule out the possibility that empagliflozin has beneficial effects through the other glomerular cells in AS, or limited to impacting lipids in podocytes in AS.
We thank the reviewer for recognizing the significance of our findings and for pointing out some additional concerns with our study. In this revised version, we have added experiments that focus on investigating the specific effect of empagliflozin on AS podocytes. We added immunofluorescence staining of AS mouse kidney sections which supports the idea that SGLT2 is expressed in podocytes. We investigated the effect of SGLT2 knockdown in AS podocyte using siRNA and compared the anti-lipotoxic effects of siSGLT2 to SGLT2i.
Reviewer #3 (Public Review):
Using cultured human podocytes the expression of SGLT2 is established using immunostaining and western blotting. An analysis of podocyte RNA wasn't performed, but the expression in cultured podocytes was comparable to that seen in human cultured proximal tubular cells. This work then paved the way for treatment of immortalized cells obtained from an Alport syndrome mouse model (Col4A3-/-), representing an autosomal recessive form of Alport syndrome. Podocytes from Alport syndrome mice showed a lipid droplet accumulation which was reduced to some extent by SGLT2 inhibition. In a series of metabolic experiments, it was shown that SGLT2 inhibition reduced the formation of pyruvate as a metabolic substrate in Alport podocytes. In vivo experiments showed an improvement in survival of Col4a3-/- mice treated with SGLT2 inhibition. When compared to ace inhibitor, SGLT2 inhibition has a similar effect on renal function and no additive effect was seen with SGLT2 inhibitor plus ace inhibitor. Like the cell assays, the in vivo treatment seemed to prevent the podocyte lipid accumulation in Alport syndrome mice.
This data in cells and animals generally supports the findings in SGLT2 inhibitor human studies, where Alport syndrome patients with proteinuria and progressive CKD seem to benefit. The work paves the way for a dedicated trial of SGLT2i in Alport patients and a reassessment of the human podocyte disease phenotype in this condition, before and after treatment. There are patients with mutations in SGLT2 with familial renal glycosuria - it would be interesting to test via urine derived podocytes whether a similar metabolic switch was occurring and its consequences to pave the way for long term treatment regimes.
We thank the reviewer for recognizing the significance of our findings. We appreciate the reviewer’s concern that podocyte SGLT2 RNA levels should be studied. In this revised version, we added the results of SGLT2 mRNA expression analysis in immortalized podocytes and tubular cells. These results were added in Figure 1E. We agree with the insightful suggestions to study the metabolic switch in familial renal glucosuria in patients with SGLT2 mutations, as well as to evaluate Col4a5 AS model. We have included these insights in our discussion.
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eLife assessment
The article is of importance to the field of glomerular diseases and rare diseases. The authors propose a link between the inhibition of SGLT2 and lipotoxicity-mediated renal injury in experimental Alport syndrome (AS) by modulation pathways linked to CKD progression, possibly through metabolic adaption in podocytes. Although there is scientific merit in the work presented, the functional analyses are incomplete to support the claim that effects pharmacological effects are mediated through podocytes in Alport Syndrome.
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Reviewer #1 (Public Review):
Ge et. al., examined sodium-glucose cotransporter-2 inhibitors (SGLT2i) in Alport syndrome (AS), and demonstrate that it was beneficial in AS through reduced lipotoxicity in podocytes as a key mechanism of action. The SGLT2i empagliflozin has been previously shown to have positive effects on hyperglycemia control, as well as on cardiovascular and renal outcomes of type II diabetes mellitus through tubuloglomerular feedback, but its effect on glomerular diseases such as AS are unknown to date. The authors have previously identified that cholesterol efflux in podocytes plays a critical pathogenic role in a diabetic kidney disease setting. The evidence that authors provide in favor of their hypothesis in a disease of non-metabolic origin such as AS, was supported as the SGLT2i was effective in reducing the …
Reviewer #1 (Public Review):
Ge et. al., examined sodium-glucose cotransporter-2 inhibitors (SGLT2i) in Alport syndrome (AS), and demonstrate that it was beneficial in AS through reduced lipotoxicity in podocytes as a key mechanism of action. The SGLT2i empagliflozin has been previously shown to have positive effects on hyperglycemia control, as well as on cardiovascular and renal outcomes of type II diabetes mellitus through tubuloglomerular feedback, but its effect on glomerular diseases such as AS are unknown to date. The authors have previously identified that cholesterol efflux in podocytes plays a critical pathogenic role in a diabetic kidney disease setting. The evidence that authors provide in favor of their hypothesis in a disease of non-metabolic origin such as AS, was supported as the SGLT2i was effective in reducing the deleterious effects of lipotoxicity in podocytes, ameliorated glomerular injury and proteinuria, and extending the life span of Col4a3 knockout mice. They further show that empagliflozin treatment mitigated AS podocytes from cell death through apoptosis, but did not impact the cell's cytotoxicity. These results support the notion that empagliflozin affects the regulation of important metabolic switch in mouse kidneys, perhaps through decreasing lipid accumulation in podocytes.
However, the authors solely rely on one IHC staining image of a human biopsy to demonstrate SGLT2 expression in podocytes in vivo. Although the authors have done several experiments which greatly increase the confidence in their findings that empagliflozin is beneficial in AS and would have clinical significance, their data does not rule out the possibility that empagliflozin has beneficial effects through the other glomerular cells in AS, or limited to impacting lipids in podocytes in AS.
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Reviewer #2 (Public Review):
The manuscript by Ge et al investigated the therapeutic benefits of the SGLT2 inhibitor empagliflozin in Alport syndrome (AS). They established the immortalized tubular cells and podocytes using wildtype (WT) mice and mice with AS. They showed that cultured human and mouse podocytes express similar levels of SGLT2 protein as compared to tubular cells. In vitro, they demonstrated that AS podocytes accumulate more lipid droplets and show increased levels of apoptosis in comparison to WT podocytes. Empagliflozin significantly reduces lipid droplets and apoptosis in AS podocytes. Furthermore, empagliflozin inhibits glucose/pyruvate-driven respiration in AS podocytes. In vivo, empagliflozin prolongs the lifespan of AS mice. Compared to untreated AS mice, empagliflozin improves kidney function and reduces the …
Reviewer #2 (Public Review):
The manuscript by Ge et al investigated the therapeutic benefits of the SGLT2 inhibitor empagliflozin in Alport syndrome (AS). They established the immortalized tubular cells and podocytes using wildtype (WT) mice and mice with AS. They showed that cultured human and mouse podocytes express similar levels of SGLT2 protein as compared to tubular cells. In vitro, they demonstrated that AS podocytes accumulate more lipid droplets and show increased levels of apoptosis in comparison to WT podocytes. Empagliflozin significantly reduces lipid droplets and apoptosis in AS podocytes. Furthermore, empagliflozin inhibits glucose/pyruvate-driven respiration in AS podocytes. In vivo, empagliflozin prolongs the lifespan of AS mice. Compared to untreated AS mice, empagliflozin improves kidney function and reduces the content of triglycerides and cholesterol esters in the kidney cortices of AS mice. Overall, the manuscript is nicely written, well-arranged, and easy to read. The experimental methods are reliable, and the conclusions are supported by the results.
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Reviewer #3 (Public Review):
Using cultured human podocytes the expression of SGLT2 is established using immunostaining and western blotting. An analysis of podocyte RNA wasn't performed, but the expression in cultured podocytes was comparable to that seen in human cultured proximal tubular cells. This work then paved the way for treatment of immortalized cells obtained from an Alport syndrome mouse model (Col4A3-/-), representing an autosomal recessive form of Alport syndrome. Podocytes from Alport syndrome mice showed a lipid droplet accumulation which was reduced to some extent by SGLT2 inhibition. In a series of metabolic experiments, it was shown that SGLT2 inhibition reduced the formation of pyruvate as a metabolic substrate in Alport podocytes. In vivo experiments showed an improvement in survival of Col4a3-/- mice treated with …
Reviewer #3 (Public Review):
Using cultured human podocytes the expression of SGLT2 is established using immunostaining and western blotting. An analysis of podocyte RNA wasn't performed, but the expression in cultured podocytes was comparable to that seen in human cultured proximal tubular cells. This work then paved the way for treatment of immortalized cells obtained from an Alport syndrome mouse model (Col4A3-/-), representing an autosomal recessive form of Alport syndrome. Podocytes from Alport syndrome mice showed a lipid droplet accumulation which was reduced to some extent by SGLT2 inhibition. In a series of metabolic experiments, it was shown that SGLT2 inhibition reduced the formation of pyruvate as a metabolic substrate in Alport podocytes. In vivo experiments showed an improvement in survival of Col4a3-/- mice treated with SGLT2 inhibition. When compared to ace inhibitor, SGLT2 inhibition has a similar effect on renal function and no additive effect was seen with SGLT2 inhibitor plus ace inhibitor. Like the cell assays, the in vivo treatment seemed to prevent the podocyte lipid accumulation in Alport syndrome mice.
This data in cells and animals generally supports the findings in SGLT2 inhibitor human studies, where Alport syndrome patients with proteinuria and progressive CKD seem to benefit. The work paves the way for a dedicated trial of SGLT2i in Alport patients and a reassessment of the human podocyte disease phenotype in this condition, before and after treatment. There are patients with mutations in SGLT2 with familial renal glycosuria - it would be interesting to test via urine derived podocytes whether a similar metabolic switch was occurring and its consequences to pave the way for long term treatment regimes.
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