SGLT2 inhibition promotes glomerular repopulation by cells of renin lineage in experimental kidney disease

Abstract

Aim: Sodium glucose co-transporter-2 (SGLT2) inhibitors stimulate renal excretion of sodium and glucose and exert renal protective effects in patients with (non-)diabetic chronic kidney disease (CKD) and may as well protect against acute kidney injury (AKI). The mechanism behind this kidney protective effect remains unclear. Juxtaglomerular cells of renin lineage (CoRL) have been demonstrated to function as progenitors for multiple adult glomerular cell types in kidney disease. This study assesses the impact of SGLT2 inhibition on the repopulation of glomerular cells by CoRL and examines their phenotypic commitment.

Methods: Experiments were performed in Ren1cre-tdTomato lineage-trace mice. Either 5/6 nephrectomy (5/6NX) modeling CKD or bilateral ischaemia reperfusion injury (bIRI) mimicking AKI was applied, while the SGLT2 inhibitor empagliflozin (10 mg/kg) was administered daily via oral gavage for 14 days.

Results: Both 5/6NX and bIRI-induced kidney injury increased the number of glomerular CoRL-derived cells. SGLT2 inhibition improved kidney function after 5/6NX, indicated by decreased blood creatinine and urea levels, but not after bIRI. In line with this, empagliflozin in 5/6NX animals resulted in less glomerulosclerosis, while it did not affect histopathological features in bIRI. Treatment with empagliflozin resulted in an increase in the number of CoRL-derived glomerular cells in both 5/6NX and bIRI conditions. Interestingly, SGLT2 inhibition led to more CoRL-derived podocytes in 5/6NX animals, whereas empagliflozin-treated bIRI mice presented with increased levels of parietal epithelial and mesangial cells derived from CoRL.

Conclusion: We conclude that SGLT2 inhibition by empagliflozin promotes CoRL-mediated glomerular repopulation with selective CoRL-derived cell types depending on the type of experimental kidney injury. These findings suggest a previously unidentified mechanism that could contribute to the renoprotective effect of SGLT2 inhibitors.

Keywords: RAAS; SGLT2 inhibition; cells of renin lineage; kidney repair; progenitor; regeneration.

Figure 1.. Different kidney injury mouse models lead to an increase in glomerular CoRL-derived cells.

a) Experimental setup of kidney injury models 5/6 nephrectomy (5/6NX) and b) bilateral ischemia reperfusion injury (bIRI) c) Blood urea values as a measurement of kidney function of kidney-injured mice during 14 day experimental time span. d) Representative pictures of tdTomato CoRL-signal within glomeruli (dashed lines) in different kidney injury models, bar=25μm, and corresponding quantification (e). Data is represented as mean ±SD, n=4-5 ‡p<0.001

Figure 2.. Homeostasis measurements on influence of SGLT2 inhibition in different experimental kidney injury models.

a) Experimental setup of kidney injury models 5/6NX and b) bIRI with a 14 day treatment. c) Weight changes during experimental window in 5/6NX and bIRI mice with control (vehicle) and SGLT2 inhibitory treatment (EMPA). d) Day 14 urinary glucose measurements in 5/6NX and bIRI exposed animals with and without SGLT2 inhibitory treatment shows effective dosage of empagliflozin. e) Blood glucose levels in 5/6NX and bIRI mice with and without SGLT2 inhibitory treatment. f) Day 14 plasma creatinine values and g) Blood urea values as a measurement of kidney function in 5/6NX and bIRI mice with control or SGLT2 inhibitory treatment. Data is represented as mean ±SD, n=6-9, *p<0.05.

Figure 3.. Injury assessments of 5/6NX and bIRI-exposed mice upon SGLT2 inhibitory treatment.

a) Representative Periodic Acid-Shiff (PAS) images indicate glomerular hypertrophy as quantified in d) in both 5/6NX and bIRI which is not further affected by SGLT2 inhibition (EMPA). b) Picro Sirius Red (PSR) stainings for collagen indicate less renal fibrosis in 5/6NX upon SGLT2 inhibition. c) CD45 stainings for leukocyte infiltration analysis. e) semi-quantitative glomerulosclerosis score based on PAS images, in which 0 means <10% of glomeruli affected, 1: 10-25% affected, 2: 26-50% affected and >50% of glomeruli affected. f) quantification of b. g) quantification of c. Bar=100μm, data are represented as mean ±SD, n=6-8 *p<0.05.

Figure 4.. Increased glomerular CoRL-derived cell number upon SGLT2 inhibition in 5/6NX and bIRI mouse models.

a) Representative images of tdTomato positive CoRL found within glomeruli (dashed lines), bar=20μm, and b) corresponding quantification. c) Representative picture of co-stainings for tdTomato (red), nephrin (green) and renin (magenta), showing that intraglomerular CoRL do not express renin. d) Representative image of co-stainings for tdTomato (red) and Ki-67 (green), indicating that intraglomerular CoRL are not proliferating. Data are represented as mean ±SD, n=6-9, ‡p<0.001.

Figure 5.. SGLT2 inhibition increases CoRL-derived podocyte number and density in 5/6 nephrectomized animals, but not in bIRI.

a) Representative images of stainings for WT1 (green) and tdTomato labeled CoRL (red). Colocalization is displayed in yellow and indicates these podocytes are derived from CoRL. Dashed lines indicate glomeruli, bar=20μm. b) Quantification of CoRL-derived podocytes in 2D sections in both disease models with and without SGLT2 inhibition. c) Corresponding estimated CoRL-derived podocyte densities. Data are represented as mean ±SD, n=6-9, †p<0.01, ‡p<0.001.

Figure 6.. Repopulation of different glomerular cell types alters upon SGLT2 inhibition depending on injury type.

a) Representative images of the stainings for parietal epithelial cells (PEC) (green) and tdTomato labeled CoRL (red). Colocalization is displayed in yellow and suggests the PEC is CoRL-derived and b) corresponding quantification. c) Representative images of stainings for mesangial cells (green) and tdTomato labeled CoRL (red). Yellow staining indicates colocalization, suggesting the particular mesangial cell derived of CoRL. d) quantification of c. Dashed lines indicate glomeruli, bar=20μm. Data are represented as mean ±SD, n=6-9, †p<0.01, ‡p<0.0001.