Megalin Knockout Reduces SGLT2 Expression and Sensitizes to Western Diet-induced Kidney Injury

Abstract Megalin (Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. Studies in our lab have shown that knockout (KO) of Lrp2 in opossum PT cells leads to a dramatic reduction in sodium–glucose co-transporter 2 (SGLT2) transcript and protein levels, as well as differential expression of genes involved in mitochondrial and metabolic function. SGLT2 transcript levels are reduced more modestly in Lrp2 KO mice. Here, we investigated the effects of Lrp2 KO on kidney function and health in mice fed regular chow (RC) or a Western-style diet (WD) high in fat and refined sugar. Despite a modest reduction in SGLT2 expression, Lrp2 KO mice on either diet showed increased glucose tolerance compared to control mice. Moreover, Lrp2 KO mice were protected against WD-induced fat gain. Surprisingly, renal function in male Lrp2 KO mice on WD was compromised, and the mice exhibited significant kidney injury compared with control mice on WD. Female Lrp2 KO mice were less susceptible to WD-induced kidney injury than male Lrp2 KO. Together, our findings reveal both positive and negative contributions of megalin expression to metabolic health, and highlight a megalin-mediated sex-dependent response to injury following WD.


Introduction
The Western-style diet (WD) is c har acterized by highly processed food rich in sugar, salt, animal protein, and saturated and trans -fats.The av era ge ann ual high-fructose corn syrup intake among Americans has increased over 120-fold over the past several decades and the prevalence of obesity has nearly doubled. 1 , 2 Obesity and diets high in processed meat and refined sugars promote the development of metabolic syndrome and chronic diseases such as car dio vascular disease and type 2 diabetes (T2D). 3 , 4he kidney has an extraordinary metabolic rate estimated to be double that of the li v er and brain. 5The majority of this energy expenditure supports transport by the proximal tubule (PT) to maintain salt and water homeostasis.Contrary to other ne phr on segments, almost no gl ycol ysis occurs in the PT; this pathway accounts for only 4% of ATP produced under aerobic conditions. 6Rather, PT cells utilize primarily lactate , pyruvate , glutamate, and free fatty acids as energy sources to dri v e oxidati v e phosphor ylation. 7In addition to maintaining its own energy needs, gluconeogenesis dri v en by the PT contributes r oughl y a quarter of serum glucose in rats under normal conditions.Under starvation conditions, this increases to nearly half of total blood glucose.It has also been shown that the PT is equally important in maintaining systemic gluconeogenesis in humans. 8 , 9n addition to generating glucose, the PT is also responsib le for r ea bsorbing glucose fr om the filter ed plasma to pr ev ent its excr etion in the urine.Glucose is r ea bsorbed via luminal sodium-glucose co-transporters 2 (SGLT2) and 1 (SGLT1), which are differentially expressed in early and later segments of the PT, r especti v el y .SGL T2 is a low-affinity high-capacity transporter that normally reabsorbs ∼90% of filtered glucose, while the high-affinity low-capacity SGLT1 r ea bsorbs the remaining ∼10%.Rather than serving as metabolic fuel for PT cells, glucose is exported to the b loodstr eam via basolateral Glucose transporter 2 (GLUT2) r ece ptors.
Inhibition of SGLT2 by phlorizin-related derivatives has become a powerful approach to maintain glycemic control in patients with T2D.These drugs pr ev ent the capture of filtered glucose by the PT, resulting in reduced plasma glucose levels.Strikingl y, n umer ous clinical trials hav e concluded that SGL T2 inhibitors (SGL T2is) ha ve benefits be y ond glucose re gulation, including impr ov ed outcomes in heart failure patients and slower pr ogr ession of chr onic kidney disease in patients with or without T2D. 10 -14The mechanisms behind these protecti v e effects are not well understood but have been suggested to include reduced mitochondrial burden in PT cells resulting from low er tr ansport needs and reduced intraglomerular pressures and reduction in proteinuria. 15 , 16n addition to glucose, the PT also r ecov ers essentiall y all proteins that escape the glomerular filtration barrier to prevent their excretion in the urine.Proteins are internalized upon binding to the multiligand receptors megalin ( Lrp2 ) and cubilin and efficiently targeted to lysosomes for de gr adation. 17 , 18Megalin is a > 600 kDa member of the low-density lipoprotein (LDL) receptor family that contains 4 ligand-binding regions, a single transmembrane domain, and a cytoplasmic tail with binding motifs that engage the clathrin adaptor protein Dab2 . 17Beyond its function as a r ece ptor, Lrp2 plays a unique role in driving membrane flux through the endocytic pathway. 19 , 20While the expression and subcellular distribution of endocytic markers appear unchanged in Lrp2 knockout ( Lrp2 KO) cells, trafficking through endocytic compartments is dramaticall y r educed when megalin is absent or its function is impaired. 19 -21egalin expr ession clearl y impacts kidney function, but its complex effects on the development and progression of kidney disease have been challenging to disentangle.In contrast, selec-ti v e kidney deletion of Lrp2 in mice results in a more modest phenotype , c har acterized primarily by urinar y excr etion of low molecular weight proteins. 22 -24Impairing megalin expression or function reduces the uptake of nephrotoxic drugs and limits their oxidati v e dama ge. 25 , 26 On the other hand, preserving megalin expression by inhibiting proprotein convertase subtilisin/kexin type 9 in pr otein uric animals is pr otecti v e in dampening r enal fibr osis and other injur y markers. 27The expression of megalin is affected in diseases such as T2D, with unknown consequences to disease pr ogr ession. 28 , 29Recentl y, our la borator y made the surprising discov er y that KO of Lrp2 in a highly differentiated PT cell line causes a dramatic ( > 80%) reduction in SGLT2 ( Slc5a2 ) mRN A tr anscripts.A more modest effect was observed in a kidney selective Lrp2 KO mouse model. 21The tr anscriptional re gulation of SGLT2 by megalin suggested the possibility that Lrp2 KO may recapitulate the glucose tolerance and/or cardiac and renal protective effects of SGLT2is.
The WD has been associated with an increased incidence of T2D, tubular injury, and chronic kidney disease in humans. 30 , 31e hypothesized that reduced expression of SGLT2 in Lrp2 KO mice would confer higher glucose tolerance, similar to that seen in SGLT2 ( Slc5a2 ) KO animals and in wild-type mice treated with SGLT2is. 32 , 33

Animal Care and Use
Mouse breeding and long-term monitoring experiments were appr ov ed by the Institutional Animal Care and Use Committee (IACUC), Uni v ersity of Pittsburgh (#19095734).Lrp2 lox/lox mice, 22 originall y obtained fr om Thomas Willnow (Max Delbr ück Center for Molecular Medicine), were bred to mice expressing Cre r ecombinase dri v en by the central nervous system-and kidneyspecific EMX promoter [generously provided by Cecilia Lo (University of Pittsburgh)].Lrp2 KO and control mice were established by heterozygous breeding.Control mice were defined as mice with genotype Lrp2 lo x/lo x Cre −/ − , and experimental Lrp2 KO mice were defined as mice with genotype Lrp2 lo x/lo x Cre −/ + .Male mice were fed a regular chow (RC) diet (Pr oLa b IsoPr o RMH 3000; kcal provided as approximately 26% protein, 14% fat, and 60% carbohydr ate , with 0.26% sodium).Following initial glucose tolerance tests (GTTs) and metabolic studies, mice were placed on a WD [pur c hased from Resear c h Diets (RD Western Diet, D12079B) and provided kcal as approximately 17% protein, 40% fat (44% saturated and 56% unsaturated), and 43% carbohydrate (70% sucrose, 30% star c h), with 0.26% sodium] for 9 wk prior to GTTs and metabolic studies.Body weight and changes in health condition were monitored weekly.Mice were maintained on WD for an additional 3 wk prior to sacrifice and collection of urine, tissue, and blood samples.The initial study was performed on a cohort of 19 male mice (11 control and 8 Lrp2 KO mice; 8-11 wk at the start of study).To investigate sex-specific differences, the effect of WD was evaluated in a separate study on a cohort of 8 female mice (5 control and 3 Lrp2 KO mice; 23 wk old at the start of study, placed on WD for 4 wk prior to GTT and metabolic studies, and maintained on WD for an additional 4 wk prior to sacrifice).Prior to sacrifice, 4 male control, 2 male Lrp2 K O , and 1 female control mouse died.

Metabolic Studies
Major determinants of whole-body energy balance were assessed in the Sa b le Systems Promethion Multiplexed Meta bolic Ca ge System.Mice wer e indi viduall y housed in a home cage setting for 72 h, during which feeding, activity, energy expenditure, drinking, and respiratory exchange ratio (RER) wer e contin uousl y monitor ed.The first 24 h were considered acclimation and not included in the analysis, such that data shown r e pr esent 48 h of data beginning on day 2  of housing.Body composition was measured by EchoMRI.GTTs were performed after a 6-h morning fast (7 am -1 pm ).Following a collection of basal blood sample ( t = 0) by tail bleed, mice r ecei v ed an intraperitoneal bolus injection of glucose at 2.0 mg/kg body mass on RC, and 1.5 mg/kg body mass on WD.

Indirect Immunofluorescence
Kidneys were fixed in formalin overnight at 4 • C and then transferred to 70% ethanol.Samples were sent to the University of Pittsburgh Biospecimen Core (PBC) for paraffin embedding and sectioning.Sections were deparaffinized using a standard protocol with xylene, 100% ethanol, and 95% ethanol.A doub le/sequential la beling pr otocol w as used to stain de paraffinized kidney sections with primary antibodies from the same host species. 34Briefly, sections wer e r ehydrated using Tris-buffer ed saline with 0.05% Tween-20 (TBST) and antigen r etriev al w as performed using heat and antigen unmasking solution (Vector, H-3300-25).Subsequently, samples w ere bloc ked in TBST containing 10% goat serum (TBST-Ser) for 1 h, and then incubated in TBST-Ser with rabbit anti-SGLT2 (1:250, ab85626) overnight.On the following day, samples wer e w ashed 3 times for 5 min each with TBST and incubated in anti-rabbit Alexa Fluor 488 in TBST-Ser for 30 min, washed 3 more times for 5 min each in TBST, and blocked in goat anti-rabbit unconjugated F(ab) fragments in TBST-Ser for 15 min.Samples were then incubated in TBST-Ser with rabbit anti-megalin antibody gener ousl y pr ovided by Dr Daniel Biemsderfer and Dr Peter Aronson (Yale Uni v ersity, 1:1000, MC-220) for 2 h, washed 3 times for 5 min each in TBST, and incubated in anti-rabbit Alexa Fluor 647 in TBST-Ser for 30 min.After washing 3 times for 5 min each in TBST, cov erslips wer e mounted onto the slides with Prolong Glass + NucBlue (Invitrogen, P36981) and sealed using clear nail polish.Samples wer e ima ged using a Leica STEL-LARIS 8 inverted confocal microscope using a 63 × oil immersion objecti v e.

Blood Chemistry Analysis
Blood was collected at the time of sacrifice, and samples were inserted in iST A T CHEM8 + cartridges (Abbot) and analyzed using a handheld iST A T 1 blood analyzer (Abbot).

Histology and Injury Scoring
F ormalin-fixed kidne ys w ere tr ansferred to 70% ethanol and sent to the Uni v ersity of Pittsburgh Biospecimen Core for paraffin embedding, sectioning, and Hematoxylin and eosin (H&E) and Masson's Trichrome staining.The entirety of the cortex and corticomedullar y r e gions of a single tr ansverse stained section at the midpoint of the kidney was imaged using a Leica LAS X Widefield microscope and scored by an investigator blinded to sample group according to the following scale: 0 = no injury, 1 = 1%-25% of tissue has injury, 2 = 26%-50% of tissue has injury, 3 = 51%-75% of tissue has injury, and 4 = 76%-100% of tissue has injur y.Injur y w as defined as any tubular injur y (dilation, sloughing, vacuolization) or inflammation.

Quantitati v e Real-Time Reverse Transcriptase Pol ymer ase Chain Reaction
Kidney samples were immediately frozen in liquid nitrogen and stored at −80

Expression of SGLT2 in Control and Lrp2 KO Mice
Western blotting and indirect immunofluorescence were used to confirm highly efficient ( > 90%) KO of megalin in EMX-Cre Lrp2 KO male mice compared with control mice ( Figure 1 A, top panel).By western blotting, we observ ed onl y a modest decrease ( ∼13%) in SGLT2 protein expression compared to their control counterparts ( Figure 1 A [bottom panel] and B).Indirect immunofluorescence staining of kidney sections r ev ealed a mor e pr onounced reduction in SGLT2 expression in Lrp2 KO mice compared with contr ols ( Figur e 1 C).

Glucose Tolerance and Metabolic Function in Control and Lrp2 KO Mice
Lrp2 KO male mice fed RC had baseline body weights, total fat mass, and total lean mass similar to their control counterparts ( Figure 2 A and B).We tested the effect of Lrp2 KO on glucose tolerance in mice on RC diets.Despite the modest reduction in SGLT2 expression, Lrp2 KO mice cleared a bolus of injected glucose mor e rapidl y than their contr ol counterparts ( Figur e 2 C and D).The magnitude of this effect is compara b le to that pr eviousl y r e ported in SGLT2 KO mice. 32ollowing GTTs, mice were placed in meta bolic ca ges and metabolic parameters (total activity, RER, energy expenditure, drinking, and feeding) wer e measur ed ov er a 48 h period.Ther e wer e no significant differ ences in meta bolic parameters between Lrp2 KO mice and control mice ( Figure 3 A-C and E), except that Lrp2 KO mice drank significantly more than control mice ( Figure 3 D).This increase in water intake is consistent with observations in SGLT2 KO mice and in wild-type mice treated with SGLT2i. 32 , 37The av era ge RER w as also lower in Lrp2 KO mice compared to controls but did not reach significance, with P -values of 0.181, 0.177, and 0.177 for the light, dark, and total cycles, r especti v el y ( Figur e 3 B).

Glucose Tolerance and Metabolic Function in Control and Lrp2 KO Mice Following a WD
Following their initial evaluation on RC, mice were fed a WD high in fat and refined sugar with normal NaCl concentration for 9 wk and evaluated as before.Remarkably, unlike control mice, which gained r oughl y 50% of their original body weight, Lrp2 KO mice did not gain weight on WD.Mor eov er, Lrp2 KO mice on WD had significantly lower % of fat compared to control counterparts ( Figure 4 A and B).Similar to our observations in mice on RC, glucose clearance in Lrp2 KO mice fed a WD was more rapid than in control mice fed a WD, with av era ge ar ea under the curv e approaching significance ( P = 0.086) ( Figure 4 C and D).Also consistent with their behavior on RC, Lrp2 KO mice on WD exhibited no significant differences in metabolic parameters compared to contr ol mice ( Figur e 5 A-C and E), except for a significant increase in water intake ( Figure 5 D).

Kidney Injury in Lrp2 KO Mice Fed a WD
Male mice fed WD were sacrificed, and their blood chemistry w as compar ed with a ge-matched Lrp2 KO and contr ol mice fed RC ( Figure 6 and Table 1 ).Blood chemistry of Lrp2 KO mice fed RC w as indistinguisha b le fr om a ge-matched contr ol mice ( Ta b le 1 ).Similarly , iST A T values for control mice on WD ( Figure 6 ) were unchanged compared with mice fed RC ( Table 1 ).By contrast, Lrp2 KO mice on WD had significantly increased levels of K + and Cl − , as well as higher blood urea nitrogen (BUN), and creatinine lev els, all suggesti v e of kidney injur y ( Figur e 6 ).Hematocrit and hemoglobin levels were reduced in Lrp2 KO mice compared to control mice.The lack of hemoconcentration suggests that volume depletion was unlikely to be driving their aberrant blood ion levels ( Figure 6 ).
To further evaluate renal injury, kidney sections from sacrificed male mice were processed for histologic staining and injur y lev el scor ed.Examination of H&E-stained kidney sections from mice on RC revealed slightly greater baseline injury in Lrp2 KO mice compared with controls ( Figure S1 ).After WD, control male mice developed enlarged tubular vacuoles, consistent with previous observations in wild-type or control mice fed a high-fat diet 38 , 39 ( Figure 7 A-C).By contrast, male Lrp2 KO mice had fewer tubular vacuoles, but exhibited dramaticall y incr eased inflammation ( Figur e 7 A-C).Reduced v acuolization in response to WD has also been r e ported in Sglt2i-treated mice. 40Changes in renal fibrosis were more striking, with Lrp2 KO mice on WD exhibiting significantl y mor e interstitial fibr osis than wild-type mice on the same diet ( Figure 7 D and E).This suggests that knocking out megalin leads to permanent and pr ogr essi v e chr onic kidney disease (CKD) in male mice fed a WD.
We used qRT-PCR to assess expression of injury marker transcripts in kidneys harv ested fr om male Lrp2 KO mice following WD ( Figure 8 ).Lrp2 KO mice had significantly increased transcript levels of acute kidney injury markers, kidney injury molecule-1 (KIM-1; Havcr1 ), and neutrophil gelatinaseassociated lipocalin (NGAL; Lcn2 ), compared with control mice.Lrp2 KO mice also had significantl y incr eased lev els of fibr osis markers fibronectin 1 ( Fn1 ) and collagen type 1 ( Col1 ), as well as significantly increased levels of inflammation markers tumor necrosis factor alpha ( Tnfa ), monocyte c hemoattr actant protein- 1 ( Mcp1 ), and interleukin 1 ( Il6 ).Though not quite significant ( P = 0.074), there was also a trend to war d increased transcript levels of inflammation marker interleukin b ( Il1b ) compared to control mice ( Figure 8 ).

Improved Glucose Tolerance and Protection in WD-fed Female Lrp2 KO Mice
We performed similar analyses to those above on a small cohort of female control and Lrp2 KO mice.Similar to male Lrp2 KO mice, female Lrp2 KO mice exhibit a modest reduction of SGLT2 pr otein expr ession ( Figur e S2 ).Like male mice, female Lrp2 KO mice also failed to gain weight on WD, and exhibited a high leanto-fat mass ratio ( Figure 9 A, B).Female Lrp2 KO mice on both RC ( Figure S3C and D ) and WD ( Figure 9 C and D) also exhibited impr ov ed glucose tolerance compared to control mice.In contrast to male Lrp2 KO mice on WD, blood chemistry of female mice was largely normal, other than reductions in HCO 3 and Ca 2 + ( Ta b le 2 ).The histology of female kidney sections following WD r ev ealed onl y slightl y incr eased kidney injur y in female Lrp2 KO mice on RC ( Figure S4 ) and on WD compared with contr ol females ( Figur e 9 E-H).qRT-PCR anal ysis also r ev ealed incr eased transcript lev els of kidney injur y biomarkers in female Lrp2 KO mice on WD compared with control females, although the changes were smaller than those in male Lrp2 KO mice, and in this small cohort, none reached significance ( Figure 9 I-P).There were no significant differences in metabolic parameters between control and Lrp2 KO female mice on either RC or WD, except for activity ( Figure S5 ).RER in Lrp2 KO female mice trended lower in the light cycle on both RC ( Figure S5B ) and WD ( Figure S5G ).

Discussion
Our studies confirm the complex role for megalin in maintaining kidney and systemic health.We found that both male and female Lrp2 KO mice had modestly reduced SGLT2 levels that translated to impr ov ed glucose tolerance.Additionally, total body weight and fat percentage in Lrp2 KO mice fed a WD were significantly lower than control.On the other hand, male Lrp2 KO mice fed a WD developed sev er e kidney damage accompanied by a berrant b lood chemistr y.Kidney function in female Lrp2 KO mice on WD appeared unchanged and kidney damage was considera b l y milder.While the critical role of PT gluconeogenesis in maintaining glucose levels under starvation conditions is well known, our study demonstrates a striking role for megalin expression in the systemic response to WD.
The metabolic phenotype of Lrp2 KO mice on RC and WD str ongl y r esemb les that of SGLT2 ( Slc5a2 )-deleted mice and SGLT2i-treated mice. 32 , 37The gl ycemic pr otection we observ ed in Lrp2 KO mice is compara b le to that observed in Slc5a2 KO mice, 32 in cana gliflozin-tr eated wild-type mice, 37 and in Sweet Pee mice that carry a truncation mutation in the Slc5a2 gene. 41Mor eov er, plasma glucose lev els wer e not r educed in Lrp2 KO mice, r e plicating observ ations in Sweet P ee and Slc5a2 KO mice. 32 , 41Lrp2 KO mice also had increased fluid intake as pr eviousl y observ ed when Slgt2 expr ession or function is impaired. 32 , 37 , 41Inacti v ation or deletion of SGLT2 r educes RER and shifts meta bolism tow ard lipid utilization. 32 , 37Although RER in Lrp2 KO mice on RC or WD trended lower than in controls, it did not reach statistical significance.Nevertheless, Lrp2 KO mice on WD exhibited lo wer bod y fat accumulation than controls, similar to observations in diabetic Slc5a2 KO and Sweet P ee mice . 32 , 41Lo wer bod y fat accum ulation has been inferr ed to mean that animals are in a catabolic state, though we have not confirmed this in our mice. 41The less dramatic effect of Lrp2 KO on RER may reflect the residual activity of SGLT2 in this model.Surprisingly, despite their lower weight gain and fat accumulation, male Lrp2 KO mice on WD developed significant kidney injury.Masson's Trichrome staining revealed significant levels of fibrosis in male Lrp2 KO mice on WD compared to control mice on the same diet, which had virtually no fibrosis.On the other hand, PT cells in control mice on WD dev eloped mor e n umer ous v acuoles compar ed with Lrp2 KO mice.This r emarka b le difference in injury profile between megalin-expressing and megalindepleted mice may explain why Lrp2 KO is pr otecti v e in some disease settings and harmful in others. 25 , 26 , 28 , 29In this regard, our data are consistent with a previous study that found reduced vacuolization in uninephrectomized Lrp2 KO mice fed a high-fat diet compared to similarly treated control mice. 42Presumably in the added presence of high levels of refined sugars, loss of megalin expression becomes deleterious to kidney health.We have found differential expression of genes involved in fructose metabolism in Lrp2 KO OK cells compared to control OK cells, though these differences have not been confirmed in our mouse model. 21Differences in fructose metabolism may contribute to the deleterious effects of megalin KO on kidney health following WD.
Inter estingl y, while glycemic protection in female Lrp2 KO mice was similar to that in males, female mice did not exhibit extensi v e kidney injur y following WD.Indeed, women and female mice are generally protected against the onset of diabetes, and female mice ar e r esistant to WD-induced weight gain.Incr eased oxidati v e str ess and mitochondrial dysfunction contribute to r enal injur y in mice on high-fat diets. 43Women have also been shown to have higher mitochondrial spare respiratory capacity and lower r eacti v e oxygen species (R OS) pr oduction than men in most tissues. 44 -46Additionally, PT energy expenditure in female rodents is thought be lower than in due to a gr eater r eliance on the distal convoluted tubule for sodium r bsorption. 47 , 48Together, these factors may contribute to the enhanced protection of female Lrp2 KO mice against WD-induced kidney injury.Understanding the relationship between diet and Lrp2 -mediated sex-dependent protection or injury may pro vide no vel tar g ets for manag ing obesity and T2D.
Our study has some limitations that limit the extent of conclusions we can draw.The cohort of female mice that we tested was small, and we may have missed differences that would be evident in larger gr oup.Additionall y, while our studies demon- strate a substantial role for megalin expression in maintaining PT function, our work here does not address how Lrp2 KO alters SGLT2 expression or activity, or how megalin defends against WD-induced kidney disease.We demonstrate an increase in pr oinflammator y cytokines in Lrp2 KO mice, and inflammation is str ongl y implicated in the development of tubular injury and fibrosis (PMC6792167).How megalin affects inflammation, and whether this inflammation is causing the kidney injury, or a result of underlying tubular injury, will require investigation in future studies.Despite these limitations, our studies raise new fundamental questions that remain to be r esolv ed.Why is the apparentl y modest r eduction in SGLT2 lev els in Lrp2 KO mice sufficient to r e pr oduce the effect of SGLT2 deletion?Although we did not observe any reduction in SGLT1 expression based on quantitation of Slc5a1 transcript levels, it is possible that SGLT1 pr otein lev els ar e also r educed in Lrp2 KO mice and contribute to the phenotype.A second question is how megalin mediates the complex response to WD.While the primary function ascribed to megalin is the endocytic r etriev al of filtered proteins, KO of megalin in PT cells also affects transcription, ion transport, and cell metabolism. 21Understanding the interplay between these di v erse functions may explain the balance of protecti v e and deleterious effects of Lrp2 KO in response to dietary conditions.

Figure 1 .
Figure 1.Lrp2 KO mice hav e r educed lev els of SGLT2.Western blot (A) of mouse kidney lysates from control and Lrp2 KO male mice on regular chow probed to detect megalin and SGLT2.(B) Quantitation of SGLT2 expression in panel A. Data analyzed by an unpaired t -test.(C) Cortical kidney sections of control and Lrp2 KO mice were stained to r ev eal SGLT2 (gr een).Mer ged panels belo w each image sho w megalin (r ed) and n uclei (b lue) to v erify efficient K O .SGLT2 exposur es and pr ocessing wer e identical for all images.Megalin staining intensity in the Lrp2 KO ima ge w as selecti v el y enhanced to ena b le better comparison of expr essing v ersus non-expr essing cells.Scale bar: 25 μm.

Figure 2 .
Figure 2. Lrp2 KO mice have improved glucose homeostasis on regular cho w.Bod y weight (A) and composition (B) control and Lrp2 KO mice are plotted.Data analyzed by an unpaired t -test with multiple comparisons correction (Holm-Sidak).(C) Temporal changes in plasma glucose following intraperitoneal injection of 2.0 mg/kg glucose in fasted control and Lrp2 KO mice.Area under the curve (AUC) for glucose calculated in panel C is shown in panel D. ( P -values denoted by asterisk: * ≤0.05.)Data analyzed by an unpaired t -test.

Figure 3 .
Figure 3. Metabolic parameters of control and Lrp2 KO mice on re gular c how.Control and Lrp2 KO mice were evaluated in metabolic cages for 48 h.Traces for each parameter are shown in the left graph of each panel (gray bars denote dark cycles), and av era ge data for each cycle and total over the 48 h period are plotted on the right.(A) Activity; (B) respiratory exc hange r atio; (C) energy expenditure; (D) drinking; and (E) feeding.( P -values denoted by asterisk: * ≤0.05.)Data analyzed by an unpaired t -test with multiple comparisons correction (Holm-Sidak).

Figure 4 .
Figure 4. Body composition and glucose homeostasis are altered in Lrp2 KO mice after Western diet.Control and Lrp2 KO mice were placed on a Western diet for 9 wk and analyzed as in Figure 2 .Body weight (A) and composition (B) of control and Lrp2 KO mice are plotted.Data analyzed by an unpaired t -test with multiple comparisons correction (Holm-Sidak).(C) Temporal changes in plasma glucose following intraperitoneal injection of 1.5 mg/kg glucose in fasted control and Lrp2 KO mice.AUC for glucose calculated in panel C is shown in (D).( P -values denoted by asterisk: * * * * ≤0.0001.)Data analyzed by an unpaired t -test.

Figure 5 .
Figure 5. Metabolic parameters of control and Lrp2 KO mice after Western diet.Control and Lrp2 KO mice fed a Western diet for 9 wk were evaluated in metabolic cages for 48 h.Traces for eac h par ameter are shown in the left graph of each panel (gray bars denote dark cycles), and av era ge data for each cycle and total over the 48 h period are plotted on the right.(A) Activity; (b) respiratory exc hange r atio; (c) energy expenditure; (d) drinking; and (e) feeding.( P -values denoted by asterisk: * * ≤0.01.)Data analyzed by an unpaired t -test with multiple comparisons correction (Holm-Sidak).

Figure 7 .
Figure 7. Lrp2 KO mice have exacerbated kidney injury after Western diet.H&E and Masson's Trichrome stained sections wer e scor ed in a blinded manner as described in the "Methods" section.(A) H&E scoring of stained sections of mice on WD. (B) Re pr esentati v e H&E stained kidney sections from control and Lrp2 KO mice after Western diet.Zoomed-in images of areas denoted with red box in (B) are enlarged in (C).Areas of proximal tubule vacuolization (yellow arrows) and inflammatory r esponses (b lack arr ows) ar e shown.(D) Masson's Trichr ome scoring of stained kidney sections from mice on WD. ( P -values denoted by asterisks: * * * * ≤0.0001.)Data analyzed by an unpaired t -test.(E) Representative Masson's Trichrome (E) stained kidney sections from control and Lrp2 KO mice after Western diet.All images shown ar e fr om mice with injur y scor es near the mean of their gr oup.

Figure 9 .
Figure 9. Effects of Western diet on glucose toler ance , metabolic par ameters, and kidney injury in female Lrp2 KO mice.Control and Lrp2 KO mice were placed on a diet for 4 wk and analyzed as in Figure 2 .Body weight (A) composition (B) of control and Lrp2 KO mice are plotted.Data analyzed by an unpaired t -test with multiple comparisons correction (Holm-Sidak).(C) Temporal changes in plasma glucose following intraperitoneal injection of 1.5 mg/kg glucose in fasted control and Lrp2 mice.AUC for glucose calculated in panel C is shown in (D).H&E scoring of stained kidney sections from mice on WD shown in (E) and Masson's scoring of stained kidney sections from mice on WD shown in (G).H&E and Masson's Trichrome stained sections wer e scor ed in a blinded manner as described in the "Methods" section.Re pr esentati v e H&E (F) and Masson's Trichr ome (H) stained kidney sections fr om contr ol and Lrp2 KO mice after Western diet.qRT-PCR analysis for injury markers (I) Havcr1 , (J) Lcn2 , (K) Fn1 , (L) Col1 , (M) Tnfa , (N) Mcp1 , (O) Il6 , and (P) Il1b .Data analyzed by an t

T able 2 .
iST A T Blood Chemical Anal ysis of Contr ol and Lrp2 KO Female Mice on Regular Chow and Western Diet • C. Kidneys were lysed using TRIzol Reagent (Ambion, Carlsbad, CA, USA) to isolate RNA.cDN A was gener ated with the RevertAid Reverse Transcrip-iST A T Blood Chemical Anal ysis of Contr ol and Lrp2 KO Mice on Regular Chow tase Kit (ThermoFisher Scientific, Pittsburgh, PA, USA).The quantitati v e r eal-time r ev erse transcriptase pol ymer ase c hain reaction (qRT-PCR) used iTAQ Uni v ersal SYBR Gr een Supermix (Bio-Rad Laboratories).Primer sets are listed in Ta b le S1 .Results were normalized to Ppia (cyclophilin A), and expr ession w as determined using the comparati v e 2( − C(T)) method.35 , 36le 1.Values are means.No significant differences detected by an unpaired t -test.SEM noted in br ac kets.