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Macula Densa SGLT1-NOS1-Tubuloglomerular Feedback Pathway, a New Mechanism for Glomerular Hyperfiltration during Hyperglycemia

Jie Zhang,1 Jin Wei,1 Shan Jiang,1 Lan Xu,2 Lei Wang,1 Feng Cheng,3 Jacentha Buggs,4 Hermann Koepsell,5 Volker Vallon,6 and Ruisheng Liu1

1Department of Molecular Pharmacology and Physiology, College of Medicine, 2Department of Biostatistics, College of Public Health, and 3Department of Pharmaceutical Science, College of Pharmacy, University of South Florida, Tampa, Florida; 4Advanced Organ Disease & Transplantation Institute, Tampa General Hospital, Tampa, Florida; 5Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany; and 6Division of Nephrology and Hypertension, Department of Medicine, University of California, San Diego, La Jolla, California

ABSTRACT Background Glomerular hyperfiltration is common in early diabetes and is considered a risk factor for later diabetic nephropathy. We propose that sodium-glucose cotransporter 1 (SGLT1) senses increases in luminal glucose at the macula densa, enhancing generation of neuronal nitric oxide synthase 1 (NOS1)– dependent nitric oxide (NO) in the macula densa and blunting the tubuloglomerular feedback (TGF) response, thereby promoting the rise in GFR. Methods We used microperfusion, micropuncture, and renal clearance of FITC–inulin to examine the effects of tubular glucose on NO generation at the macula densa, TGF, and GFR in wild-type and macula densa–specificNOS1knockoutmice. Results Acute intravenous injection of glucose induced hyperglycemia and glucosuria with increased GFR in mice. We found that tubular glucose blunts the TGF response in vivo and in vitro and stimulates NO generation at the macula densa. We also showed that SGLT1 is expressed at the macula densa; in the presence of tubular glucose, SGLT1 inhibits TGF and NO generation, but this action is blocked when the SGLT1 inhibitor KGA-2727 is present. In addition, we demonstrated that glucose increases NOS1 expression and NOS1 phosphorylation at Ser1417 in mouse and cultured human tissue. In macula densa–specific NOS1 knockout mice, glucose had no effect on NO generation, TGF, and GFR. Conclusions We identified a novel mechanism of acute hyperglycemia–induced hyperfiltration wherein increases in luminal glucose at the macula densa upregulate the expression and activity of NOS1 via SGLT1, blunting the TGF response and promoting glomerular hyperfiltration.

J Am Soc Nephrol 30: 578–593, 2019. doi: https://doi.org/10.1681/ASN.2018080844

More than 30 million Americans have diabetes. Di- Received August 20, 2018. Accepted January 27, 2019. abetic nephropathy is a major complication of di- abetes mellitus1–3 and the leading cause of ESRD. An J.Z. and J.W. contributed equally to this work. increase in GFR or glomerular hyperfiltration has Published online ahead of print. Publication date available at www.jasn.org. been observed in about 70% of patients with type 1 diabetes4,5 and 50% of patients with type 2 diabe- Correspondence: Dr. Jie Zhang, Department of Molecular Pharmacology and Physiology, University of South Florida Col- 5–8 tes, andisassociatedwithanincreasedrisk lege of Medicine, 12901 Bruce B. Downs Boulevard, MDC 8 for diabetic nephropathy and worse prognosis.4,7–9 Tampa, FL 33612. Email: [email protected] The pathogenesis of glomerular hyperfiltration in Copyright © 2019 by the American Society of Nephrology

578 ISSN : 1046-6673/3004-578 J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH diabetes has not been fully elucidated. Several mechanisms have Significance Statement been implicated, primarily including vascular and tubular theo- ries. According to the vascular mechanism, glomerular hyperfil- Although glomerular hyperfiltration is common in early diabetes and tration results from an imbalance between vasoconstrictive fac- considered a risk factor for later diabetic nephropathy, the mech- fi tors and vasodilatory factors.4,5,10 The tubular theory proposes anisms underlying glomerular hyper ltration have not been fully clarified. The authors identified a novel mechanism of acute hy- that tubular growth and the upregulated sodium-glucose co- perglycemia–induced hyperfiltration in which increases in luminal transporter 2 (SGLT2) enhance proximal tubular reabsorp- glucose at the macula densa upregulate the expression and activity tion, which reduces sodium chloride (NaCl) delivery to the of neuronal nitric oxide synthase 1 (NOS1) via sodium-glucose co- macula densa and increases GFR via the tubuloglomerular transporter 1 (SGLT1); this blunts the tubuloglomerular feedback fi feedback (TGF) response (SGLT2-NaCl pathway).11–13 (TGF) response and promotes glomerular hyper ltration. This novel SGLT1-NOS1-TGF pathway mediates the glomerular hyperfiltration The TGF response describes a mechanism by which an in- observed in response to acute hyperglycemia. These findings estab- crease in NaCl delivery to the macula densa promotes the re- lish a critical role of macula densa NOS1 and SGLT1 as key determi- lease and formation of ATP and/or adenosine,14–17 which then nants of, and potential therapeutic targets for, acute hyperglycemia– constricts the afferent arteriole (Af-Art) and induces a tonic associated glomerular hyperfiltration, and possibly for diabetes as well. inhibition of single- GFR.18–20 neuronal nitric oxide fl fl synthase (NOS1) is the predominant nitric oxide synthase knockout (MD-NOS1KO) mice (NKCC2cre/NOS1 ox/ ox) 21,22 (NOS) isoform expressed in macula densa cells, and nitric were generated by crossing an NKCC2-Cre line with oxide (NO) generated by the macula densa blunts TGF re- NOS1-floxed mice as described previously.28 All protocols 23–26 sponse. Recently, several studies from our laboratory were approved by the Institutional Animal Care and Use demonstrated the decisive role of macula densa NOS1 in the Committee at the University of South Florida, College of TGF response and the long-term control of GFR, sodium ex- Medicine (IS00003816 and IS00004119). All chemicals 27–29 cretion, and BP. Mice with deletion of NOS1 from the were purchased from Sigma (St. Louis, MO), except as macula densa exhibit enhanced TGF responsiveness and de- indicated. velop salt-sensitive hypertension.28 Although many previous studies have assessed the TGF response and renal NO produc- Induction of Acute Hyperglycemia tion in diabetes,11,30–33 whether macula densa NOS1 is a To determine the intravenous dose of glucose that is able to causal factor for diabetic hyperfiltration remains elusive. increase blood glucose levels over the renal threshold of glucose More than 99% of filteredglucose in the kidney is reabsorbed by (corresponding to blood glucose concentration of approxi- sodium-glucose cotransporter 1 (SGLT1) and SGLT2 in the prox- mately .180 mg/dl in humans and .250 mg/dl in ro- imal tubules. SGLT2 is present in the S1 and S2 segments of dents37,38), we applied a bolus injection of 50 ml of glucose proximal tubules and mediates approximately 97% of glucose (2.5 or 5 M in saline) in lightly anesthetized mice via the retro- resorption, whereas SGLT1 is present in the S3 segment of prox- orbital venous sinus, and the blood glucose levels were measured imal tubules and accounts for the remaining 2%–3% of the at 3, 10, 20, 40, 60, and 80 minutes after glucose injection, by filtered glucose.34 The luminal glucose concentration at the mac- Precision Xtra Glucose Meter K (Fisher Scientific, Waltham, ula densa is usually negligible under normoglycemic conditions. MA). Mice injected with 50 ml saline (0 M glucose) served as Luminal glucose concentration at the macula densa rises, how- controls. ever, when the amount of filtered glucose exceeds the maximal capacity of reabsorption by proximal tubules in hyperglycemic Measurement of Glucose Concentration in Urine and states. Moreover, SGLT1has recently been detected on the apical Early Distal Tubule membrane of macula densa cells in mouse35 and rat kidneys36 To determine the effect of hyperglycemia on glucose concen- with a custom-made antibody. However, the role of macula tration at macula densa, the glucose concentration in urine and densa SGLT1 in the control of TGF and GFR in a hyperglycemic early distal tubule fluid were measured. Micropuncture prep- setting is not known and has not been investigated. aration was performed as previously described.28,39,40 Briefly, In this study, we tested a novel hypothesis that the increase in mice were anesthetized with inactin (80 mg/ml, intraperito- luminal glucose concentration at the macula densa enhances neal injection) and ketamine (50 mg/ml, intramuscular injec- NOS1-dependent NO formation via SGLT1, thereby inhibiting tion). A tracheostoma was placed to facilitate breathing and TGF responsiveness and promoting glomerular hyperfiltration femoral artery was catheterized for BP measurement. The in hyperglycemia (SGLT1-NOS1 pathway). femoral vein was catheterized for infusion of saline with 1% BSA at the rate of 1 ml/h per 100 g body wt throughout the experiment. After an abdominal incision, the left was METHODS catheterized for the collection of urine and the left kidney was exposed and immobilized in a kidney holder cup. The Animal kidney orientation was positioned so that superficial tubules C57BL/6 mice (male, 13–15 weeks old) were purchased from could be clearly visualized under the microscope (SZX16; Jackson Laboratory. The macula densa–specific NOS1 Olympus, Tokyo, Japan). The segment of early distal tubule

J Am Soc Nephrol 30: 578–593, 2019 Macula Densa SGLT1-NOS1-TGF Pathway 579 BASIC RESEARCH www.jasn.org was identified by the movement of green dye along the same was set at 0 nl/min and 40 nl/min, each for 3–5 minutes. The nephron, after a bolus injection of artificial tubular fluid change of Psf (DPsf) was used as an index of the TGF re- (ATF) with 5% fast green into a random proximal segment. sponse. To determine the effect of glucose on TGF response After 30 minutes of equilibration, urine was collected in vivo, DPsf was measured twice consecutively in the same through a catheterized ureter for 20 minutes, and the fluid nephron: first DPsf was measured with glucose-free ATF, in early distal tubule was collected by oil-filled pipette for and second DPsf was measured using ATF with 16.7 mM 3–5 minutes after a blockade of oil droplet as basal. Then, 50 glucose. Second DPsf measurements using glucose-free ml glucose solution (5 M in saline) was intravenously in- ATF, ATF with 16.7 mM L-glucose, or ATF with 16.7 mM jected via retro-orbital venous sinus. Urine was collected mannitol served as control groups. In addition, because during 0–20, 20–40, 40–60, and 60–80 minutes after the in- twice consecutive measurements of DPsf inthesameneph- jection of glucose solution, and the fluid in early distal tubule ron may introduce a systematic error, a single measurement was collected at 20, 40, 60, and 80 minutes. The glucose of DPsf was also performed directly under high-glucose con- concentration in the urine and early distal tubular fluid sam- ditions, using ATF with 16.7 mM glucose. Measurements ples was measured with mouse glucose assay kit (Crystal with glucose-free ATF, ATF containing 16.7 mM L-glucose, Chem, Elk Grove Village, IL). or ATF containing 16.7 mM mannitol served as control groups. Measurement of GFR in Conscious Mice GFR was measured by the clearance of plasma FITC–inulin Measurement of TGF in vitro with Microperfusion with a single bolus injection in conscious mice as previ- Mice were anesthetized with isoflurane and kidneys were ously described.28,41,42 Mice were lightly anesthetized with removed and sliced. Kidney slices were placed in ice-cold isoflurane and injected with FITC–inulin solution (3.74 ml/g DMEM containing 5% BSA. A single, superficial Af-Art body wt) through the retro-orbital venous sinus. Blood (10 ml) and its intact were microdissected together was collected into heparinized capillary tubes through the with adherent tubular segments consisting of the thick as- retro-orbital venous sinus at 3, 7, 10, 15, 35, 55, and 75 min- cending limb (TAL), macula densa, and early distal tubule utes after inulin injection. The blood samples were centrifuged under a stereomicroscope (SMZ1500; Nikon, Yuko, Japan), at 8000 rpm for 5 minutes at 4°C. Plasma (1 ml) was collected as described previously.28,39 The microdissection was com- from each sample. FITC–inulin concentration of the plasma pleted within 60 minutes and the dissected sample was was measured using a plate reader (Cytation3; BioTek, transferred to a temperature-regulated chamber mounted Winooski, VT) with 485 nm excitation and 538 nm emission. on an inverted microscope (Axiovert 100TV; ZEISS). The GFR were calculated with GraphPad Prism 6 (GraphPad bath solution (containing 5.5 mM glucose) in the chamber Software, San Diego, CA). (total volume of 1.5 ml) was exchanged continuously at a To determine the effect of hyperglycemia on GFR, we first rate of 1 ml/min and the temperature of the bath was main- measured the basal GFR in mice, and then 7 days later, the mice tained at 37°C throughout the experiment. The Af-Art were intravenously injected with 50 ml glucose solution (5 M was cannulated with one glass pipette and perfused with in saline) together with FITC–inulin (3.74 ml/g body wt) DMEM, and intraluminal pressure of Af-Art was maintained through the retro-orbital venous sinus for repeated measure- at 60 mm Hg throughout the experiment. The TAL was ment of GFR. To determine whether osmolality affects GFR, cannulated with another glass pipette and perfused with we repeated the experiments by replacing D-glucose with glucose-free macula densa solution containing, in mM: L-glucose (50 ml,5Minsaline)ormannitol(50ml, 5 M in 10 HEPES, 1.0 CaCO3,0.5K2HPO4, 4.0 KHCO3,1.2 saline). MgSO4, 0.5 Na acetate, 0.5 Na lactate, and either 10 NaCl or 80 NaCl; pH 7.4. The imaging system consisted of a mi- Measurement of TGF in vivo with Micropuncture croscope (Eclipse Ti; Nikon), a digital charge–coupled de- The preparation of micropuncture in mouse kidney was vice camera (CoolSnap; Photometrics, Tucson, AZ), xenon performed as described above. Tubular flow of a selected light (LB-LS/30; Shutter Instruments), and optical filter with multiple visible loops was obstructed changer (Lambda 10–3; Shutter Instruments). Images with a grease block. Stop-flow pressure (Psf)inproximal were displayed and analyzed with NIS-Elements imaging tubule upstream of the grease block was measured with the software (Nikon). servo-nulling method (Model 900A; World Precision In- After a 20-minute equilibration period, macula densa per- struments, Sarasota, FL). The micropipette for pressure fusate was switched from 10 to 80 mM NaCl, and luminal measurement with a tip diameter of 2–3 mmwasfilled diameter of the Af-Art was observed for at least 5 minutes. with 2 M KCl colored with 1% fast green. The proximal The TGF response was determined by the average change in the tubule segment distal to the grease block was perfused luminal diameter of the Af-Art. To determine the effect of with ATF (containing in mM: 4 NaHCO3,5KCl,2 luminal glucose on TGF response in vitro,theTGFre- CaCl2,7 urea, 2 MgCl2,128NaCl,and1%fastgreen;pH sponse was measured twice consecutively in the same isolated 7.4; pH 7.4). Psf was measured when tubular perfusion rate juxtaglomerular apparatus (JGA). First TGF response was

580 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH measured by switching macula densa perfusate from to determine whether the osmolality has any effect on NO 10 to 80 mM NaCl in the absence of glucose. In the second generation in the macula densa. TGF response measurement, macula densa was perfused To determine the effect of glucose at the basolateral side with 10 mM NaCl macula densa solution plus 16.7 mM glu- of the macula densa on NO generation, we measured the cose for 15 minutes, followed by switching macula densa NO generation in the macula densa when the glucose con- perfusate from 10 to 80 mM NaCl while maintaining the centration in the bath solution was increased from 5.5 same glucose level. to 16.7 mM when the tubular perfusate was maintained To determine whether the osmolality has any effect on TGF glucose-free. response in vitro, we repeated the above experiments, replac- In separate experiments, to determine the significance ing D-glucose with L-glucose (16.7 mM) or mannitol of SGLT1 in the effect of glucose on the NO generation at (16.7 mM). the macula densa, the macula densa of isolated JGA To determine the effect of glucose in the basolateral side from mouse kidney was treated with the selective SGLT1 of macula densa on TGF response in vitro, we changed the inhibitor KGA-2727 via the tubular lumen perfusate for glucose concentration in the bath solution while maintain- 30 minutes before the NO generation was measured by the ing the glucose-free tubular perfusate. The first TGF re- above protocol. sponse was measured with normal bath solution (containing 5.5 mM glucose), and the second TGF response was mea- Analysis of the Single-Cell RNA-Sequencing Profile sured with glucose concentration in the bath solution We analyzed the single-cell gene expression profiles of kidney increased to 16.7 mM. samples (normal 1, normal 2, normal 3, and normal 4) from To determine the effect of luminal glucose on the TGF re- C57BL/6 mice that were recently reported by Park et al.45 The sponse under constant and low tubular NaCl at the macula datasets were accessed in National Center for Biotechnology densa, as observed in diabetic rats, the diameter of the Af-Art Information Gene Expression Omnibus (GEO) database was assessed when tubular glucose concentrationwas increased (accession no. GSE107585). We identified and selected the from 0 to 16.7 mM while the NaCl concentration was main- macula densa cells from 12,090 kidney cells, using the tran- tained at 15 mM.12 scriptional copies of both marker genes “Na/K/2Cl cotrans- In separate experiments, to determine the significance of porter (Slc12a1)” and “nitric oxide synthase 1 (Nos1)” .0as SGLT1 in the effect of glucose on the TGF response, the cut-off (Table 1). Then, we compared the mRNA expression macula densa of isolated JGA from mouse kidney was treat- levels of glucose transporters (Slc2a1, Slc2a2, Slc2a3, Slc2a4, 2 ed with the selective SGLT1 inhibitor KGA-2727 (10 6 M) Slc2a5, Slc2a6, Slc2a8, Slc2a9, Slc2a12, Slc2a13, Slc5a1, and (Kissei Pharmaceutical Co., Ltd., Nagano, Japan)43 via the Slc5a2) in these selected macula densa cells. All statistical anal- tubular lumen perfusate for 30 minutes before the TGF re- yses were performed using R (version 3.4.3). sponse was measured by the above protocol. Immunofluorescence Measurement of NO Generation at the Macula Densa Double-immunofluorescence staining of SGLT1 as well as in Isolated Perfused Juxtaglomerular Apparatus NOS1 to localize the macula densa in human kidney was The preparation of the mouse Af-Art and attached macula performed as previously reported.28,39 The human kidney densa was performed as described for the measurement biopsy samples were collected from cadaveric kidney do- of TGF in vitro with microperfusion. After a 30-minute nors in Tampa General Hospital, Florida. The samples equilibration period, the macula densa was loaded with were fixed overnight with 4% paraformaldehyde and then fluorescent NO probe 4-amino-5-methylamino-2’,7’- embedded in paraffin. After deparaffinization and antigen difluorofluorescein diacetate (DAF-2 DA; 10 mMplus0.1% unmasking, 2 mm sections were blocked with 10% normal pluronic acid) from the tubular lumen for 30 minutes, then goat serum and 0.1% Tween-20 in PBS for 30 minutes. For washed for 10 minutes with macula densa perfusate. DAF-2 DA the detection of SGLT1, the slices were incubated overnight was excited at 490 nm with a xenon light, and the emitted with a SGLT1 antibody (rabbit polyclonal IgG; 1:500).35,36 fluorescence was recorded at wavelengths of 510–550 nm. After washing, the sections were incubated for 1 hour with The rate of increase in fluorescence intensity of DAF-2 DA a fluorescent secondary antibody (Alexa Fluor 594 goat anti- was used to determine NO generation by the macula densa.28,44 To determine the effect of luminal glucose on the fi NO generation in the macula densa, basal NO generation Table 1. Identi cation and selection of macula densa cells was measured with glucose-free macula densa perfusate for from single kidney cells 5 minutes, followed by measurement of NO generation in Condition Normal 1 Normal 2 Normal 3 Normal 4 Total the macula densa as the glucose concentration in the tubular Subtotal 2943 5060 1383 2704 12,090 perfusate was increased to 16.7 mM. In addition, we repeated Slc12a1.0 268 268 165 279 980 the above experiments, replacing D-glucose with L-glucose Nos1.0 7681940 . . (16.7 mM) or mannitol (16.7 mM) as the osmotic controls Slc12a1 0&Nos1 0 6581938

J Am Soc Nephrol 30: 578–593, 2019 Macula Densa SGLT1-NOS1-TGF Pathway 581 BASIC RESEARCH www.jasn.org rabbit, 1:1000; Abcam, Cambridge, MA). For the detection of by enhanced chemiluminescence detection on Hyperfilm NOS1, the slices were incubated overnight with a (Amersham Pharmacia Biotech, Piscataway, NJ). NOS1 antibody (A-11; mouse monoclonal IgG, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) followed by the fluores- Statistical Analyses cent secondary antibody (Alexa Fluor 488 goat anti-mouse; Statistical analyses were performed using SPSS 13.0 (IBM, 1:1000). Negative controls included sections incubated with- Armonk, NY). The effects of interest were tested using t test, out primary and/or secondary antibodies. All slices were or ANOVA, followed by multiple comparisons post hoc test mounted with VECTASHIELD antifade medium with DAPI when appropriate. Data are presented as mean6SEM, and (Vector Laboratories, Burlingame, CA), and images were cap- P,0.05 was considered statistically significant. tured with Nikon Eclipse E600FN Confocal Microscope equip- ped with a Cascade 131 512F digital camera (Photometrics, Tucson, AZ). RESULTS

Human Kidney Tissue Culture A Bolus Intravenous Injection of Glucose Induced The human kidney biopsy samples were cultured with the Hyperglycemia and Glucosuria with Increased GFR hanging drop technique, as recently described.46 The cul- A bolus intravenous injection of 50 mlof2.5Mglucose ture medium contained DMEM (Invitrogen, Carlsbad, increased blood glucose immediately after injection, but CA); 0.1 mM MEM nonessential amino acids (Invitrogen); the blood glucose quickly returned to normal levels at 2mMsodiumpyruvate;2nML-glutamine;0.01mg/ml 20 minutes (Figure 1A). An intravenous injection of 50 ml insulin, 5.5 mg/ml transferrin, and 5 mg/ml selenium sup- of 5 M glucose raised blood glucose over 200 mg/dl and plement; 100 U/ml penicillin; 100 mg/ml streptomycin; induced significant glucosuria as well as increased glucose and 10% FBS. A drop of medium (approximately 30 ml) concentration in the early distal tubule for 80 minutes was prepared on the lid of a petri dish. A piece of human (Figure 1B). kidney biopsy tissue (1 mm3) was placed into the drop, Using the latter glucose dose, we found that an intravenous and the dish was carefully inverted to keep the drop intact injection of glucose significantly increased GFR by 21.265.3%. with the tissue suspended. PBS was added to the bottom of However, neither L-glucose nor mannitol had any effect on the dish to prevent dehydration. The kidney biopsy tissue GFR. The data demonstrates that an increase in blood glucose pieces were cultured at 37°C in a humidified atmosphere over the renal threshold raises GFR (Figure 1C). of 5% CO2 and 95% room air. Because most of the NOS1 in the renal cortex comes from macula densa cells, to de- Tubular Glucose Blunts TGF Response in vivo and in termine whether glucose alters macula densa NOS1 ex- vitro pression or phosphorylation, we cultured human renal TGF responses in vivo were measured twice consecutively in cortex tissue in hanging drop medium with either each nephron using ATF without and with glucose. In the 5.5 mM glucose or 16.7 mM glucose for 30 minutes, and first measurement, when tubular perfusion rate of ATF with- then measured NOS1 expression and phosphorylation by outglucosewasincreasedfrom0to40nl/min,Psf decreased Western blotting. from 38.461.9 to 33.262.5 mm Hg (DPsf=5.261.0 mm Hg), whereas the mean arterial pressure was 88.963.9 mm Western Blotting Hg during measurement. In the second measurement with An intravenous injection of 50 ml of 5 M glucose was applied ATF containing 16.7 mM glucose, Psf decreased from 38.26 in mice to induce hyperglycemia as described above, and 1.6–34.461.8 mm Hg (DPsf =3.860.9 mm Hg), whereas the after 60 minutes,47 we harvested the kidneys and measured mean arterial pressure was 87.264.2 mm Hg (Figure 2A). NOS1 expression and phosphorylation in the renal cortex When we used glucose-free ATF, ATF with L-glucose or ATF where most of the NOS1 comes from macula densa cells. with mannitol in the second measurement, there were no Kidney tissue protein extracts (50 mg per lane) were sepa- significant differences between the first and second TGF re- rated on a 7.5% SDS-PAGE gel as described previously.28,41 sponse measurements (Figure 2, B–D). These data demon- After blocking for 1 hour at room temperature with 5% strate that tubular glucose inhibits the TGF response in vivo skim milk, the membranes were incubated overnight at 4° (Figure 2E). C with a C-terminal NOS1 antibody28,48 (mouse polyclonal We also measured the TGF response in vitro in isolated and IgG; 1:3000; BD Biosciences, San Jose, CA) or an antibody of double-perfused JGAs. In the absence of glucose in the tubular NOS1 phosphorylated at Ser141749–51 (P-NOS1) (rabbit perfusate, the TGF response indicated by the average change polyclonal IgG; 1:500; Abcam), respectively. The mem- of Af-Art diameter was 3.860.2 mm. In the presence of glu- branes were then incubated with horseradish peroxidase– cose in the tubular perfusate, the TGF response was reduced conjugated secondary antibody (goat anti-mouse, IgG; to 2.460.2 mm (Figure 2F). When D-glucose was replaced 1:300,000; Bio-Rad, Hercules, CA; or goat anti-rabbit, by L-glucose or mannitol, no significant differences were IgG; 1:300,000). The immunoreactive bands were revealed observed between the first and second TGF response

582 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH

AB 600 1400 0 M glucose * Urine 2.5 M glucose 1200 Distal 500 * 5 M glucose * * 1000 400 * 800 * 300 600 * * * * * 400 200 * 200 Blood glucose (mg/dl) * * 100

Glucose concentration (mg/dl) 0 0 03 10 20 40 60 80 0 20406080 Time (min) Time (min)

C 350 Baseline * 5 M 300

250 GFR (ul/min) 200

0 Glucose L-glucose Mannitol

Figure 1. Intravenous injection of glucose induces hyperglycemia and glucosuria with increased GFR. (A) The blood glucose con- centration in response to different doses of intravenous glucose injection; n=10–12; *P,0.01 versus baseline. (B) The glucose con- centration in urine and early distal tubular fluid in response to an intravenous injection of 50 ml of 5 M glucose; n=5–7; *P,0.01 versus baseline. (C) An intravenous injection of 50 ml of 5 M glucose increased GFR by 21.265.3%; n=10; *P,0.01 versus baseline. In- travenous injection of L-glucose or mannitol had no significant effect on the GFR; n=9–11. Statistical difference was calculated by two- way ANOVA followed by multiple comparisons post hoc test. measurements (Figure 2, G and H). These data demonstrate Tubular Glucose Inhibits TGF Response via SGLT1 that tubular glucose inhibits TGF response in vitro. Toexplore the expression of glucose transporters in the macula In contrast, the TGF response was not significantly changed densa, we identified 38 macula densa cells that express both when the glucose concentration in the bath solution was in- Slc12a1 and Nos1 out of 12,090 kidney cells from C57BL/ creased from 5.5 to 16.7 mM while the tubule was perfused 6 mice, and compared the mRNA expression levels of glucose with a glucose-free solution (Figure 2I). These data demon- transporters in these selected macula densa cells. Our analysis strate that it is the glucose in the lumen rather than at the showed the highest transcriptional level of Slc5a1 among the basolateral side of the macula densa that blunts the TGF glucose transporters, suggesting that SGLT1 might be the pri- response (Figure 2J). mary glucose transporter in the macula densa cells (Table 2). In addition, under conditions of constant, low tubular To confirm the expression of SGLT1 at the macula densa in NaCl of 15 mM, the diameter of Af-Art increased from the human kidney, we performed double-immunofluores- 14.660.3 to 15.760.4 mm when D-glucose concentration cence staining of SGLT1 and NOS1 in slices of human kidney in tubular perfusate was increased from 0 to 16.7 mM, biopsy specimens. We found that SGLT1 (red) was clearly de- whereas the Af-Art diameter was not significantly changed tected on the apical membrane of macula densa cells marked when D-glucose in the tubular perfusate was replaced by with an NOS1 antibody (green) using confocal microscopy L-glucose (Figure 2K). (Figure 3A).

J Am Soc Nephrol 30: 578–593, 2019 Macula Densa SGLT1-NOS1-TGF Pathway 583 BASIC RESEARCH www.jasn.org

A B 10 10 9 9 8 * 8 7 7 6 6 5 5 4 4 3 3 pressure (mmHg) pressure (mmHg) Change of stop flow Change of stop flow 2 2 1 1 0 0 ATF Glucose ATF L-glucose CD 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 pressure (mmHg) pressure (mmHg) Change of stop flow 2 Change of stop flow 2 1 1 0 0 ATF Mannitol ATF ATF

E F 0 mM glucose 16.7 mM glucose 18 10 Baseline 16.7 mM 16 8

* 6 14

4 12 pressure (mmHg) Af-Art diameter ( μ m) Change of stop flow 2 10

0 0 Glucose-free Glucose L-glucose Mannitol 1080 10 80 mM Tubular NaCl concentration GH 0 mM glucose 16.7 mM L-glucose0 mM glucose 16.7 mM mannitol 18 18

16 16

14 14

12 12

Af-Art diameter ( μ m) 10 Af-Art diameter ( μ m) 10

0 0 1080 10 80 mM 1080 10 80 mM Tubular NaCl concentration Tubular NaCl concentration

Figure 2. Tubular glucose blunts TGF response in vivo and in vitro. The TGF responses in vivo were measured twice consecutively in each nephron with micropuncture using ATF without and with glucose. TGF response was indicated by the maximum DPsf when the tubular perfusion rate was increased from 0 to 40 nl/min. (A) In the presence of glucose, DPsf was reduced from 5.261.0 to 3.86 0.9 mm Hg; n=5 mice per 15 tubules; *P,0.05 versus baseline. (B–E) None of ATF with L-glucose, ATF with mannitol, or glucose-free

ATF significantly changed DPsf; n=3–5miceper12–14 tubules; *P,0.05 versus baseline. (F–J) TGF response in vitro was determined by the change of Af-Art diameter when switching the tubular perfusate from 10 to 80 mmol/L of NaCl, and in the presence of 16.7 mM glucose in the tubular perfusate, TGF response was reduced from 3.860.2 to 2.460.2 mm. Neither L-glucose nor mannitol signifi- cantly altered TGF response in vitro. When the glucose concentration in the bath solution was increased from 5.5 to 16.7 mM while the tubule was perfused with a glucose-free solution, the TGF response in vitro was not significantly changed; n=7–12; *P,0.01 versus 0 mM glucose. (K) Under conditions of constant low tubular NaCl of 15 mM, the diameter of Af-Art increased from 14.660.3 to 15.760.4 mm when glucose concentration in tubular perfusate was increased from 0 to 16.7 mM; n=5–7; *P,0.05 versus 0 mM glucose. Statistical difference in (A–D) was calculated by paired t test. Statistical difference in (E, J, and K) was calculated by two-way ANOVA followed by multiple comparisons post hoc test. Statistical difference in (F–I) was calculated by one-way ANOVA.

584 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH

IJ 5.5 mM glucose 16.7 mM glucose 18 6 16

14 4 * 12 2

diameter (um) Baseline Change of Af-Art

Af-Art diameter ( μ m) 10 16.7 mM

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K 20 15 mM NaCl 15 mM NaCl 18

16 *

14

12 0 mM Af-Art diameter (um) 10 16.7 mM 0 Glucose L-glucose

Figure 2. Continued.

In the presence of the selective SGLT1 inhibitor, the TGF There was a 1.860.3-fold increase in the protein level of response in vitro was not significantly attenuated by increasing NOS1 (Figure 5, E and F) and a 1.960.1-fold increase in the the luminal glucose concentration (Figure 3B). These data protein level of P-NOS1 in human renal cortex tissue cultured demonstrate that the SGLT1 inhibitor blocks the inhibitory with 16.7 mM glucose compared with 5.5 mM glucose effect of luminal glucose on the TGF response (Figure 3C). (Figure 5, G and H).

Tubular Glucose Stimulates NO Generation in the Glucose-Induced Hyperfiltration Is Mediated by Macula Macula Densa Adding 16.7 mM glucose to the tubular perfusate increased NO Densa NOS1 Todetermine the significance of macula densa NOS1 in glucose- generation in the macula densa of isolated JGAs by 117.26 induced hyperfiltration, we repeated the experiments above in 13.4%. In contrast, macula densa NO generation in the mac- MD-NOS1KO mice. Adding glucose to the macula densa per- ula densa was not significantly changed by luminal L-glucose, fusate had no effect on the NO generation in MD-NOS1KO mice luminal mannitol, or glucose in the bath solution (Figure 4A). (Figure 6A). The TGF in vitro (Figure 6, B and C) or TGF in vivo These data indicated that it is the glucose in the lumen rather (Figure 6D) was not significantly changed in MD-NOS1KO than bath that stimulates macula densa NO generation. mice after adding glucose to tubular perfusate. Furthermore, Additionally, in the presence of the SGLT1 inhibitor, NO – fi generation in the macula densa was not significantly changed acute hyperglycemia induced elevation in GFR was signi cantly attenuated in MD-NOS1KO mice compared with wild-type after adding 16.7 mM glucose in the tubular perfusate (Figure 4B), mice (Figure 6E). These results indicated that macula densa indicating that luminal glucose induces NO generation in the NOS1 mediates the glucose-induced hyperfiltration. macula densa via SGLT1. Glucose Increases Macula Densa NOS1 Expression and NOS1 Phosphorylation at Ser1417 DISCUSSION Hyperglycemia resulted in a 1.660.2-fold increase in protein level of NOS1 (Figure 5, A and B) and a 1.860.2-fold increase in pro- This study provides evidence for a novel mechanism of hyper- tein level of P-NOS1 (Figure 5, C and D) in mouse renal cortex. glycemia-induced glomerular hyperfiltration in nondiabetic

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Table 2. The transcriptional copy of glucose transporters in blunts the TGF vasoconstrictor response, (3) this effect is de- the macula densa pendent on intact SGLT1 and NOS1 in the macula densa, Gene Transcriptional Level and (4) macula densa NOS1 mediates the acute hyperglycemia– Slc5a1 0.5526360.175681 induced increase in GFR. Slc2a1 0.1315860.055572 The significance of blood glucose levels in diabetic hyper- Slc2a4 0.1052660.050452 filtration has been recognized in studies that reported a low- Slc2a8 0.0789560.044332 ering of glomerular hyperfiltration in patients with diabetes Slc2a2 0.0526360.036709 after effective insulin therapy, but further increases with wors- Slc5a2 0.0526360.036709 ening of plasma glucose control.4,52 In accordance, acute intra- Slc2a13 0.0263260.026316 venous infusion of glucose can increase GFR in both individuals 6 Slc2a3 0 0 with and without diabetes,53–55 as well as in experimental an- 6 Slc2a5 0 0 imals.56,57 The renal threshold for glucose reabsorption corre- Slc2a6 060 sponds to blood glucose concentrations of approximately Slc2a9 060 58–60 Slc2a12 060 180 mg/dl. In this study, we determined that an intravenous dose of glucose is able to induce a sustained increase in blood glucose levels over the renal threshold, along with significant mice by showing that (1) luminal glucose at the macula densa glucosuria and increased glucose concentration in early distal is sensed via SGLT1 and thereby increases the activity and tubular fluid in C57BL/6 mice. Because there is only fluid reab- expression of NOS1, (2) luminal glucose at the macula densa sorption but no more glucose reabsorption from the beginning

A

B C 6 0 mM glucose 18 0 mM glucose 16.7 mM glucose 16.7 mM glucose

16 4 14 * 12 2

Af-Aft diameter ( μ m) 10 Control

SGLT1 inhibiton Change of Af-Art diameter (um) 0 0 1080 10 80 mM Control SGLT1 inhibiton Tubular NaCl concentration

Figure 3. Tubular glucose-induced TGF inhibition is mediated via macula densa SGLT1. SGLT1 on macula densa cells was identified by immunofluorescence staining in human kidney biopsy specimens. (A) Positive staining of SGLT1 (red) was observed in macula densa cells marked with NOS1 antibody (green). (B and C) SGLT1 inhibitor KGA-2727 blocked the inhibitory effect of luminal glucose on the TGF response in vitro in isolated perfused JGA from mouse kidney; n=10–12; *P,0.01 versus baseline. Statistical difference was calculated by two-way ANOVA followed by multiple comparisons post hoc test.

586 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH

AB 300 300 0 mM glucose * Baseline * 16.7 mM glucose 16.7 mM

200 200

100 100 NO generation (unites/min) NO generation (unites/min)

0 0 Glucose L-glucose Mannitol Glucose Control SGLT1 inhibition Tubular Perfusate Bath

Figure 4. Tubular glucose stimulates NO generation in the macula densa. The macula densa NO generation was measured in isolated perfused JGA with DAF-2 DA. In the presence of glucose in tubular perfusate, NO generation in the macula densa was increased from 118.367.7 to 25769.2 U/min. NO generation was not significantly altered by luminal L-glucose or mannitol. (A) Increased glucose concentration in the bath had no significant effect on NO generation in the macula densa; n=13–15; *P,0.01 versus baseline. (B) SGLT1 inhibitor KGA-2727 blocked the glucose-induced NO generation at the macula densa in isolated perfused JGA from mouse kidney; n=10–15; *P,0.01 versus baseline. Statistical difference was calculated by two-way ANOVA followed by multiple comparisons post hoc test. of distal tubule to the end of nephron, the glucose concen- study, we found an increase in luminal concentration of tration in the urine was much higher than that in distal D-glucose rather than L-glucose or osmolarity enhanced NO tubule. In addition, consistent with the acute effect of hy- generation in the macula densa. Moreover, the basolateral in- perglycemia on GFR, this hyperglycemia model demon- crease of glucose concentration in the macula densa had no stratedthatanintravenousinjectionofD-glucose,butnot significant effect on NO production, which suggests that it is L-glucose or mannitol, significantly increased GFR in non- the luminal increase in glucose concentration at the macula diabetic mice. densa that enhances NO generation. Previous studies have suggested a critical role of the TGF Toexplore the expression of glucose transporters in mac- mechanism in glomerular hyperfiltration in response to mod- ula densa cells, we analyzed the single-cell RNA-sequencing erate levels of hyperglycemia.13 The glucose infusion in- profile of mouse kidneys that was deposited in the GEO duced GFR elevation in nondiabetic dogs was absent in database in a recently reported study.45 We defined macula animals with nonfiltering kidneys, in which the TGF response densa cells by the expression of the two marker genes NOS1 was interrupted.57 In addition, the TGF response has been and NKCC2, and compared the mRNA expression levels of extensively studied in db/db mice and STZ-induced diabetic established glucose transporters in these macula densa cells. rats via measurement of proximal tubular Psf, proximal-distal We found that the glucose transporter with highest tran- differences in single-nephron GFR, or freeflow perturbation scriptional level was SGLT1. SGLT1 mediates most of analysis of TGF efficiency at the natural operating point. In the sodium-dependent glucose uptake across apical cell both type 1 and type 2 diabetic animal models, the TGF re- membranes in the small intestine and the reabsorption of sponse was found to be inhibited or reset.11,12,33,61,62 However, approximately 3% of the filtered glucose in the kidney, pre- it remained unclear whether glucose per se has a significant dominantly in the S2 and S3 segment of the proximal tu- effect on the TGF response. Our study demonstrated that an bule.34,63 More recently, the expression of SGLT1 was also increase in the concentration of D-glucose, but not L-glucose detected on the apical domain of the TAL of the loop of or mannitol, in the tubular perfusate attenuated the TGF re- Henle and the macula densa in both mouse35 and rat kid- sponse. Moreover, there was no significant effect when the neys36, but it was not detected in human macula densa cells glucose was applied in the bath, indicating it is the glucose with a specific antibody made by H.K.64 Because the localization at the apical rather than basolateral sides of the macula densa and identification of the macula densa cells in the human kidney that inhibits the TGF response. tissue in the latter study appeared ambiguous, we requested and NO generated by NOS1 in the macula densa is a major used the same antibody to perform double-immunofluores- modulator of the TGF response, which buffers or attenuates cence staining of SGLT1 as well as NOS1 to localize the macula the vasoconstrictor TGF tone.21–26 However, the effect of glu- densa in human kidney biopsy specimens that had been imme- cose on macula densa NO generation remained unclear. In this diately fixed after harvest. Our results confirmed the expression

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AB 2.5

2.0 Normoglycemia Hyperglycemia * 1.5 Mouse NOS1

1.0 β-actin .5 Relative NOS1 protein levels 0.0 Normoglycemia Hyperglycemia

CD 2.5

* Normoglycemia Hyperglycemia 2.0

Mouse P-NOS1 1.5

1.0 β-actin .5

Relative P-NOS1 protein levels 0.0 Normoglycemia Hyperglycemia

EF 2.5 * 5.5 mM glucose 16.7 mM glucose 2.0

Human NOS1 1.5

1.0 β-actin .5

Relative NOS1 protein levels 0.0 5.5 mM 16.7 mM Glucose concentration GH 2.5

5.5 mM glucose 16.7 mM glucose * 2.0

Human P-NOS1 1.5

1.0

β-actin .5

Relative P-NOS1 protein levels 0.0 5.5 mM 16.7 mM Glucose concentration

588 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH of SGLT1 in human macula densa, which provides clinical rel- that macula densa NOS1 mediates the acute luminal glu- evance and significance to the SGLT1 aspect of our study. More- cose–induced increase in NO generation in the macula densa over, when the selective SGLT1 inhibitor KGA-2727 was added and TGF inhibition, as well as the acute hyperglycemia- to the tubular perfusate, the glucose-induced macula densa induced glomerular hyperfiltration. NO generation and TGF inhibition were blocked. These results The major limitation of our study is that no functional indicate that an increase in glucose concentration at the macula TGF measurements are available in humans to show the densa blunts the TGF response and enhances NO generation via clinical relevance of the proposed macula densa SGLT1- SGLT1. Besides SGLT1, the second most highly expressed glu- NOS1-TGF pathway. Although the SGLT1-NOS1 signaling cose transporter in the macula densa cells was GLUT1. For pathwayisthefocusofthisstudy,weexpectthattheeffects mammalian cells, GLUT1 is the most prominent transporter of the SGLT1-NOS1 and SGLT2-NaCl pathways on TGF for glucose influx with a manner of passive and facilitative dif- and GFR are separate mechanisms operating in the diabetic fusion after concentration gradient. In the proximal tubule kidney. We assume that hyperglycemia increases glucose epithelial cells, GLUT1 also mediates the efflux of intracellu- filtration and enhances SGLT2-mediated sodium-glucose lar glucose in the basolateral membrane because the influx of reabsorption, which decreases NaCl delivery to the macula luminal glucose via SGLTs elevates intracellular glucose concen- densa and inhibits the vasoconstrictor TGF tone.12,34,75 At tration above the interstitium. Accordingly, we speculated that the same time, the increased luminal glucose at the macula under normoglycemic conditions, GLUT1 in the basolateral densa is sensed by SGLT1, which upregulates NOS1 expres- membrane may mediate the influx of interstitial glucose as an sion and activity, and further reduces the vasoconstrictor energy source because of the minimal luminal glucose at the TGF tone. Thus, these two signaling pathways additively macula densa; however, under hyperglycemic conditions, promote the glomerular hyperfiltration in diabetes. Beyond GLUT1 may facilitate the efflux of intracellular glucose to inter- these two TGF-related mechanisms, high glucose-induced stitium through basolateral membrane because of the active glu- vasodilation of Af-Art should be a possible vascular mecha- cose influx via luminal SGLT1. nism that cannot be discounted for diabetic hyperfiltration. We This study also demonstrated that acute hyperglycemia was recently demonstrated that an increase in luminal glucose con- associated with the upregulation of protein levels of NOS1, centration dilates the mouse Af-Art by stimulation of the endo- along with increased phosphorylation of NOS1 at Ser1417 in thelium-derived NO production via GLUT1.76 Another study mouse renal cortex, where most of the NOS1 comes from by Toma et al.77 also showed that high glucose in vascular per- macula densa cells. Moreover, in cultured human renal cortex fusate rather than bath solution dilates the rabbit Af-Art, which tissue, hanging drop medium with a high concentration of involves the local accumulation of succinate and activation of glucose increased NOS1 expression and stimulated the G protein–coupled metabolic receptor GPR91. Additionally, NOS1 phosphorylation at Ser1417. Regarding potential mech- acute hyperglycemia stimulates b-cells to produce and secret anisms, on the basis of the related literature, we speculate that insulin. It has been demonstrated that insulin per se could induce glucose upregulates NOS1 expression and phosphorylates an increase in GFR,78,79 which is associated with the direct renal NOS1 via the PI3K/Akt65–69 and/or cAMP/PKA70–74 vasodilation80,81 and sodium retention by enhanced reabsorp- pathways. tion in distal tubule.80,82–84 Therefore, the elevation of insulin Recently, our laboratory developed the MD-NOS1KO level might be another potential mechanism for the increases of mouse strain by crossing NKCC2-Cre mice with NOS1- GFR in response to acute hyperglycemia. floxed mice, in which all of the splice variants of NOS1 In conclusion, our study indicates that an acute increase in were deleted from macula densa cells.28 Because the expres- glucose concentration at the luminal side of the macula densa sion of NOS1 is negligible in TAL of compared enhances the expression and activity of NOS1 via SGLT1, fl fl with that in the macula densa, this NKCC2cre/NOS1 ox/ ox which blunts the TGF response and promotes the rise in mouse strain could be considered as a macula densa–selec- GFR (Figure 7). This novel SGLT1-NOS1-TGF pathway me- tive NOS1 knockout. Thus, in this study, the MD-NOS1KO diates the glomerular hyperfiltration observed in response to mice were utilized to determine the significance of macula acute hyperglycemia. These findings establish a critical role densa NOS1 in the effects of glucose on macula densa NO of macula densa NOS1 and SGLT1 as key determinants and generation, TGF response, and GFR. We found that all of the potential novel therapeutic targets for glomerular hyperfiltra- glucose-induced effects that were observed in wild-type tion associated with acute hyperglycemia and thus, potentially, mice were absent in MD-NOS1KO mice, which suggests diabetes.

Figure 5. Glucose upregulates macula densa NOS1 expression and phosphorylation. The protein levels of (A and B) NOS1and (C and D) P-NOS1 in the renal cortex. Hyperglycemia was induced by an intravenous injection of 50 ml of 5 M glucose in mice; n=4–5; *P,0.05 versus normoglycemia. The protein levels of (E and F) NOS1 and (G and H) P-NOS1 in human renal cortex tissue cultured with different glucose concentrations; n=4; *P,0.05 versus 5.5 mM glucose group. Statistical difference was calculated by t test.

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A 300 0 mM glucose * 16.7 mM glucose

200

100

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* 6 *# 250

4 GFR (ul/min) 200 2

Change of stop flow pressure (mmHg) 0 0 WT MD-NOS1KO WT MD-NOS1KO

Figure 6. Glucose-induced hyperfiltration is mediated by macula densa NOS1. (A) The effects of tubular glucose on NO generation in the macula densa (n=13–15), (B and C) TGF response in vitro (n=10–12), d an(D) TGF response in vivo (n=3–5miceper11–15 tubules) were measured in MD-NOS1KO mice and compared with wild-type (WT) mice. *P,0.05 versus baseline; #P,0.05 versus WT. (E) The change of GFR in response to an intravenous injection of 50 ml of 5 M glucose was measured in MD-NOS1KO mice and compared with WT mice; n=9–10; *P,0.05 versus baseline; #P,0.05 versus WT. The data of WT were the same data presented in Figure 1C, Figure 2, A and F, Figure 3C, and Figure 4A. Statistical difference was calculated by two-way ANOVA followed by multiple comparisons post hoc test.

590 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 578–593, 2019 www.jasn.org BASIC RESEARCH

response in vivo was also assessed directly under high-glucose con- Hyperglycemia ditions. The DPsf measured with ATF containing 16.7 mM glucose was significantly lower than the control measurements with glucose- Tubular glucose at macula densa free ATF, ATF containing 16.7 mM L-glucose, or ATF containing 16.7 mM mannitol. Supplemental Figure 2. The complete gels for the Western blot of SGLT1 NOS1 and P-NOS1 in the (A and B) mouse renal cortex and (C and D) human kidney biopsy tissue. NOS1 expression and activity at macula densa

REFERENCES NO generation and release at macula densa 1. Selby JV, FitzSimmons SC, Newman JM, Katz PP, Sepe S, Showstack J: The natural history and epidemiology of diabetic nephropathy. TGF vasoconstrictor tone Implications for prevention and control. JAMA 263: 1954–1960, 1990 2. Gross JL, de Azevedo MJ, Silveiro SP, Canani LH, Caramori ML, GFR Zelmanovitz T: Diabetic nephropathy: Diagnosis, prevention, and treatment. Diabetes Care 28: 164–176, 2005 3. Tervaert TW, Mooyaart AL, Amann K, Cohen AH, Cook HT, Figure 7. Macula densa SGLT1-NOS1-TGF pathway mediates fi fi Drachenberg CB, et al.: Renal Pathology Society: Pathologic classi - the glomerular hyper ltration during hyperglycemia. An increase cation of diabetic nephropathy. J Am Soc Nephrol 21: 556–563, 2010 in glucose concentration at the luminal side of the macula 4. Bank N: Mechanisms of diabetic hyperfiltration. Kidney Int 40: 792– densa upregulates the expression and activity of NOS1 via 807, 1991 SGLT1, which blunts the TGF response and thereby promotes 5. Levine DZ: Can rodent models of diabetic kidney disease clarify the theriseinGFR. significance of early hyperfiltration?: Recognizing clinical and experi- mental uncertainties. Clin Sci (Lond) 114: 109–118, 2008 6. Vora JP, Dolben J, Dean JD, Thomas D, Williams JD, Owens DR, et al.: Renal hemodynamics in newly presenting non-insulin dependent di- ACKNOWLEDGMENTS abetes mellitus. Kidney Int 41: 829–835, 1992 7. Nelson RG, Bennett PH, Beck GJ, Tan M, Knowler WC, Mitch WE, et al.: J.Z., J.W., and R.L. designed the study. J.Z., J.W., and S.J. performed Diabetic Renal Disease Study Group: Development and progression of theexperiments.J.Z.,J.W.,L.X.,andF.C.analyzedthedata.J.Z.and renal disease in Pima Indians with non-insulin-dependent diabetes mellitus. NEnglJMed335: 1636–1642, 1996 J.W. drafted the manuscript. L.W., J.B., H.K., V.V., and R.L. revised 8. Keller CK, Bergis KH, Fliser D, Ritz E: Renal findings in patients with the manuscript. All authors approved the final version of the short-term type 2 diabetes. JAmSocNephrol7: 2627–2635, 1996 manuscript. 9. Helal I, Fick-Brosnahan GM, Reed-Gitomer B, Schrier RW: Glomerular This work was supported by American Society of Nephrology hyperfiltration: Definitions, mechanisms and clinical implications. Nat – Ben J. Lipps Research Fellowship Awards (to J.Z. and J.W.), an American Rev Nephrol 8: 293 300, 2012 10. Anderson S, Vora JP: Current concepts of renal hemodynamics in di- Heart Association Career Development Award 18CDA34110441 abetes. J Diabetes Complications 9: 304–307, 1995 (to L.W.), and the National Institutes of Health (grants DK112042 11. Thomson SC, Deng A, Bao D, Satriano J, Blantz RC, Vallon V: Ornithine and DK106102 to V.V.; and grants DK099276, HL142814, and decarboxylase, kidney size, and the tubular hypothesis of glomerular HL137987 to R.L.). hyperfiltration in experimental diabetes. JClinInvest107: 217–224, 2001 12. Vallon V, Richter K, Blantz RC, Thomson S, Osswald H: Glomerular hyperfiltration in experimental diabetes mellitus: Potential role of tu- DISCLOSURES bular reabsorption. J Am Soc Nephrol 10: 2569–2576, 1999 Over the past 36 months, V.V. has served as a consultant and received 13. Vallon V, Thomson SC: Renal function in diabetic disease models: The honoraria from Bayer, Boehringer Ingelheim, Eli Lilly, Janssen Pharmaceutical, tubular system in the pathophysiology of the diabetic kidney. Annu Rev and Merck, and received grant support for investigator-initiated research from Physiol 74: 351–375, 2012 Astra-Zeneca, Bayer, Boehringer Ingelheim, Fresenius, and Janssen 14. Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, et al.: Pharmaceutical. Mediation of tubuloglomerular feedback by adenosine: Evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci U S A 98: 9983– 9988, 2001 15. Ren Y, Arima S, Carretero OA, Ito S: Possible role of adenosine in SUPPLEMENTAL MATERIALS macula densa control of glomerular hemodynamics. Kidney Int 61: 169–176, 2002 This article contains the following supplemental material online at 16. Ren Y, Garvin JL, Liu R, Carretero OA: Role of macula densa adenosine – http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018080844/-/ triphosphate (ATP) in tubuloglomerular feedback. Kidney Int 66: 1479 1485, 2004 DCSupplemental. 17. Thomson S, Bao D, Deng A, Vallon V: Adenosine formed by 59- Supplemental Figure 1. To avoid any systematic error introduced nucleotidase mediates tubuloglomerular feedback. J Clin Invest – by consecutive measurements of DPsf in the same nephron, TGF 106: 289 298, 2000

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