Metabolic Responses of Normal Rat Kidneys to a High Salt Intake

Abstract In this study, novel methods were developed, which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague–Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to 4.0% NaCl diet. In addition, targeted mRNA expression analysis of cortical segments was performed. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. A novel finding was the increased expression of glycolysis-related genes in Cx and isolated proximal tubular segments in response to an HS diet, consistent with increased release of pyruvate and lactate from the kidney to the renal venous blood. Data suggests that aerobic glycolysis (eg, Warburg effect) may contribute to energy production under these circumstances. The study provides evidence that kidney metabolism responds to an HS diet enabling enhanced energy production while protecting from oxidative stress and injury. Metabolomic and transcriptomic analysis of kidneys of Sprague-Dawley rats fed a high salt diet.


Introduction
The conversion of daily food intake into energy and biomass is a complex process with some mechanisms operating relati v el y inefficientl y as essential for short-term survi v al with others operating in more efficient and sustained ways required in face of sustained chronic stressors. [1][2][3] Although malnourishment is one such chronic stressor, industrialized societies are largely faced with dietary excesses including condiments such as ta b le salt (NaCl). Excess dietary salt is a well-recognized cardiovascular risk factor especially in those genetically susceptible to hypertension ("salt-sensitivity"). Half of hypertensi v e patients ar e b lood pr essur e (BP) salt-sensiti v e 4-6 who suffer nearl y 3times greater risk of chronic kidney disease (CKD). [7][8][9] The underlying genetic and physiological determinants of BP salt-sensiti vity r emain incompletel y understood although general pr ogr ess has been made in understanding the contributions of kidney function, and of the neural and endocrine controllers of car dio vascular system determinants of sodium and water excretion. 10 , 11 Recently, kidney metabolism has begun to emerge as a novel and important determinant of BP in salt-sensitive forms of hypertension. 12 , 13 However, the dearth of studies examining kidney intermediar y meta bolism in hypertension is r emarka b le gi v en the gr eat importance of this organ in the r egulation of salt and water in the long-term control of BP. A great deal of energy is r equir ed b y the kidne y to support renal tubular transport of sodium and other substances, which are all linked to the activity of Na + -K + -ATPase and H + pumps on the basolater al membr anes of renal tubules. [14][15][16][17] Kidneys have one of the highest specific metabolic rates among all organs estimated in humans to be over 400 kcal/kg tissue/d, which is the same as the heart, twice as high as the li v er and the brain, and much higher than other organs. 12 , 15 The housekeeping role of intermediary meta bolic pr ocesses r equir ed to conv ert n utriti v e substances to energy, cellular components, and w aste pr oducts is well understood. However, it has been increasingly recognized that these meta bolic pathw ays and intermediate pr oducts can influence gene expression, signal transduction, and other regulatory pathw ays. 18 Intermediar y meta bolism and r elated mitochondrial and cellular functions are now recognized to play an essential role in the development of acute kidney injury and CKD. 19 , 20 Relati v el y few studies have focused on these aspects of kidney function in response to excess dietary salt. T raditionally , studies of organ meta bolism hav e been limited to examination of a r elati v el y few metabolites. 21 , 22 With the emergence of largescale mass spectr ometr y and anal ysis tools ( aka meta bolomics), it is curr entl y possib le to identify and prioritize sev eral thousands of detected features providing a comprehensive analysis in a tissue specimen of the changes in metabolism that occur acr oss v arious organs of the body as r ecentl y demonstrated by Jang et al. 23 who analyzed the arterial and venous blood of 11 organs in fasted pigs. Relevant to the current study, Rinschen et al. 13 examined the effects of a high salt (HS) diet upon the glomerular and cortical tissue of Dahl salt-sensiti v e rats. Howev er, with meta bolomics yet in an infant stage, no one has even y et c har acterized kidney meta bolism r esponses of a normal nonhypertensi v e r odent model to an HS diet. The pr esent study utilized the commonly used outbr ed Spra gue-Da wle y (SD) rat model. By definition, the metabolome represents the complete set of metabolites found in a biological sample built upon the genetic b lue print of an organism. As shown in the present study, even the most advanced mass spectrometry techniques as used in the present study can detect from 5000 to 6000 compounds and only about a sixth of those can be linked to Meta boanal yst data base. This is in contrast to a transcriptome RNAseq analysis that provides 20-25 000 protein coding genes whose function is at least partiall y alr ead y kno wn.
To overcome the current limitations of metabolomic analysis, a multi-omics approach was used in the present study in whic h RN A-seq tr anscriptional data w ere obtained in par allel to provide the metabolic pathway templates upon which to integr ate c hang es of both g ene expr ession and meta bolites. Also, a nov el system w as dev eloped, whic h allow ed for the intermittent sampling of arterial and r enal v enous b lood (and urine collection) together with continuous measurement (24 h/d) of renal blood flow (RBF) and arterial BP in fr eel y moving rats before and for 3 wk following an increase of salt diet. Glomerular filtration r ates (GFR) w er e similarl y obtained fr om other gr oup of unanesthetized SD rats. These data ena b led the first determination of solute mass balances (metabolic fluxes) of O 2 and metabolic substances in unanesthetized unrestr ained SD r ats. In par allel studies, renal cortical and medullary tissue samples were obtained from other groups of rats fed a 0.4% NaCl (LS) and at days 7, 14, and 21 after switching to a 4.0% NaCl (HS) diet. In addition, targeted mRNA expression analysis was performed in isolated cortical tubular segments and glomeruli. The results of the study show that even normal SD rats undergo enormous shifts in transcriptomic and metabolomic profiles in response to eating an HS diet. These adaptations appear necessary to sustain vital physiological functions of the kidney and to pr ev ent injur y in face a gr eat incr ease of the meta bolic workload placed upon the kidney when subjected to sustained HS diets.

Animals
Male SD rats were purchased from Envigo (Indianapolis, IN) and housed in envir onmentall y contr olled r ooms with a 12-h light/dark cycle. Rats had free access to 0.4% NaCl AIN-76A diet (LS) (Dyets, Bethleham, PA) and water ad libitum. All protocols wer e appr ov ed by the Medical College of Wisconsin Institutional Animal Care and Use Committee (AUA00000851).

Chronic RBF Measurement and Blood Sample Collection
Rats ( n = 7, 10-11 wk of age) were performed RBF probe (Transonic, Ithaca, NY) implantation and femoral arterial catheterization as pr eviousl y described. 24 , 25 Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted as pr eviousl y described. [26][27][28] Following an abdominal incision, RBF probe was implanted on left r enal arter y and the ca b le w as exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter (MRE025, BRAINTREE, MA) w as inserted thr ough the femoral v ein and placed in the left r enal v ein and secur ed to the luminal wall with 10-0 nylon (Figure S1). Three % heparinized saline was infused at a rate of 100 μL/h through arterial and renal venous catheter throughout the study. RBF and BP via arterial line wer e measur ed by conscious fr eel y moving rats and recorded on av era ge of ev er y min ute for 24 h/d. After 7-10 d of r ecov er y period, 200 μL of arterial and r enal v enous b lood wer e sampled and that blood was replaced from donor rats before and following 7, 14, and 21 d after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet. The 4.0% salt diet was chosen in this study because our previous studies have shown that it produces little change of blood pressure and kidney injury in SD rats or other "salt-resistant" strains of rats but does pr oduce r obust h ypertension and kidne y injury in Dahl SS rats in a few weeks. 29 Blood gases (pO 2 and pCO 2 ; mmHg), electr ol ytes, total hemoglobin concentration (Hb; g/dL), and oxyhemoglobin saturation (SHbO 2 ; %) were immediately measured by radiometer (ABL800 FLEX, Brea, CA). Overnight urine (18 h) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 d of HS (HS14) or 21 d of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys ( n = 5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine, and tissue were stored in −80 • C until further analysis. RBF in rats is often normalized b y kidne y weight, but it is impossib le to r e peatedl y measur e the kidney weight of the same rats and even measuring body weight r e peatedl y is difficult in this model. As salt did not alter the kidney weight of surgical sham control rats ( n = 5 for LS group, n = 6 for HS group) (Table  S1), data normalization to body weight was performed.
Since glomerular filtration rate (GFR) experiments and blood dr aws w ere performed during the da ytime, the a v era ge RBF ov er a 12-h period during the daytime (6 am -6 pm ) was used for the following calculations.

Chronic GFR Measurement
GFR w as measur ed by se parate gr oup of rats ( n = 6) by transcutaneous measurement of FITC-sinistrin as pr eviousl y described. 24 , 30 , 31 Briefly, an indwelling inferior vena cava catheter was implanted 7-10 d before GFR measurement via femoral vein. An abdominal median incision was performed to be considered as a surgical sham of the other gr oup. GFR w as measur ed befor e and follo wing 7, 14, and 21 d after the s witch in diet from LS to HS. GFR (mL/min/100 g body wt) was defined as 21.33 mL/100 g body wt, the conversion factor calculated by Friedemann et al., 32 divided by FITC-sinistrin half-life (min).
From the GFR and RBF from the other group, filtration fraction was calculated by the formula below: Filtr ation fr action = GFR/(2 × RBF × (1-Hct)), where Hct is the hematocrit.
Tubular r ea bsorption of sodium w as estimated as below 33 : GFR × whole blood Na + (measured in RBF group rats by radiometer)-Urine flow × urinary Na + (measured in RBF group rats b y r adiometer).

Plasma/Urine Metabolite Extraction
Meta bolites wer e extracted fr om 20 μL of plasma and 20 μL of urine from each SD rat in the study according to standard operating pr ocedur es in the Mass Spectr ometr y and Pr otein Chemistr y Service at The J ackson La borator y. 34 Meta bolites wer e extracted using 500 μL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H 2 O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ μL in the extraction buffer as internal standards. Each sample was then vortexed for 30 s on the highest setting, subject to 1 min of mixing with the Tissue Lyser II in prechilled cassettes, and then sonicated at 30 Hz for 5 min of 30 s on 30 s off in an ice water bath. Samples were then placed in the −20 • C fr eezer ov ernight (16 h) for extraction. Following the extr action, samples w ere centrifuged at 21 000 × g at 4 • C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant w as di vided into 5 equal v olume aliquots, one for each of the 4 modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at −80 • C until further use.

Tissue Metabolite Extraction
Meta bolites wer e extracted fr om 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating pr ocedur es in the Mass Spectr ometr y and Pr otein Chemistry Service at The Jackson Laboratory 34 as described for the plasma and urine samples with slight modification. Meta bolites wer e extracted using 1000 μL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H 2 O) buffer containing internal standards as a bov e per 20 mg of sample to ensure the extraction equi v alents wer e normalized. Each sample had a 5mm stainless steel bead added, then were pulverized in extraction buffer for 2 min using Tissue Lyser II. Samples were then placed in the −20 • C freezer overnight (16 h) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into 5 equal volume aliquots, one for each of the 4 modes and the rest to create equal r e pr esentation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at −80 • C until further use.
Each sample w as r econstituted in 25 μL of 95% H 2 O/5% ACN for C18 modes and 95% ACN/5% H 2 O for HILIC modes. The sample run sequence was randomized (Random.org) and 2 technical r e plicates for each sample were injected at 10 μL (represents ∼2 μL of fluid samples or 2 mg of tissue sample starting volume weight, r especti v el y, per run). Quality contr ol pooled samples r e pr esenting all samples within the specific fluid/tissue were run at the beginning and end of the run set at concentrations equi v alent to the samples. These pooled samples were used for normalization through a quality control batch correction of the runs over time to account for tec hnical variance . All instrument settings were set as described in a previous study. 34

Metabolomics Data Analysis
The RAW data files (consisting of MS1 and MS2 spectra collected) wer e anal yzed using Thermo Compound Discov er er (v3.2.0.421) according to supplementary methods from a previous study at The J ackson La borator y. 34 Spectra in the data were subject to a blank background subtraction to remove contaminant peaks (S/N threshold = 2). Additionally, all data were subject to a quality control correction selecting for peaks only consistently detected in the pool for normalization. The MS1 and MS2 data w ere sear c hed a gainst the Thermo mzCloud data base, ChemSpider data base, Meta bolika Pathw ays, and mzLogic predicted composition in the Compound Discov er er workflow. In this workflow, the data were compared against standard databases containing MS2 spectra for high-confidence matching, wher e onl y those with MS2 matches in the database passed filtering. All data were then filtered for quality of MS2 spectr al matc hing using an MS2 FISH cov era ge filter ≥ 10 in Compound Discov er er. This filtering allows for higher confidence identification and minimizes the false identification of metabolites. Additionally, metabolites that were focused on as key targets (eg, metabolites in the mass balance analysis) were c hec ked for spectr a matc hing bey ond just FISH scoring as in Figure S2. Differential comparisons were performed comparing normalized abundances and P -values were calculated using the Tukey HSD test (posthoc) after an ANOVA test. From there the P -values were adjusted for stringency in the multiple testing using the Benajmini-Hochberg algorithm. The data also were run through The Jackson Laboratory inhouse MetID Conversion tool created in R by the Computational Sciences Service to add additional identification numbers and metadata making it easier for other resear c hers to convert the nomenclature of metabolites used in this study in the future. These data are provided in the Supplemental F ile . Further analysis was performed using a combination of Compound Discoverer, custom R analysis, and MetaboAnalyst as needed.

Lactate Assay
Tissue and plasma lactate concentration was validated by a commerciall y av aila b le kit according to the man ufactur er's instructions (PicoProbe Lactate Fluorometric Assay Kit, Bio Vision Cat# K638-100).

mRN Aseq Anal ysis
RN A was extr acted fr om snap fr ozen tissues by Trizol r ea gent and cleaned up by RNAeasy MinElute Cleanup kit (Qiagen). RNAseq analysis and data anal ysis wer e performed at Novogene (Durham, NC). Detailed methods, quality contr ol (Ta b le S2), mapping r esults (Ta b les S3 and S4), and used softw ar e (Ta b le S5) are shown in the supplement.

Isolation of Nephron Segments and Quantification of RNA Expression Levels
The methods of the ne phr on segments isolation and qPCR wer e pr eviousl y described 35 , 36 and summarized in supplement. Primer information is shown in Ta b le S6.

Determination of Kidney O 2 and Metabolites Extraction Ratio
Whole blood O 2 extraction was calculated as previously described 37 : Oxygen content (mL/dL) = (1. 31

Data Analysis of Metabolomics
Compound names were converted to the corresponding names av aila b le in the Meta boanal yst 5.0 database ( https://www.meta boanalyst.ca ) (analyzed September-December 2022) by "Compound ID Conversion" and those compounds are used as "refer ence meta bolome ," whic h can be detected based on our analytical platform. Sparse partial least squares discriminant analysis (sPLS-DA) for all named compounds in each of the 4 modes (C18 + / − and HILIC + / −) was performed. The number of components was fixed at 5 and v aria b les per component at 20. Heat map with the hier ar c hical clustering analysis was performed for significantly changed compounds between LS, HS7, HS14, and HS21 (ANOVA Fisher's LSD P < .05) with a Euclidean distance measure and by the Ward algorithm. Enrichment analysis for tissue meta bolites w as performed for significant differ ences by t -test ( P < .05) in each of HS14 and HS21 compared to LS. Enrichment analysis for plasma metabolites was performed for significant differ ent meta bolites by linear models with covariate adjustments ( P < .05) in arterial and venous differences. Those data analyses of metabolomics were performed using Metaboanalyst. 39

Statistical Analysis
Contin uous v alues ar e pr esented as the means ± SEM. Statistical comparisons were made using a t -test for 2-group comparisons, and analysis of variance (ANOVA) followed by Holm Sidak's posthoc test for multiple between-group comparisons. A P < .05 was considered significant. ROUT test was performed for outlier test ( Q = 5%). The error in the values obtained by combining values in tubular reabsorption of Na + calculation with errors w as indir ectl y estimated based on the err or pr opa gation form ula sho wn belo w ( M : Mean, e : error) 40 :

Effects of HS Diet on Arterial Pressure, RBF, and O 2 Extraction.
Av era ge 24 h mean arterial pr essur e (MAP) of SD rats slightly but significantl y incr eased fr om 114 ± 2 to 119 ± 2 mmHg ( P < .05) in 3 d after switching the diet from LS to HS and maintained at that level throughout the study ( Figure 1 A). Average 24 h RBF r ose nearl y 20% during the first 3 d from 10.0 ± 0.6 to 11.6 ± 0.6 mL/min ( P < .05; Figure 1 B) and was sustained at this ele vated le vel throughout the 21 d of the HS diet. Normalized by the body weight determined in the surgical sham control rats (Ta b le S7), the increase in RBF with the HS diet remained statistically significant at HS7, HS14, and HS21 (Ta b le S8). Renal vascular resistance (RVR) did not change by HS ( P = .75). An example of an SD rat in which arterial pr essur e and RBF were recorded contin uousl y (24/7) before and 21 d following the switch to the HS diet is illustrated in Figure S3. A similar rise of RBF was observed in ev er y rat studied and a similar increase in the magnitude of the diurnal rhythm of the RBF w as observ ed in all rats. Shown in Figure S4, total urinary Na + excretion increased 10-times (from 0.9 ± 0.1 to 10.3 ± 0.4 μmol/min) consistent with the 10-times increase in the % of NaCl in the diet when switch from 0.4% salt to 4.0% NaCl.
With the increase of RBF, the O 2 deli v er y to the left kidney incr eased pr oportionall y to RBF due to the unchanged O 2 content in artery (Table S9)

Effects of HS Diet on GFR and Calculated Filtration Fraction.
In a separate group of rats, the total body GFR was determined in unanesthetized r ats, whic h underw ent the same surgery (sham) as those with implanted renal flow probes and subjected to the same dietary protocol. As summarized in Table S7 and Figure 1 F, GFR incr eased fr om 0.64 ± 0.04 at LS to 0.83 ± 0.05 at HS7 (mL/min/100 g body weight) ( P < .05) and was thereafter maintained at that level throughout the study. The filtration fraction ( Figure 1 G), calculated using the RBF determined from the contin uousl y r ecorded rat gr oup, w as incr eased fr om 0.18 ± 0.01 at LS to 0.22 ± 0.02 at HS7 and remained elevated at this level throughout the study. As determined from these data, a strong positi v e corr elation w as found between total tubular r ea bsorption of Na + and O 2 consumption as presented in Figure 1 H.  Figure S5B). P -value adjustments (Benjamini-Hochberg) reduced the chance of making Type-I errors and it was found that 138 named metabolites were significantly changed at HS14 compared to LS (adjusted P < .05) in Cx and 229 in the OM. At HS21, 218 were significantly changed in the Cx and 225 in the OM.

Effects of HS Diet on Untargeted Metabolomic Profiles of Cortical and Outer Medullary Tissue
sPLS-DA was carried out for these named compounds to visualize and assess similarities and differences of metabolites in response to the HS diet. For each of the 4 modes (C18 + / − and HILIC + / −), 5 components with 20 v aria b les per rat were analyzed. As shown in Figure S6A , the analysis revealed that separate metabolic states were distinguishable within the Cx and OM tissues in response to the HS diet at both days 14 and 21. A clear distinction is observed between LS and HS14 and HS21 days of feeding.
The significantly altered metabolites over time (ANOVA Fisher's LSD P < .05) determined in the C18 + mode were 321 of 1114 in Cx and 151/668 in OM. The significantly altered metabolites in the C18 − mode were Cx 161/649 and OM 225/804; in the HILIC + mode were Cx 241/565 and OM 129/805; and in the HILIC − mode were Cx 63/564 and OM 91/470. The heat maps of those meta bolites ar e shown in Figur e S6B in which the distincti v e patterns observed by sPLS-DA were validated in the hierar c hical clustering, which was performed with a Euclidean distance measure and by the Ward algorithm.
A metabolite enrichment analysis was performed for those meta bolites that wer e significantl y changed ( P < .05) to prioritize and place them into known biological pathways as described by the small molecule pathway database (SMPDB). As shown in Figure S7, the "ar ac hidonic acid metabolism" was significantly enriched in the Cx at HS14. In the OM, the "tyrosine meta bolism," the "l ysine de gr adation," and the "beta alanine meta bolism" wer e significantl y enriched at HS14. Nota b l y, at HS21, no enrichment of any of the meta bolomic pathw ays w as found either in the Cx or in the OM.

Effects of HS Upon Cortical and Outer Medullary mRNA Expression (mRNAseq Analysis)
mRNAseq analysis was performed on the same tissue anal yzed for meta bolites as described a bov e to v alidate identification of enriched meta bolomic pathw ays using a denser genomic scale dataset (eg, ∼3000 named metabolites vs ∼30 000 gene transcripts). Ther e wer e 32 545 genes identified by the mRNAseq analysis, 22 293 were protein coding genes. Within that, adjusted P < .05 by Benjamini and Hochberg's test, comparing LS to HS14, 497 significantly increased and 422 decreased in Cx. Comparing LS to HS21, 3044 increased and 2917 decreased in Cx. In the OM, comparing LS to HS14, 91 increased and 22 decreased and comparing LS to HS21, 555 increased and 165 decreased.
The genes that stand out among those that were significantly changed by the HS diet are those related to the inflammatory system [ie, C3 (log 2 HS21/LS = 2.47), RT1-Db1(2.32), Itgal (2.32), etc]. In addition to these, in Cx the Mthfr ( −2.27) gene was significantly changed whose polymorphisms are related to hypertension, 41 Nrep ( −2.24) also known as P311 stimulates translation of TGF β and is related to tissue fibrosis, 42 and Slc16a1 ( −2.16) also known as Mct2 is a proton-linked monocarboxylate transporter. 43 Immune system-related genes were also upregulated in OM.
The pathway analysis of mRNAseq data (KEGG) ( Figure 2 ) found that genes most upregulated in the Cx tissue of SD rats fed HS diet were those related to the "Signaling molecules and interaction pathway," the "Immune system pathway" (red bars), and r elated pathw ays including "Cytokines r ece ptors," "Chemokine signaling," "NF-kappa B signaling," "Th17 cell differentiation," "T cell signaling," etc. Those pathways most downregulated were those related to metabolism (blue bars) including "TC A c ycle," "Fatty acid de gr adation," "Valine , Leucine , and Isoleucine de gr adation," "Glycine , Serine , and Thr eonine meta bolism," "Carbon metabolism," etc. In the OM tissue, the upregulated pathways wer e also largel y r elated to the immune system, but interestingly fewer pathways of metabolism were found to be downregulated with the HS diet.

mRNA Expression of Cortical Tubular Transporters
Of special mechanistic interest were the changes in gene expression of tubular transporters affected by the HS diet. Figure  S8 shows the Cx tissue mRNA expression of genes encoding transporters for glucose and amino acids that tended to be downregulated at HS14 with most of these reaching statistical significance by HS21. This includes amino acid transporters ( Slc1 , Slc3 , Slc7 ), sodium-glucose transporters (SGLT isoforms Slc5a1 , Slc5a2 ), urate ( Slc22a12 ), and lactate transporters ( Slc5a12 , Slc5a8 ). It was found that the glucose transporter 2 (GLUT isoforms Slc2a2 ) were downregulated at HS21 while expression of Slc2a1 was increased. Although it is unclear which of these would pr edominate functionall y, it is evident that the upregulation of the GLUT transporters would be consistent with a greater release of glucose and glycolysis products being released into the interstitial space. Also of note, monocarboxylate transporters (MCT) and Na + -K + -ATPases wer e upr egulated by the HS diet, which is consistent with enhanced proximal tubule (PT) Na + r ea bsorption necessitating greater utilization of ATP for active transport. Sodium transporters and channels including NHE3 ( Slc9a3 ), NKCC2 ( Slc12a1 ), NCC ( Slc12a3 ), and ENaC ( Scnn1 ) were upregulated. On the other hand, the effect of HS on the OM transporter genes was modest ( Figure S9).
In addition, pr oteol ysis-r elated genes ar e pic ked up in F igure S8 as well. There are myriads of proteolytic enzymes expressed in the kidney, 44 , 45 and those are effected by HS diet in Cx tissue. Genes encoding pr otease-acti v ated r ece ptors ( F2r ) 46 ar e upr egulated by HS, whereas megalin ( Lrp2 ) and clathrin ( Cltc ) were r educed. Inter estingl y, within cathe psin-encoding genes, Ctsa and Ctsb , which mainly expressed at PT S1 were downregulated and Ctsc and Ctsd , which mainl y expr essed at distal convoluted tubule (DCT) and connecting tubule (CNT) were upregulated.

Omic Integr a tion With Sta tistical Mapping and Validation of Metabolomic and mRNAseq Data
Data obtained from the metabolomics and mRNAseq analysis can be effecti v el y utilized in several ways. First, to validate against each other the predicted pathways that appear to be most affecting metabolic functions when fed an HS diet. Second, to identify pathways that were not of obvious importance from the meta bolomic anal ysis gi v en the limited n umber of compounds that can curr entl y be identified by a global mass spec analysis. Figure 3 summarizes the integrated metabolomic and mRNA expr ession data r elated to mitochondrial energy pr oduction in the Cx sample. By HS21, both metabolites and genes encoding the major enzymes of the TC A c ycle were found to be generall y downr egulated including reductions of citric acid, succinic acid, and fumar ate . While mRN As-encoding enzymes in the TC A c ycle were do wnregulated o ver all, some of mRN Asencoding proteins that make up the electron-transfer complex were not ( Figure 3 ). Especially, many genes encoding for proteins of complex V including Mt-atp8 wer e upr egulated at HS21 while the expression of others was reduced. It was also found that the gene encoding uncoupling protein 2 ( Ucp2 ) w as upr egulated by HS.
Several other important pathways were enriched in the metabolomic enrichment analysis ( Figure S10). Specifically, the "Ar ac hidonic acid metabolism" pathway in the Cx ( Figure S10A) and the "Lysine de gr adation" pathway in the OM ( Figure S10B) wer e alter ed by the HS diet. As shown in Figure S10A, the meta bolomic anal ysis in the Cx found that ar ac hidonic acid w as significantl y incr eased at HS14, together with downstream metabolites 8-HETE and thromboxane B2 (TXB2). The general upregulation of this pathway is reinforced by enhanced expression of most of the genes on ar ac hidonic acid metabolism pathway, which were upregulated on HS14 and HS21 compared to expression observed with the LS diet. The notable exception to this were the Cyp-4 genes important in the production of 20-HETE, which has both pro-and antihypertensive actions r esulting fr om modulation of v ascular and tubular functions of the kidney. 47 Figure S10B illustrates the "Lysine degradation" pathway in which many key elements were found in the meta bolomic anal ysis to be downr egulated in the OM as found at days HS14 and HS21. This included reduction of tissue lysine itself and reduced levels of α-ketoglutar ate , glutamate , and allysine. However, since lysine was significantly reduced, a reduction in the metabolites derived from lysine would be expected to be also reduced with no significant changes in the enzymes in this pathw ay wer e observ ed by the mRNAseq analysis.

Effects of the HS Diet Upon Gl ycol ysis in the Renal Cortex
As illustrated in Figure 4 A , the Cx levels of glucose tended to be reduced by day 14 of the HS diet and were significantly reduced b y da y 21 of the HS diet. A seemingl y compensator y r esponse is reflected by the observation that many genes encoding glycolytic enzymes w ere upre gulated at HS14 and even more so at HS21. Significant increases ( P < .05) were found in hexokinase isoforms ( Hk ) r equir ed for conv ersion of glucose to glucose 6-P, and in phosphofructokinase isoforms ( Pfk ), r equir ed to convert glucose 6-P to fructose 1,6-BP. A reduction of glyceraldehyde 3-P and 3-phosphoglycerate was found at HS21 and several isoforms of aldo-keto reductase ( Aldo ) and gly er aldehyde-3-phosphate dehydrogenase ( Gapdh ) were found to be reduced at HS21. Incr eases wer e also found in pyruvate kinase ( Pkm ). The Cx pyruvate levels tended to be increased at HS14 but did not differ from levels observed with LS at HS21. Lactate   dehydrogenases ( Ldh ) were also upregulated, but the tissue lactate did not differ from levels observed with LS at either HS14 or HS21.
The relationship of glycolysis with the oxidati v e pentose phosphate pathway (PPP) is also illustrated in Figure 4 A. A moderate increase ( P < .05) of ribose-5-phosphate was found at HS14 indicating a greater oxidation of glucose via this pathway which at this time would yield greater NADPH to scavenge ROS. However, at HS21 ribose-5-phosphate was no longer found to be elevated. The lactate to pyruvate ratio in cortical tissue tended to incr ease fr om 4.0 ± 0.6 at LS to 5.6 ± 0.6 at HS21 but did not reach statistical significance ( P = .11) ( Figure 4 B). As acti v ation of gl ycol ysis w as of particular inter est, lactate concentration in the Cx tissue was validated by fluorescent lactate analysis, which showed the similar trend as metabolomics data ( Figure 4 C). Further arteriovenous solute mass balance analyses are discussed below.
In general, it appears that metabolites of the gl ycol ytic pathway in the Cx may be initiall y elev ated at HS14 but by HS21 appear to be reduced to levels similar to or less than that observed when fed the LS diet. As noted in Figure S8, gene expression of the PT apical membr ane SGLT tr ansporter Scl5a1 isoform w as incr eased at HS14 ( P < .05) but not at HS21. A significant reduction of the Scl5a2 isoform was found at HS21 suggesting there could be a reduced luminal uptake of glucose in the PTs, perhaps contributing to the reduced Cx glucose levels. Gluconeogenesis may be expected to be suppressed since phosphoenolpyruvate carboxykinase 1 ( Pck1 ) that acts as the rate limiting enzyme in gluconeogenesis was reduced ( Figure 4 A).
Many genes inv olv ed in the gl ycol ytic and TC A c ycle were altered in Cx, while less significant changes were observed in OM ( Figure S11). Figure S12 summarizes the genes and meta bolites r elated to the malate-aspartate shuttle. The mRNAseq analysis confirmed that this pathway exists in the Cx, but since gene expression was not assessed separately in the cytoplasm and mitochondria one cannot determine whether the expression of enzymes that dir ectl y suppl y pr otons, suc h as Mdh , w er e alter ed. Malate lev els wer e r educed at HS21 ( P < .05) as was Slc25a11 gene expression, which codes for the mitochondrial carrier that transports malate across the IMM. It is interesting that Slc25a12 , which codes for the protein that transports aspartate across the IMM to the intermembrane space was significantly increased at HS21, but the r elev ance of this is unclear. Figure S13 summarizes the genes and meta bolites r elated to urea cycle and nitric oxide production in the renal Cx. It was found in the Cx that citrulline and arginine were significantly reduced together with a reduction in Nos1 mRNA expression at HS14. Aspartate was found to be elevated perhaps representing a compensatory response. By HS21, arginine had returned to lev els observ ed with LS and w as associated with elev ations of Nos3 , which appears to r e pr esent a compensator y r esponse. Arg2 mRNA expression was also elevated at HS21, which could also dri v e an incr ease of ur ea pr oduction although ur ea w as not measured by the mass spec analysis.

Targeted mRNA Expression Analysis of Isolated Cortical Tubular Segments and Glomeruli
The purity of isolated tubules was determined by comparing expr ession differ ences of Nphs2 (podicin in glomeruli), A pq1 (aquaporin I in PT), Nkcc2 ( Slc12a1 ) (Na + /K + /2Cl − co-transporter in cTAL), and Scnn1a (alpha subunit of ENaC in cortical collecting duct). As shown in Figure 5 A, Nphs2 could be detected only in the isolated glomeruli, whereas it was scarcely detected in isolated PT, which ov erwhelmingl y expr essed Aqp1 . Nkcc2 and Scnn1a wer e clearl y expr essed both in the cTAL and cortical collecting duct non-PT segments but were absent in PT and glomeruli. Ther e w as no differ ence betw een LS and HS21 in any tissue , except that Scnn1a was decreased in non-PT HS21. Since we were una b le to cleanl y distinguish cTAL segments fr om cortical collecting duct segments, these were analyzed together and designated as non-PT segments. Consistent with the mRNAseq analysis of cortical tissue significant increases in expression of hexokinase 1 ( Hk1 ) and Pkm (pyruvate kinase) were found in PT of HS fed rats consistent with an increases of activity of the glycolytic pathw ay ( Figur e 5 B). Although Hk1 expr ession lev els ar e r elati v el y low in the PT, the HS diet resulted in a significant increase while Hk2 and Hk3 expressed at much lower levels remained unchanged as was the case for Ldha (lactate dehydrogenase-A). No mRNA expr ession differ ences to the HS diet of Hk1 , Hk2 , Hk3 , Pkm , or Ldha were observed in either the glomeruli or the nonproximal tubular segments ( Figure 5 B). Standard curves of Hk1 and Pkm primers are shown in Figure S14A. All samples were within detection limits. Figure S14B shows an example of an amplification plot with the Hk1 primer in glomerular samples and PT samples.

Arterial and Venous Metabolites and Solute Mass Balance Determinations
As shown in Figure S15, a total of 3137 of compounds in plasma were detected when the detections from all 4 modes were combined. Of these,1531 are named compounds.
We first analyzed the metabolomic profiles in the artery (Art) and renal vein (RV) separately. It was found that the Art ( Figure  S16A) and RV meta bolomes (Figur e S16B) as anal yzed by sPLS-DA exhibited a clear shift in the metabolic states at HS7 and HS14. It is also evident that by HS21, the metabolic state w as mor e similar to that found when r ats w ere fed the LS diet r e pr esenting a return to "normal" although clear differences are seen in component 2 metabolites in both Art and RV samples. This return of the meta bolomic pr ofiles tow ard that of the LS state after 21 d of the HS diet is also clearly seen in the associated heat maps r e pr esenting those metabolites that were significantly changed by the HS diet for each of the designated modes (ANOVA Fisher's LSD P < .05). Specifically, the number of significantly altered metabolites from the total metabolites, which are detected in different modes are as follows: C18 + Art 96/808, C18 + RV 65/808, C18 − Then, the difference of metabolites between Art and RV was examined. The distribution of the ratio of RV to Art metabolites is shown in Figure S17. Focusing the analysis on the differences between the Art and RV of those named compounds in response to the HS diet it is seen in Figure S18A that by sPLS-DA anal ysis that se par ations w ere found betw een Art and Rv metabolites in the C18 + and HILIC + modes comparing LS and HS7, HS14 and HS21. However, there was no clear separation found between the days of the HS diet. As illustrated in Figure S15B, within the named 1531 compounds, 546 compounds were contained in the Metaboanalyst 5.0 database. Of these, 131 compounds (Ta b le S10) wer e significantl y (raw P < .05) changed over time by HS as determined using linear models with covariate adjustments 48 on Meta boanal yst 5.0. Enrichment analysis of those metabolites revealed the significant enrichment of "oxidation of br anc hed c hain fatty acids" and "carnitine synthesis" ( Figure S18B) including the metabolites such as carnitine, acetylcarnitine , lysine , and α-ketoglutar ate .
The urine metabolomic features are summarized in Figure  S19 showing that a total of 10 241 compounds were identified in the urine sample from the four modes (C18 + / − and HILIC + / −). Of these, 4980 were named compounds and after removing duplications and those compounds not found in plasma, there remained 749 compounds. Of these, only 367 were found to match the Meta boanal yst 5.0 database, which were used for the final solute mass balance analysis. Those metabolites in the urine for which the urine e xcretion e xceeded the total kidney filtr ation fr action are shown in Ta b le S11 . With LS feeding, 19 meta bolites wer e found to be excr eted in excess of the filtration fraction. Inter estingl y, the number of metabolites excreted more than filtration fraction ke pt incr easing ov er time (29 at HS7, 37 at HS14, and to 39 at HS21) and began to include uremic toxins such as creatinine, indoxyl sulfate, and hippuric acid. The clearances of those 367 metabolites were calculated and expressed in scatter plot in Figure S20. Carbohydrate and amino acids were specifically analyzed to determine changes over time, whic h w ere gr aphed as shown in F igur e S21. It w as found that most of the clearances of metabolites were increased by the HS.
Calculated metabolite solute mass balances comparing those determined in rats fed LS to those at HS7, HS14, and HS21 are shown in Figure S22 and Figure 6 . In these individual graphs in Figure 6 , metabolite solute mass balances are represented fr om two perspecti v es. First, whether the meta bolites wer e net consumed (N.C.) or net produced (N.P.). For this purpose, the 95% CI of the mean was calculated and if "0" was not contained within this confidence interval it indicates a compound is either net consumed or net produced b y kidne y ( P < .05, highlighted in red in Figure 6 ). Second, the graphs reflect whether the solute mass balances changes over time, for which a one-way r e peated measur es ANOVA post hoc Holm-Sidak was performed comparing all times to the LS fed state ( P < .05 indicated by * ). The analysis indicates that the solute mass balance of some of the important carbohydrates and their deri v ati v e utilized for both gl ycol ysis and the TC A c ycle were modified by HS intake (see graphs in blue boxes). Specifically, it is seen that in the LS state glucose was being net produced in the kidney ( P < .05), which was not observed during the days of HS feeding. Lactate net production became significant ( P < .05) at days 14 and 21 of the HS diet with a similar trend in pyruvate. Plasma lactate concentration w as v alidated by fluorescent assay kit and a similar tendency was observed (Ta b le S12). Ther e w as a gr eater net consumption of the important TC A c ycle intermediate α-ketoglutarate ( P < .05) in the LS fed r ats, whic h w as no longer appar ent with HS feeding. A net consumption of citrate was found with the LS diet and this positi v e net consumption was sustained throughout most of the periods of HS feeding ( P < .05) except at HS14. Gluconeogenic amino acids (see graphs in orange boxes) including aspar agine , tryptophan, phenylalanine , valine , arginine , glutamate , histidine , and proline w er e net pr oduced in significantl y gr eater amounts ( P < .05) during various days of the HS diet. The ketogenic amino acid lysine was also net pr oduced fr om the kidney at HS14 (see graph in gr een box).

Discussion
The kidne ys pla y a crucial role in eliminating excess salt in the diet and maintaining homeostasis in the body. In saltsensiti v e indi viduals with r educed sodium excr etor y function BP increases when fed an HS diet, which in turn leads to vascular, cardiac and kidney dysfunction, and injury. Although excess salt intake is less likely to produce hypertension in individuals with LS sensiti vity, 49-51 the pr esent study finds that an HS diet does have a significant effect on kidney metabolism even in normal SD rats in which minimal hypertension is observed when fed an HS diet.

Effect of Excess Salt on Renal Hemodynamics and O 2 Utilization in SD Rats
As determined by continuous 24 h/d monitoring, the SD rats as expected showed only a slight increase in the av era ge dail y MAP ( ∼5 mmHg) in response to the HS diet. By comparison, a r elati v el y m uch larger incr ease in RBF w as observ ed. An ev en gr eater incr ease of GFR w as observ ed consistent with the previous observations 52 although the mechanism for this are unclear. Nevertheless, this resulted in the significant increase in the calculated filtration fraction (FF). Although high GFR salt-sensitivity is thought to be associated with greater susceptibility to progression of renal dysfunction, [53][54][55] it is clear that SD rats possess compensatory mechanisms that ena b le them to compensate and pr ev ent the injurious effects of an HS diet. This is in stark contrast to Dahl SS rats, which were generated by selecti v e br eeding of SD rats and whose GFR is reduced by the second week of HS feeding. 30 Increased FFs with HS intake have also been observed in salt-sensitive humans (increase in MAP by 8 mmHg, increase in FF by 0.04) and women using oral contrace pti v es (incr ease in MAP by 1-2 mmHg, increase in FF by 0.02). 56 , 57 As O 2 content in arterial blood did not change over time with the HS diet, O 2 deli v er y incr eased in pr oportion to RBF. On the other hand, O 2 consumption increased proportionally greater than the increase in the delivery. Although an HS diet w as r e ported to decr ease the tubular O 2 consumption in isolated micr odissected r enal tubules fr om mice, 58 this does not appear to reflect in vivo responses where tubular O 2 consumption is altered by many factors including GFR and RBF that must be taken into account. 59 Although the correlation between Na + r ea bsorption and O 2 consumption in the kidney is well recognized as determined under a variety of conditions, 33,60 to the best of our knowledge, this is the first r e port that has evaluated this relationship in the unanesthetized fr eel y moving animal r e peatedl y. In pr evious in vi v o e xperiments with do gs, renal Na + r ea bsorption w as r e ported to be 20 mol per mol of O 2.

, 60
Our data at LS are 20.7 Na/O 2 (assuming 22.4 L/mol of O 2 ), which is close to the pr eviousl y r e ported v alue . It is notew orthy that the value of Na/O 2 did not significantly change even under the HS (18.2 at HS21). Gi v en that 99% of the filtered Na + is reabsorbed, the tubular load is primarily dictated by increase in GFR so an increase of GFR would be expected to increase the tubular workload and O 2 consumption.
Salt administration altered the expression of many geneencoding cortical tubular Na + transporters, which was especially evident at HS21. The enhanced expression of Na + transporters in the cortex ma y ha ve affected metabolic changes. It was found that Na + transporters and channels were generally upr egulated wher eas sugar and amino acids tr ansporters w ere found to be downregulated. As illustrated in Figure S8, in cortical tissue (Cx), increased gene expression was found of Nkcc2 ( Slc12a1 ), which is expressed in cTAL, NCC ( Slc12a3 ), which is expressed in the aldosterone sensitive distal collecting tubules (DCT), ENaCa ( Scnn1a ), which is expressed in the DCT and cortical collecting ducts (CCD), and NHE3 ( Slc9a3 ), which is expressed in PT . 61 T o gether, the increased mRNA e xpressions of the tr ansporters w ould be expected to increase both proximal and distal tubular Na + r ea bsorption. 62 , 63 These observations are consistent with the conclusions reached by Udwan et al. 58 that the fractional r ea bsorption of Na + is distributed differ entl y along the tubule as determined by dietary Na + intake.

Change in Metabolites and Gene Expression by the HS Diet
Despite the current progress in mass spectrometry and the ability to detect more than 5000 metabolites within biological samples, the number of annotated compounds reduces that number to several thousand and of those the assignment to known biochemical pathways is a limiting factor when compared to those obtained from mRNAseq analysis in which more than 20 000 known protein-coding genes can be mapped to less than 1000 biochemical meta bolites r e pr esented in the KEGG pathway maps. In the present study, we have utilized the combined strength of large-scale transcriptome sequencing (mRNAseq) with global profiling of metabolites in which the integrated analysis has identified man y pathwa ys of metabolism in which statisticall y significant differ ences to salt diet were obtained. Even those which did not reach statistically significant differences for the metabolomic analysis were of great utility when c hanges w er e consistent with pathw ays found of importance in the mRNAseq analysis.

Compensatory Mechanisms to Protect From Hypertension and Kidney Injury in SD Rats
One of the important underlying questions is what are the underl ying compensator y mechanisms that protect the SD rat from hypertension and kidney injury when fed an HS diet.
Although the specific answer to this question is not provided by the present analysis, enormous changes are occurring in the kidney metabolism and in many of the molecular and biochemical pathways that affect major functions of the kidney and inflammator y pathw ays of tissue injur y. Inflammator y pathw ays ar e stim ulated by R OS whic h is gener ated during the process of oxidati v e phosphor ylation. Sev eral pathw ays work as R OS scav engers ar e found in this study. First, oxidati v e PPP might be upre gulated, whic h is evident fr om incr ease in meta bolites, Ribulose-5-phosphate . PPP gener ate N ADPH, whic h is r equir ed for antioxidant system. 64 Second, Ucp2 mRNA expression elevated by the HS diet, which is also an R OS scav enger. 65 Acti v ation of Nos3 mRNA expression was also found. NOS3 generate nitric oxide and work as an R OS scav enger. 66 These might interact with each other to scavenge ROS and protect kidneys from damage.
Although the relationship between inflammation in the kidne y and h ypertension has been studied intensel y ov er the last decade, 67-69 the effect of salt on kidney without hypertension has been bar el y studied. It is interesting to note that the present study suggests that salt intake may acti v ate the inflammator y system in the kidney, even if the increase in blood pressure is slight ( ∼5 mmHg). In the present study, the HS diet increased renal oxygen consumption, and the associated ROS generation ma y ha v e contributed to the acti v ation of NFkB and other inflammator y systems. 70 Additionall y, the observ ed incr ease in GFR in the current study suggests increases of tubular flow, as confirmed in microperfusion studies, 71 , 72 which increase shear stress or hoop stress on tubules 73 , 74 activates mTORC1 36 thereby altering cellular metabolism. The mechanism of the inflammator y r esponse inde pendent of changes in b lood pr essur e is an intriguing finding, and further studies are needed to elucidate the mechanism.
The results of this study show that the profile of metabolites both in the kidneys and in systemic (ie, arterial plasma) changed over time in response to the HS diet as it was found that considera b le changes occurred in both arterial and renal venous blood meta bolic pr ofiles. It is v er y inter esting, howev er, that although significant changes were observed in the plasma metabolic profiles at HS days 7 and 14, the general meta bolic pr ofile r eturned in each of these rats to one similar to that observed with the LS diet b y HS da y 21. This is in contrast to the profiles obtained from the Cx and OM tissue analysis, whic h w er e markedl y changed over the 21 d of the HS diet and did not return to LS levels. This raises the interesting question of whether extrarenal changes in metabolic function might play an important role in normally protecting the kidneys from the injurious effects of an HS diet and from organs these signals might arise. There are reports that an HS diet alters the gut microbiome 75 , 76 and li v er meta bolism, 77 which need to be explored in greater depth.

Effects of the HS Diet on Arachidonic Acid Metabolism Pathway
Important effects of an HS diet on the ar ac hidonic acid (AA) pathw ay wer e identified by both metabolomic and mRNAseq analysis, which found the upregulation of many elements of this pathway significantly altered in the Cx at HS14 with a tendency to return toward LS levels at HS21 ( Figure S10). It is well recognized that ar ac hidonic acid is a major component of cell membrane phospholipids in the kidney, which is metabolized by c yclooxygenase (CO X), c ytochrome P450 monooxygenase (CYP450), lipoxygenase (LOX), and leukotrienes (LTs) enzymes. COX pr oduction of pr osta glandins (PG) and LTs leads to inflammator y injur y in the kidney. CYP450 production of hydroxyeicosatetraenoic acids (19-HETE and 20-HETE) play important roles in tubular ion transport and in modulating tubuloglomerular feedback to regulate the load on the glomerulus. 78 It is interesting that despite increased expression of AA in the Cx, reduction of Cyp4a1 and Cyp4a2 genes was observed. This may r educe expr ession of 20-HETE, which is known to r educe r enal vasoconstriction and renal vascular responses to angiotensin II, endothelin, nor e pine phrine , nitric oxide , and carbonmonoxide 79 and play a key role in kidney damage during the inflammator y pr ocess. The effects of HS on the AA pathw a y ha ve been found to contribute importantly to tubular transport, BP salt-sensitivity, and kidney injury in the Dahl SS rat model of hypertension. [80][81][82][83] We also observed a significant increase of TXB2 in the renal Cx at HS14, which appeared to be attenuated by HS21. TXB2 is an inacti v e meta bolite of thr omboxane A2 (TXA2), which is a potent vasoconstrictor and can lead to loss of renal structural integrity and inflammatory damage to the kidney. 84 , 85

HS Do wnregula tes the TC A Cycle and Upregula tes Gl ycol ysis
Ther e w as a marked differ ence between the effects of the HS diet upon the metabolomic profiles of the Cx and OM. The Cx clearly showed major changes in the metabolic profiles while few c hanges w ere seen in the OM in response to the HS diet ( Figure 2 ). One of the most interesting changes found in the Cx w as r elated to the TC A c ycle which at HS21 exhibited reductions in citr ate , pyruvate , α-ketoglutar ate , succinate , and in the mRN A expression of nearly all of the enzymes controlling the activity of the TC A c ycle. Conv ersel y, gl ycol ysis appears to be upregulated indicated by related enzymes including hexokinases, pyruvate kinases, and lactate dehydrogenases.
PTs are thought to have limited capacity for glycolysis with energy needs being met by oxidati v e mitochondrial meta bolism making them susce ptib le to damage with acute reductions of kidney perfusion. 86 Although not absent, the expression of HK in PTs is considered to be small. 16 , 87 , 88 In the present study, a novel finding was the increased expression of glycolysis-related genes in Cx in response to an HS diet. As determined from the qPCR analysis of the isolated proximal tubular segments of rats fed the HS diet, the incr eased expr ession of Hk1 and Pkm are consistent with increased activity of the glycolytic pathway ( Figures 4  and 5 B). Inter estingl y, similar downregulation of the TCA cycle and upregulation of glycolysis have also been documented in Dahl SS rats following an HS diet. 89 , 90 These findings suggest that upregulation of glycolysis in response to an HS diet may be a normal physiological response, which may be exaggerated in SS rats, potentially due to the absence of pr otecti v e counterr egulator y pathw ays that pr otect the kidneys a gainst the detrimental effects of HS diets. Despite such metabolic changes, it is presumed that fatty acids continue to serve as the primary energy source in the PT. Further resear c h will be r equir ed to compr ehensi v el y decipher the intricate dynamics of energy production and alterations in substrate pr efer ence in the PT under HS conditions.
It is r elev ant that the HS diet did not produce major changes in the meta bolomic pr ofiles of the OM of SD rats. This strain to maintain normal levels of renal medullary blood perfusion. SS rats have been found to exhibit a rapid 30% reduction of medullar y b lood perfusion during the first week of an HS diet 30 and we have found in a proteomic study of isolated mitochondria of these rats a downregulation, which is not observed in salt-insensiti v e consomic SS.13 BN rats. 91 SS rats also exhibit a significant increase in total RVR when fed an HS diet while saltinsensiti v e rats (consomic SS.1 BN rats) in contrast to a rather reduction in RVR in salt-insensitive SS.1 BN and SD rats in the pr esent study. 92 Pr otection fr om r enal ischemia, especiall y in the renal OM of SD rats may pr eserv e meta bolic functions of the mTAL as suggested by an absence of a down regulation of the metabolism pathways when fed an HS diet ( Figure 2 ). The downregulation of the TCA cycle proteins has been observed in mitochondria of isolated mTAL of SS rats. 91 Several studies from our la borator y hav e shown that r eduction of medullar y b lood flow in the SD rat with chronic medullary infusion of H 2 O 2 or an SOD inhibitor (DETC) result in a salt-sensiti v e form of hypertension. 93 , 94 So too, reduction of renal medullary oxidative stress in SS rats by intrarenal infusion of L-arginine reduces salt induced hypertension in SS rats. 95 The lack of significant changes in meta bolism-r elated genes in OM may reflect salt insensitivity in SD.

Par adoxical Rela tionship Betw een Kidne y O 2 Consumption and TCA Cycle Activity
One of the most interesting observations of the present study w as the seemingl y paradoxical phenomenon of an increase in kidney O 2 consumption and energy usage in face of a reduction in the TC A c ycle activity. What is the source of this additional energy production and O 2 usage? The data indicate that with the downregulation of the TCA cycle with the HS diet, glycolysis became a dominant source of energy production despite incr eased RBF incr eased O 2 extraction. The incr ease of r enal venous lactate ( Figure 6 ) is consistent with increased activity of the gl ycol ytic pathw ay. Aer obic gl ycol ysis which w as originall y described in cancer cells by Warburg in 1921 96 is also indicated to occur in the kidney 14 , 97 and has more recently been suggested to be inv olv ed in the meta bolic ev ents observ ed in dia betic kidney disease and ageing. 98 , 99 Although the biochemistry of the Warburg effect is not fully understood, this phenomenon is consistent with our current observations. NADH produced by the activation of glycolysis is not only used for lactate generation, but is also oxidized by the NADH shuttle (eg, the malate-aspartate shuttle). The increased release of pyruvate into the renal vein after HS suggests that not all of the excess NADH in the cytoplasm produced by acti v ation of the gl ycol ytic system is used for lactate production. Enzymes of malate-aspartate shuttle have been identified in the kidney 100 and gene expression of key enzymes in this pathway was observed in our study. The malate-aspartate shuttle has largely studied in cancer cells, and some suggest that the glucose fermentation (ie, Warburg effect) is a secondary consequence of saturation of the shuttle. 101 The malate-aspartate shuttle can be stimulated by an increase in glutamine uptake, 102 , 103 which we observed ( Figure 6 ). The key enzymes, oxaloacetate transaminase (GOT) and malate dehydrogenase (MDH) acti v ate the shuttle by forming a complex with acetylation 104 and we observed an increase in Got mRNA expression. Given the recognized limitations of predicting the activity of the shuttle from gene expression levels, the data are consistent with the idea that the malateaspartate shuttle is re gener ating N ADH inside of the mitoc hondrial matrix and sustaining oxidati v e phosphor ylation.

The HS Diet Alters Amino Acid Metabolism
The kidne ys pla y a major role in the homeostasis of the body amino acid pools through the synthesis, de gr adation, filtr ation, r ea bsorption, and urinar y excr etion of these compounds. Studies carried out in fasted swine found glutamine and proline from the arterial blood are largely disposed of by the kidneys and other amino acids such as serine, tyrosine, and arginine generated and r eleased fr om the kidneys for export to other tissues. 23 The current study carried out in unanesthetized nonfasted rats found glutamine was not taken up at LS state but a strong tendency for an increased uptake of glutamine ( P = .08) was observed during HS feeding. The kidneys also play an important role in protein metabolism, which is filtered by the glomerulus 105 is taken up into the lysosomes of the tubules and de gr aded to amino acids. 106 , 107 Even the OM of the kidney appears to participate in amino acid metabolic function in the SD rat. Specifically, a clear reduction was observed in the metabolites related to the de gr adation of lysine in the OM at HS14 (Figure S10B), which then tended to return to war d LS levels at HS21. This included reductions of α-ketoglutar ate , glutamate , and allysine in the Lysine de gr adation pathw ay. Lysine has r ecentl y attracted attention for its ability to suppr ess salt-sensiti v e hypertension. 108 Although lysine is one of the essential amino acids, we found it was released from the SD rat kidney after HS ( Figure 6 ), consistent with observations by Jang et al. 23 in pig study. This is likely explained by the de gr adation of protein either by glomerular epithelial or tubular lysosomes during renal passage. [109][110][111] Mor eov er, we observ ed almost all the amino acids wer e r eleased from kidney into renal vein in SD rats fed LS and further release w as observ ed in most of detected amino acids when fed HS. In general, the data indicate that the kidney produces amino acids fr om pr otein de gr adation faster than their utilization by the kidney and that the HS diet enhanced pr oteol ysis. Ther e wer e, howe ver, se veral notable exceptions to this such as glutamine, which w as r eleased fr om the kidney at LS and tended to be taken up after HS. This uptake of glutamine may be inv olv ed in the acti v ation of the malate-aspartate shuttle pathway as discussed earlier.
It was also found that the megalin ( Lrp2 ) and clathrin ( Cltc ) mRNA expr ession lev els wer e r educed with the HS diet (Figure S8) whereas several proteases and plasmid partitioning (PAR genes) were upregulated in Cx. Megalin and clathrin are key players in apical endocytosis in PT and reduction of these proteins is related to a reduction in albumin endocytosis. 111 , 112 Megalin is downregulated with HS diets even in salt insensiti v e Wistar or SD rats and in the absence of increased urinary albumin excretion. 113 , 114 However , as discussed earlier , the fact that the expression of transporter genes in the renal cortical PTs appear to be decreased while the expression of genes distal to the TAL is increased suggests that proteolysis may be shifted to the distal tubules. For example, Ctsa and Ctsb , which ar e pr edominantl y expr essed in the PTs, ar e downr egulated, while Ctsc and Ctsd , which are highly expressed in the DCT and other areas, 61 are upregulated after salt loading. Lrp2 and Cltc expressed in PT but not in DCT. It is known that DCT performs endocytosis of proteins, but it is not known which proteins play a key role, and further segment-specific studies are needed.

Limitations and Ultimate Goals
The present study provides a unique data set obtained in unanesthetized normal SD rats in which the metabolomic and genomic transcriptional responses to an HS diet with each rat serving as their own control, before and following the switch of diet. While avoiding the stress of surgery and anesthesia, by its nature this approach was limited to obtaining only global "solute mass balance" data reflecting metabolism of the whole kidney and was unable to distinguish between the cortex and medulla of the kidney. For this reason, parallel groups of rats were studied to obtain cortical and OM tissue for analysis at comparable days of HS loading. As such, it is recognized that the limitation of these data are biased to some extent by the unavoida b le effects of anesthesia-surgery and procedures required to obtain these tissues. The isolation of tubules for such analyses is yet another step removed from the "normal" physiological state but the results of the targeted mRNA analysis appear to validate conclusions drawn from the mRNAseq analysis of cortical tissue. Regarding the purity concern for microdissection, it is shown in Figure 5 A that there is a 1000 times difference in Nkcc2 between isolated PT and non-PT. This indicates that even a difference of 1/1000 would be detecta b le if contamination were to occur in one group. Although 100% purity of PT is unlikely, contamination from non-PT segments or glomeruli found in the present study could not account for the doubling of Hk1 expression in the PT segments of SD rats fed an HS diet. It should also be noted that we have not obtained absolute copy number per cell from our analysis of the isolated PTs and only relative changes are represented.
Other limitations of the present study must also be consider ed. RBF w as normalized by total body weight although conv entionall y r e ported in terms of v olume flow per kidney weight. This was necessary since with RBF is continuously measured ov er nearl y 4-wk so normalization to kidney weight could not be done for the intermittent time points of the study since kidneys w ere w eighted only at the end of the 21-d period of the HS diet.
Although in other studies of SD rats that we have carried out, we have found no difference in kidney size comparing SD rats fed LS or HS for 21 d, RBF in the present study was nonetheless normalized by body weight. Another limitation of the present study is that although all samples were collected at the same time of day to avoid diurnal effects (both from the unanesthetized and anesthetized rats) the rats were not fasted so ad lib eating/drinking could increase the variance of the data.
Finally, the limitations imposed by the evolving field of untargeted global meta bolomics ar e a consideration for all such studies in this field. Despite the solid pr ogr ess that has been made in metabolite identification and the sensitivity of the mass spectr ometr y techniques, the capability of detection and identification of compounds is far fr om compr ehensi v e. Verification is important especially for compounds with low FISH scores, and it is evident that continuing efforts must be made in the development of the databases and informatics to link substrates, enzymes, and metabolites to biochemical and physiological pathways. The present study provides only directional changes in metabolite concentrations and it is evident that gr eater n umbers of pur e standards will be needed to ena b le large scale quantitati v e anal ysis. Going forw ard, it will also be important to trace the fate of administered isotope-labeled meta bolites to v alidate the hypotheses that ar e generated based on metabolic flux studies since we are currently only able to calculate net consumption or net production in the kidney (eg, solute mass balances). Finally, sex differences of kidney meta bolism wer e not assessed as the chronic instrumentation of the smaller female rats when age matched proved to be overly daunting.

Conclusion
The kidneys of even normal SD rats with low blood pressure saltsensitivity exhibited significant changes in the metabolomic profiles in order to sustain the increased transport workloads and energy needs of the kidneys. The temporal patterns identified unique metabolic changes in the first 14 d of HS followed by what appear to be compensatory response required to sustain the energy r equir ements of the kidney. It seems that, at least at the mRNA level, the gl ycol ysis w as enhanced, and the production of pyruvate and lactate increased despite the increased oxygen consumption, while the TC A c ycle was do wn regulated by the HS diet in SD rat's kidney cortex. NADH produced during the process of increased glycolysis is used for lactate production in the cytoplasm and is inv olv ed in oxidati v e phosphorylation in mitochondria via malate-aspartate shuttle, which could potentially contribute to oxygen consumption. Besides the acti v ation of gl ycol ysis, the oxidati v e PPP, uncoupling protein 2 and nitric oxide synthetase 3 wer e upr e gulated eac h of which scav enge R OS and pr otect a gainst kidney dama ge in SD rat's kidney. The pr ogr essi v e incr ease of energy r equir ed for the HS diet in face of a reduction in TC A c ycle activity may be sustained by a "Warburg-like" effect whereby glucose and other 6-carbon sugars are converted by glycolysis into cellular energy and the metabolite lactate, which we found elevated in the renal venous blood (eg, lactic acid fermentation). Although not pr eviousl y identified in kidney cells, it is r ecognized that lactic acid fermentation can occur in muscle cells undergoing intense activity enabling ATP and NAD + production to continue gl ycol ysis. 115 Finall y, although kidney pr oteol ysis appears to be enhanced by the HS diet, the metabolic consequences of this is unclear.

Ac kno wledgments
All meta bolomics pr otocols wer e dev eloped in and the mass spectr ometr y data wer e collected in the Mass Spectr ometr y and Pr otein Chemistr y La b within Pr otein Sciences at The J ackson La borator y. We would also like to thank Miao Yu in the Computational Sciences Service at The J ackson La borator y for cr eation of the MetID converter to add additional metadata to each metabolite identified. Additionally, w e w ould like to thank Youg Liu in the Medical College of Wisconsin for assistance in the interpretation of the mRNAseq data.