Targeted Deletion of Klotho in Kidney Distal TubuleDisrupts Mineral Metabolism
Hannes Olauson,* Karolina Lindberg,* Risul Amin,* Ting Jia,* Annika Wernerson,†
Göran Andersson,† and Tobias E. Larsson*‡
*Division of Renal Medicine, Department of Clinical Science, Intervention, and Technology, Karolinska Institutet,Stockholm, Sweden; †Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet and KarolinskaUniversity Hospital, Stockholm, Sweden; and ‡Department of Nephrology, Karolinska University Hospital, Stockholm,Sweden
ABSTRACTRenal Klotho controls mineral metabolism by directly modulating tubular reabsorption of phosphate andcalcium and by acting as a co-receptor for the phosphaturic and vitamin D–regulating hormone fibroblastgrowth factor-23 (FGF23).Klotho nullmice have amarkedly abnormal phenotype.We sought to determineeffects of renal-specific and partial deletion of Klotho to facilitate investigation of its roles in health and dis-ease. We generated amousemodel with partial deletion of Klotho in distal tubular segments (Ksp-KL2/2).In contrast to Klotho null mice, Ksp-KL2/2 mice were fertile, had a normal gross phenotype, and did nothave vascular or tubular calcification on renal histology. However, Ksp-KL2/2 mice were hyperphos-phatemic with elevated FGF23 levels and abundant expression of the sodium-phosphate cotransporterNpt2a at the brush border membrane. Serum calcium and 1,25-dihydroxyvitamin D3 levels were normalbut parathyroid hormone levels were decreased. TRPV5 protein was reduced with a parallel mild increasein urinary calcium excretion. Renal expression of vitamin D regulatory enzymes and vitaminD receptor washigher in Ksp-KL2/2 mice than controls, suggesting increased turnover of vitamin D metabolites and afunctional increase in vitamin D signaling. There was a threshold effect of residual renal Klotho expressionon FGF23: deletion of.70%of Klotho resulted in FGF23 levels 30–250 times higher than inwild-typemice.A subgroup of Ksp-KL2/2 mice with normal phosphate levels had elevated FGF23, suggesting a Klotho-derived renal-bone feedback loop. Taken together, renal FGF23-Klotho signaling, which is disrupted inCKD, is essential for homeostatic control of mineral metabolism.
J Am Soc Nephrol 23: ccc–ccc, 2012. doi: 10.1681/ASN.2012010048
Type I membrane-bound a-Klotho (Klotho) is ex-pressed in tissues requiring abundant calciumtransport, such as the kidneys and parathyroidglands. It was originally described as a senescence-related protein, because mice lacking functionalKlotho protein develop a syndrome resembling hu-man aging, including shortened life span, atheroscle-rosis, vascular calcification, and osteoporosis.1
Klotho regulates mineral metabolism by pro-moting renal calcium reabsorption through stabi-lization of the transient receptor potential vanilloid-5(TRPV5) channel in distal tubules2 and inhibits in-organic phosphate reabsorption through decreasedexpression and activity of the sodium-dependentphosphate cotransporter type 2a and c (Npt2a,Npt2c) in proximal tubules.3 Furthermore, Klotho
forms a specific receptor complex with fibroblastgrowth factor receptor 1c (FGFR1c) that transmitssignaling of the circulating hormone FGF23.4 RenalFGF23-Klotho signaling leads to internalization ofNpt2a and Npt2c5–7 and alterations in Cyp27B1and Cyp24A1 transcripts encoding regulatory
Received January 19, 2012. Accepted June 25, 2012.
Published online ahead of print. Publication date available atwww.jasn.org.
Correspondence: Dr. Tobias E. Larsson, Clinical Research Cen-ter, Sixth Floor, Novumhuset, Karolinska University Hospital inHuddinge, SE-14186 Stockholm, Sweden. Email: [email protected]
enzymes in vitamin D metabolism.8 At the systemic level,this translates into a reduction of serum phosphate and 1,25-dihydroxyvitamin D3 (1,25(OH)2D).
The importance of Klotho as a renal coreceptor for FGF23is evidenced by Klotho and Fg23 null mice that share nearlyidentical biochemical phenotypes consistent with dismantledFGF23 signaling, including hyperphosphatemia, elevated 1,25(OH)2D, and hypercalcemia.1,9 Notably, the aging character-istics of Klotho and Fgf23 null mice are attenuated by dietaryrestrictions of phosphate and/or 1,25(OH)2D, or by eliminat-ing vitaminD effects through ablation of its receptor (VDR) orthe enzyme responsible for its activation (Cyp27B1).10–13 Col-lectively, this suggests that the aging phenotype is due to bothsystemic toxicity of mineral metabolites and perturbedFGF23-Klotho signaling.
The FGF23 level gradually increases during progressionof CKD, whereas expression of Klotho decreases.14,15 Distur-bances in FGF23-Klotho activity presumably contribute to theaccelerated aging process and cardiovascular disease evidentin CKD patients.16,17 Given the profound phenotype ofKlotho null mice, we wanted to determine the effect of a renal-specific and partial deletion of Klotho. Such a model wouldshed light on several issues, including dose-dependent ef-fects and kidney-derived systemic influence of Klotho ongrowth development and life span. Furthermore, it wouldallow the investigation of cell-specific autocrine or paracrineeffects of renal Klotho involved in the pathophysiology ofCKD-related complications such as interstitial fibrosis andectopic calcification.
Herein, we generated a novel mouse model with a kidney-specific Klotho deletion mainly in distal tubular segments andprovide genetic evidence favoring a pivotal role of FGF23-Klotho signaling in controlling mineral metabolism.
RESULTS
Generation of Ksp-KL2/2 MiceThe targeting vector of floxed Klotho mice and genotypingresults are shown in Figure 1A. To obtain the desired Klothodeletion, we used Ksp-cadherin-Cre mice, which express Creexclusively in tubular epithelial cells in the mature and devel-oping kidney and in the developing genitourinary tract.18 Apartial deletion of renal Klotho was confirmed with immuno-histochemistry (Figure 1B). The relative level of remnant me-dian Klotho transcripts in 15 screened kidney homogenateswas 0.69 (range, 0.26–1.0) compared with wild-type controls.Protein levels of Klotho quantified by Western blotting corre-lated highly to transcript levels (Figure 1C).
Gross Phenotype of Adult Ksp-KL2/2 MiceKsp-KL2/2mice were viable, fertile, and did not differ in size ordisplay any gross physical or behavioral abnormalities (Figure 2).There was no correlation between residual Klotho expressionand body size.
Generation of b-KL2/2 MiceTo confirm functionality of the floxed Klotho allele, we gen-erated a global deletion of Klotho (b-KL2/2) using miceexpressing Cre under the control of the human b-actin pro-moter. In stark contrast to Ksp-KL2/2 mice, b-KL2/2 micerecapitulated the phenotype of existing Klotho null mice,1 in-cluding severe growth retardation, kyphosis, lessened activity,and significantly reduced life span (Figure 2). In agreement,b-KL2/2mice (n=2, mean6 SEM) were hyperphosphatemic(5.360.06 mmol/L), hypercalcemic (2.8060.06 mmol/L),with extremely elevated FGF23 levels (234,630630,000 pg/ml)despite normal serum creatinine (44.861.9 mmol/L).
Biochemistries of Ksp-KL2/2 MiceSerum biochemistries were examined in adult mice at 8 weeksof age (Figure 3A). Ksp-KL2/2mice were hyperphosphatemicwith elevated FGF23. Parathyroid hormone (PTH) was de-creased in Ksp-KL2/2 mice, whereas 1,25(OH)2D, calcium,and creatinine levels were unaltered. Urinary calcium excre-tion was significantly increased as determined by calcium/creatinine ratio (Figure 3B) and fractional excretion of cal-cium (1.82 versus 0.67; P,0.05), but no difference was foundin urinary phosphate/creatinine ratio (Figure 3B) or fractionalexcretion of phosphate (28.4 versus 35.0; P=0.44).
Ksp-KL2/2 Mice and Dietary Phosphate LoadingSerum biochemistries of 8-week-old Ksp-KL2/2 and wild-type mice challenged with a high phosphate diet areshown in Figure 3C. Both groups developed hyperphosphate-mia, albeit more pronounced in Ksp-KL2 /2 mice. TheFGF23 response to phosphate loading was accentuated inKsp-KL2/2 mice. Ksp-KL2/2 mice had higher PTH, contrast-ing the reduced PTH level when fed a regular diet (Figure 3A).In linear regression analysis, phosphate was the only assessedbiochemical parameter that correlated to FGF23 in mice on aregular diet (r2=0.29; P,0.01), whereas phosphate (r2=0.29,P,0.05) and PTH (r2=0.41, P,0.005) correlated with FGF23in mice on a high phosphate diet.
Comparison of Ksp-KL2/2 and Wild-Type Mice withMatched Serum Phosphate LevelsIn this subgroup analysis, Ksp-KL2/2 mice had significantlyhigher FGF23 despite similar phosphate values (Figure 3D).No differences in calcium, phosphate, PTH, 1,25(OH)2D, orcreatinine were noted (Table 1). In the same mice, transcriptlevel of Cyp27B1was increased (P,0.001) with a similar trendfor Cyp24A1 (P=0.08).
Serum Biochemistries as a Function of Residual KlothoExpressionBecause Klotho was deleted with variable efficacy, we ana-lyzed the Klotho transcript level as a continuous variable.FGF23 was the only serum parameter significantly correlat-ing to Klotho (r2=0.23; P,0.05), whereas no correlations werefound for phosphate (P=0.09), calcium (P=0.13), PTH (P=0.13),
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or 1,25(OH)2D (P=0.72). To determine possible thresholdeffects, we screened 15 Ksp-KL2/2 mice and arbitrarily cate-gorized them into the following three groups based ontheir residual Klotho level: Klotho level .70% of wild-typecounterparts (mean 88%; n=7); Klotho level of 30%–70%(mean 59%; n=6); and Klotho ,30% (mean 28%; n=2).Phosphate and calcium levels gradually increased, whereasPTH levels decreased with lower Klotho expression (Table2). In contrast, a marked threshold effect was establishedfor FGF23 when relative Klotho expression was ,30%,
leading to levels 30–250 times higher than in wild-type mice(Figure 4).
HistologyNo differences were found with regard to general renalmorphology, calcifications, or fibrosis in Ksp-KL2/2 mice(Figure 5). The parathyroid glands were grossly normal withsimilar Klotho expression as in wild-type mice (data notshown). In contrast, b-KL2/2 mice had reduced kidney sizeand cortex height, higher cell density, extensive vascular and
Figure 1. Generation of floxed Klotho allele and targeted deletion of Klotho in Ksp-KL2/2 and b-KL2/2 mice. (A) Left panel showsschematic representation of wild-type allele (top), targeting vector (middle), and floxed allele with deleted Neo cassette (bottom).LoxP sites are flanking exon 2, thus enabling targeted deletion of the Klotho gene by Cre recombination. Right panel shows ge-notyping of mice. Representative PCR products from Klothoflox/flox (0.47 kB), Klothoflox/+ (0.47 and 0.37 kB), and wild-type (0.37 kB)mice. (B) Immunohistochemical staining of kidneys revealed partial deletion of Klotho in Ksp-KL2/2 mice, and complete deletion ofKlotho in b-KL2/2 mice. (C) Western blotting of whole kidney extracts from wild-type, Ksp-KL2/2, and b-KL2/2 mice. Numbers in-dicate relative transcript levels of Klotho measured by real-time qPCR, and for Ksp-KL2/2 these were arbitrarily categorized in sep-arate groups based on degree on the efficacy of Klotho deletion. Klotho protein level quantified by Western blotting correlated highlyto its transcript level.
J Am Soc Nephrol 23: ccc–ccc, 2012 Renal Klotho and Mineral Metabolism 3
Renal Protein ExpressionImmunohistochemical analysis revealed abundant expressionof Npt2a at the brush border membrane in Ksp-KL2/2 micecompared with wild-type controls. The vitamin D receptor(VDR) had a more heterogeneous pattern in Ksp-KL2/2
mice. There was no difference in cell proliferation rate as de-termined by Ki67 index (1.0% versus 0.95%; P=0.66; n$5 ofeach genotype) (Figure 6A).
Dual immunofluorescence staining in wild-type miceshowed that Klotho predominantly colocalized with the distaltubuli marker TRPV5, and very weakly with the proximalmarkerNpt2a. A similar pattern of residual Klotho proteinwasfound inKsp-KL2/2mice. The signal intensity of Klotho in theproximal tubuli appeared unaltered in Ksp-KL2/2 comparedwith wild-type mice, confirming a distal rather than proximaldeletion of Klotho (Figure 6B).
Western blotting showed increased VDR and decreasedTRPV5 protein in Ksp-KL2/2 mice (Figure 6C). However,immunostaining did not show a cell-specific downregulationof TRPV5 in the tubular segments where Klotho was deleted(Figure 6B).
Renal Gene ExpressionTranscript level of renal Cyp27B1 was increased in Ksp-KL2/2
mice, whereas VDR, Npt2a, Npt2c, FGFR1, CaSR, and TRPV5were unaltered (Table 3). Significant correlations were foundbetween Klotho and Cyp27B1, VDR, Npt2a, and FGFR1 (Fig-ure 7A). Renal transcript levels of mice challenged with a highphosphate diet are also shown in Table 3. Similarly, Cyp27B1
expression was higher in Ksp-KL2/2 mice whereas VDR,Npt2a, and CaSR were reduced. The correlations betweenKlotho to VDR and Npt2a were strengthened by dietaryphosphate loading (Figure 7B).
DISCUSSION
We present a novel mouse model (Ksp-KL2/2) harboring a par-tial deletion of Klotho predominantly in distal tubular segmentsof the kidney, demonstrating a pivotal role of Klotho in regula-tion ofmineralmetabolism.Ksp-KL2/2mice provide additionalvaluable information compared with general Klotho null mice.First, the variable efficacy of Klotho deletion allowed testing forpotential dose-dependent effects, revealing a graded increase inserum phosphate and FGF23 levels with lower Klotho levels,corroborating with the established phosphaturic action ofFGF23-Klotho. There was also a trend toward an increase inserum calcium level that paralleled the Klotho reduction. Thereason for this remains unclear, especially given that fractionalexcretion of calciumwas increased and PTH somewhat lower inKsp-KL2/2 compared with wild-type mice. One explanationmay be a functional increase in VDR signaling because VDRprotein was quantitatively increased in the face of an unalteredsystemic 1,25(OH)2D level. We also found it rather surprisingthat the 1,25(OH)2D level remained normal given that FGF23-Klotho signaling is a counter-regulatory hormone system for1,25(OH)2D. Mechanistically, there was an increase inCyp27B1 and a borderline significant increase in Cyp24A1 atthe transcript level, which presumably translates into both anincreased synthesis and degradation of active vitaminD. The risein Cyp27B1 is in agreement with attenuated FGF23-Klotho
Figure 2. Weight and gross appearance of Ksp-KL2/2 and b-KL2/2 mice. (Left panel) Ksp-KL2/2 mice did not differ in body weightcompared with wild-type controls. Conversely, b-KL2/2 mice were markedly smaller than wild-type littermates already at 4 weeks ofage. (Right panel) Ksp-KL2/2 mice were viable, fertile, and had a normal gross phenotype. In contrast, b-KL2/2 mice displayed severegrowth retardation, kyphosis, and a reduced life span with spontaneous deaths occurring around 7 weeks of age. †††Wild-type versusb-KL2/2 males; #Wild-type versus b-KL2/2 females. #P,0.05; †††###P,0.001.
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signaling, whereas the rise in Cyp24A1 con-tradicts the established physiology of FGF23.The exact cause remains elusive but mayspeculatively be due to differences in distalversus proximal tubuli signaling of FGF23-Klotho.
The combination of hyperphosphatemiaand high FGF23 levels in Ksp-KL2/2 micestrongly indicates a partial renal FGF23 re-sistance, supported by the fact that FGF23was the only biochemical variable related toKlotho expression and the observed expo-nential increase in FGF23when renal Klothodrop below approximately 30% of normallevels. Although not entirely surprising,this is an important in vivo proof of prin-ciple suggesting that initial onset of FGF23-Klotho dysregulation in CKD at least in partcould be attributed to a renal FGF23 resis-tance. It may also explain the large interin-dividual FGF23 variation observed in ESRDpatients,16 which simply cannot be attrib-uted to differences in the circulating min-eral metabolites. However, this study isclearly limited in terms of investigating thedynamics of FGF23-Klotho in early CKD,and this important question should be ad-dressed in future studies.
A fundamental question is how a renalFGF23 resistance due to Klotho deficiencytranslates into increased FGF23 productionin bone. Secondary changes inmineral meta-bolism, including hyperphosphatemia, areplausible. Indeed, we found a gradual rise inserumphosphate and FGF23 with decliningKlotho levels, anddietary phosphate loading
Figure 3. Serum and urine biochemistry in adult Ksp-KL2/2 mice. (A) At 8 weeks ofage, Ksp-KL2/2 mice displayed hyperphosphatemia and elevated FGF23 levels.Conversely, serum PTH was lower in Ksp-KL2/2 mice than in controls. Calcium, cre-atinine, and 1,25(OH)2D were similar in the two groups (*P,0.05; n=10 in each group
for all analysis except 1,25(OH)2D wheren$5). (B) Analysis of urine biochemistry re-vealed hypercalciuria in Ksp-KL2/2 mice. Uri-nary phosphate excretion was unchanged(*P,0.05; n=9–10 per group). (C) Serum bio-chemistry in Ksp-KL2/2 and wild-type miceon a high phosphate diet (1.65% phosphatefor 10 days). Both groups developed hyper-phosphatemia and elevated FGF23, althoughmorepronounced inKsp-KL2/2mice.Ksp-KL2/2
mice on a high phosphate diet had higher se-rum PTH than wild-type controls (*P,0.05;**P,0.01; n$5 per group). (D) In a subgroupanalysis of Ksp-KL2/2 and wild-type mice withmatched serum phosphate levels (2.56 versus2.60 mmol/L; P=0.90; n=5 in each group),FGF23 was significantly higher in Ksp-KL2/2
mice (140.4 versus 80.6 pg/ml; P,0.05).
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further increased FGF23. However, FGF23 was higher in Ksp-KL2/2 mice compared with wild-type mice with similar serumphosphate levels, suggesting that additional mechanisms arepresent. Speculatively, soluble Klotho may antagonize FGF re-ceptor signaling in bone, which has been implied as an impor-tant transcriptional regulator of FGF23,19 or interact with otheryet unidentified FGF23 regulatory elements. These possibilitiesneed to be explored in additional experimental studies.
In contrast to general Klotho nullmice, Ksp-KL2/2mice hada normal growth development and did not differ in body sizecompared with wild-type littermates. This contradicts that re-nal Klotho insufficiency causes systemic toxicity, although itmay also be explained by the incomplete knockout efficacy (i.e.,the residual Klotho amounts are sufficient to maintain a nor-mal growth status and body weight). Similarly, the biochemicalabnormalities in Ksp-KL2/2 mice may be too subtle to evoke
systemic toxicity as in general Klotho nullmice, favored by pre-vious studies showing that a low calcium- and phosphate-con-taining rescue diet attenuates the phenotype in animalmodelsof absent FGF23-Klotho signaling11
Another distinct difference compared with general Klothonull mice is the absence of overt renal abnormalities such asinterstitial fibrosis and ectopic calcifications in Ksp-KL2/2
mice. This was unexpected given that Klotho has been impli-cated as an antifibrotic agent by blocking TGF-b signaling20
and by acting as an endogenous inhibitor of vascular calcifi-cation.15 This argues against dominant cell-specific autocrineor paracrine effects of renal tubular Klotho, at least in theshort term. Additional long-term studies are warranted todetermine whether renal-specific Klotho deletion affectsthese phenotypes, especially in CKD.
Both FGF23 and PTH are phosphaturic hormones, actingon the same phosphate transporters in thekidney, namely Npt2a and Npt2c.7,21,22 Inthe face of hyperphosphatemia, serumPTH was suppressed, whereas FGF23 wasincreased in Ksp-KL2/2 mice on a regulardiet, suggesting that FGF23 may either re-spond earlier or play a more dominant rolethan PTH in regulating phosphate transportduring chronic hyperphosphatemia. The re-duction in PTH found in Ksp-KL2/2 micecould could either be caused by higher sys-temic calcium levels or the fact that FGF23inhibits PTH synthesis and secretion by di-rectly targeting the parathyroid glands.23,24
In contrast, Ksp-KL2/2 mice challengedwith a high-phosphate diet had higherPTH thanwild-type littermates. The underly-ing mechanisms remain unclear. One possi-bility is that parathyroid Klotho expressionis differentially regulated inKsp-KL2/2miceand that dietary phosphate loading evokes aKlotho-dependent parathyroid FGF23 resis-tance. Irrespectively, this phenomenon mayrepresent a rescue mechanism against phos-phate toxicity when FGF23 signaling is in-appropriately low in relation to the degreeof phosphate challenge.
Table 1. Serum biochemistry and gene expression in wild-type and Ksp-KL2/2
FGF23 was the only serum variable differing between wild-type and Ksp-KL2/2 mice in a subgroupanalysis ofmicewithmatched serumphosphate levels. In gene expression analysis, therewas amarkedincrease of Cyp27B1 in the Ksp-KL2/2 mice. Values are median (interquartile range).
Table 2. Serum biochemistry as a function of residual Klotho expression
FGF23 (pg/mL) 83.3 (43.3–122.8) 115.3 (74.3–193.3) 160.4 (78.9–182.0) 12,076 (3234–20,917)Phosphate (mmol/L) 2.71 (1.16–3.02) 2.63 (1.80–3.20) 3.09 (2.61–3.46) 3.69 (3.07–4.32)Calcium (mmol/L) 1.74 (1.67–1.89) 1.80 (1.67–1.92) 1.86 (1.65–1.93) 1.97 (1.84–2.10)PTH (pg/mL) 87.2 (65.8–188.9) 76.5 (54.2–185.1) 63.0 (53.2–79.4) 55.2 (53.2–57.1)1,25(OH)2D (pmol/L) 176.8 (102.0–168.9) 134.8 (81.1–215.3) 161.0 (66.5–382.4) 130.9 (n=1)Creatinine (mmol/L) 39.5 (28.5–41.0) 37.5 (33.5–44.5) 37.3 (32.5–41.0) 36.5 (34.5–38.5)In wild-type and Ksp-KL2/2 mice, categorized into groups based on residual Klotho level, there was a gradual increase in serum FGF23, phosphate, and calciumlevels and a reciprocal reduction in PTH level as Klotho expression declined.
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The proximal tubule is the principal sitefor renal phosphate reabsorption.22 Inagreement with a previous report, wefound that Klotho expression is largelyconfined to the distal tubules3 and thatthe Cre recombination driven by the Ksp-cadherin promoter is functional mainly indistal tubular cells. Although immunoflu-orescence staining suggested a low Klothoexpression also in proximal segments, itis still likely that distal Klotho plays animportant role in proximal phosphatetransport.
Hyperphosphatemic Ksp-KL2/2 micehad increased Npt2a protein expression atthe brush-border membrane. In contrast,the Npt2a transcript level was positivelycorrelated to the renal Klotho, indicatinguncoupling of Npt2a transcription totranslation. Because the observed reduc-tion in the Npt2a transcript level is anadequate compensatory response to hyper-phosphatemia, our data suggest that dis-mantling of renal FGF23-Klotho signalingmay result in aberrant Npt2a protein process-ing or degradation rather than transcrip-tional dysregulation. A similar uncoupling,possibly an adaptive response, was observedfor VDR.
Urinary calcium excretionwas increasedin Ksp-KL2/2 mice consistent with theknown action of renal Klotho from previ-ous in vitro studies2 and the observed re-duction in TRPV5 protein. In contrast, wedid not detect any difference in urinaryphosphate excretion despite hyperphos-phatemia and increased abundance ofNpt2a in Ksp-KL2/2 mice. We speculatethat this may be due to intermittent periodsof hypophosphaturia that were not cap-tured by our spot urinary analysis.
In summary, deletion of Klotho in dis-tal tubular segments reveals a fundamentalrole of Klotho in renal homeostatic con-trol of mineral metabolism and as a de-terminant of circulating FGF23.
CONCISE METHODS
Generation of Kidney-Specific KlothoKnockout MiceMice with a kidney-specific Klotho deletion
were generated using Cre-Lox recombination.
Briefly, the sequence of mouse chromosome 5
Figure 4. Serum phosphate and FGF23 as a function of renal Klotho expression.Klotho transcript levels were normalized to 1 in wild-type mice, and Ksp-KL2/2 micewere arbitrarily divided into three groups based on relative Klotho level: .0.7 (meanlevel 0.88; n=7); 0.30–0.7 (mean 0.59; n=6); and ,0.3 (mean 0.28; n=2). Serumphosphate level increased linearly after the decreased Klotho expression. In contrast,a threshold was found for FGF23 when relative Klotho expression was below 0.3,leading to levels 30–250 times higher than in control mice.
Figure 5. Renal histology in Ksp-KL2/2 and b-KL2/2 mice. Hematoxylin and eosinstaining (top) showed a normal renal morphology in Ksp-KL2/2 mice contrastingb-KL2/2 mice, which had reduced cortex height and higher cell density. In von Kossastaining (middle), b-KL2/2 mice displayed vascular and tubular calcifications (arrows Aand B, respectively). No calcifications were seen in wild-type or Ksp-KL2/2 mice. Asubtle increase in fibrosis was noted in kidneys from b-KL2/2 mice when stained withSirius red for collagen (bottom). Sirius red staining was similar in Ksp-KL2/2 mice andwild-type littermates. Number of kidneys examined was n=5 (wild-type); n=6 (Ksp-KL2/2); n=2 (b-KL2/2).
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Figure 6. Renal protein expression of Ki67, VDR, TRPV5 and Npt2a in Ksp-KL2/2 mice. (A) Proliferation rate as determined by im-munostaining for Ki67 did not differ between Ksp-KL2/2 and wild-type mice (top) (1.0% versus 0.95%; P=0.66). Staining for VDR(middle) revealed a more heterogeneous expression pattern in Ksp-KL2/2 mice compared with wild-type littermates. Npt2a proteinwas abundantly expressed at the brush border membrane in Ksp-KL2/2 mice compared with wild-type controls (bottom). Number ofkidneys examined was n=5 (wild-type); n=6 (Ksp-KL2/2); n=2 (b-KL2/2). (B) Dual immunofluorescence staining for Klotho and the distaltubuli specific marker TRPV5 revealed complete colocalization in wild-type mice, and partial colocalization in Ksp-KL2/2 mice. Therewas no apparent difference in TRPV5 staining intensity between distal segments with or without Klotho expression. Klotho was onlyweakly expressed in the proximal tubuli, and dual immunofluorescence staining for Klotho and the proximal tubuli specific markerNpt2a showed almost no colocalization. Npt2a expression at the brush border membrane was markedly higher in Ksp-KL2/2 micecompared with wild-type controls. (C) Western blotting of whole kidney extracts from wild-type and Ksp-KL2/2 mice revealed a gradualdecrease of TRPV5 (left panel), whereas VDR protein (right panel) level increased with declining Klotho level. Original magnification,340 in top panel of B; 310 in bottom panel of B.
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was retrieved from the Ensembl database (http://www.ensembl.org).
The RP23–434H9 BAC clone was used for generation of homology
arms and the conditional knockout region of the targeting vector.
The fragments were cloned in the LoxFtNwCD or pCR4.0 vector
and electroporated into C57BL/6 embryonic stem cells. Male chi-
meras were generated and subsequently bred with wild-type females
to generate Klotho-LoxP heterozygotes (Klothoflox/+). Klothoflox/+
were crossed with mice expressing Cre recombinase under the
Ksp-cadherin promoter (B6.Cg-Tg(Cdh16-cre)91Igr/J; Jackson
Laboratory, Bar Harbor, ME). Homozygous mice without Cre
(Klothoflox/flox) served as wild-type controls. To minimize the intra-
litter variability, mice were intercrossed, making 50% of the offspring
kidney-specific Klotho null mice and 50% controls that were
subsequently analyzed. Mice with a systemic Klotho deletion were gen-
erated using mice expressing Cre under the human b-actin promotor
(FVB/N-Tg(ACTB-cre)2Mrt/J, Jackson Laboratory). Generation of the
targeting vector, cloning, and electroporation into embryonic stem cells
was carried out by Caliper Life Sciences (Hopkinton, MA).
GenotypingTotal DNA was extracted from tail biopsies using DirectPCR Lysis
Reagent (Viagen, Los Angeles, CA). PCR amplification was carried
out on a 2720 Thermal Cycler (Applied Biosystems, Carlsbad, CA)
using HotStarTaq DNA Polymerase (Qiagen, Venlo, Netherlands).
The PCR products were visualized on a 1% agarose gel with GelRed
Nucleic Acid Gel Stain (Biotium, Hayward, CA). Sequences of the
primers used for genotyping are listed in Sup-
plemental Table 1.
Animal HousingMice were fed standard rodent chow (RM1;
SDS, Essex, UK) containing 0.73% calcium and
0.52% phosphate. In the high phosphate exper-
iment mice were fed a diet containing 1.0% cal-
cium and 1.65% phosphate (TD.88345; Harlan
Laboratories, Indianapolis, IN) for 10 days, start-
ing at 8 weeks of age. Blood sampling was per-
formed by tail vein incision at intermediate time
points and by bleeding the axillary artery
at sacrifice. All experiments were conducted in
compliance with the guidelines of animal experi-
ments at Karolinska Institutet and approved by
the regional ethical board.
Figure 7. Gene expression in kidneys of Ksp-KL2/2 mice. (A) Klotho transcript level correlated significantly to Cyp27B1, VDR, Npt2a,and FGFR1. (B) The correlations between Klotho, VDR, and Npt2a were accentuated in mice on a high phosphate diet.
Table 3. Renal gene expression in Ksp-KL2/2 mice on a regular and highphosphate diet
Data are mean 6 SD. Transcript level of Cyp27B1 was significantly higher in Ksp-KL2/2 mice ona regular diet. Cyp27B1, VDR,Npt2a, andCaSRwere significantly changed inKsp-KL2/2mice on a highphosphate diet in Ksp-KL2/2 mice.
J Am Soc Nephrol 23: ccc–ccc, 2012 Renal Klotho and Mineral Metabolism 9
Santa Cruz Biotechnology), and rabbit polyclonal anti-Npt2a
(NPT27; Alpha Diagnostics, San Antonio, TX). Proliferation index
(%) was calculated as Ki67 positive cells/total number of cells in at
least four viewing fields (at 320 magnification) from two sections
of each assessed mouse.
HistologyParaffin sections from wild-type, Ksp-KL2/2, and b-KL2/2 kidneys
were stained with hematoxylin and eosin, periodic acid–Schiff , von
Kossa, and Sirius red according to standard histologic protocol. Sec-
tions were examined blinded by an experienced renal pathologist
(A.W.). Glomeruli, tubuli, interstitium, and vessels were systemati-
cally evaluated for all samples.
Western BlottingKidney extracts were homogenized in PBS-TDS buffer (Sigma-
Aldrich Co., St Louis, MO) using a Tissue Lyzer LT. Extracts were
incubated on ice for 30 minutes, and centrifuged at 10,000 rpm for
10 minutes at 4°C. Supernatants were collected for further analysis.
Protein quantification was carried out using a BCA protein assay kit
(Thermo Scientific, Rockford, IL). Forty micrograms of protein was
separated on a 7.5% SDS-PAGE and electrotransferred to a polyvi-
nylidene fluoride membrane (Bio-Rad). After blocking with Casein
blocking solution (Thermo Scientific), membranes were sequentially
incubated with primary and secondary antibodies. Primary antibod-
ies were anti-Klotho (KM2076), anti-VDR (sc-13133), anti-TRPV5
(SC-30187), and anti-b-tubulin (Santa Cruz). The secondary anti-
body was IRDyes 680LT (LI-COR Biosciences, Lincoln, NE). Visual-
ization was carried out using the ODYSSEY Infrared imaging system
(LI-COR Biosciences). The amount of Klotho protein was nor-
malized to the amount of b-tubulin in each sample. Image studio
2.0 (LI-COR Biosciences) was used for densitometry and results are
presented as arbitrary densitometric units.
Statistical AnalysesIn comparisons between Ksp-KL2/2 and wild-type mice, Ksp-KL2/2
mice with relative Klotho levels .70% were excluded. In correla-
tion tests, all mice were included. GraphPad Prism 5.0 software
(GraphPad Software Inc, La Jolla, CA) was used for statistical analy-
sis. Gaussian distribution was tested using the D’Agostino and Pear-
son omnibus normality test. Variables fulfilling the criteria for normal
distribution were tested with the two-tailed t test. Non-normally dis-
tributed variables were compared using the Mann–Whitney test. Cor-
relationswere testedwith linear regression analysis.P values,0.05were
considered statistically significant. A minimum of five mice of each
genotype was analyzed in all experiments, unless otherwise stated.
ACKNOWLEDGMENTS
This study was funded by grants from the Swedish Foundation for
Strategic Research, Swedish Research Council, Swedish Kidney Foun-
dation, and Karolinska Institutet.
DISCLOSUREST.E.L. served as a consultant and/or received honoraria from Genzyme,
Sanofi-Aventis, Shire, Amgen, Abbott, Astellas, Fresenius, and AstraZeneca.
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