tamm-horsfall protein regulates granulopoiesis and...

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Tamm-Horsfall Protein Regulates Granulopoiesis andSystemic Neutrophil Homeostasis

Radmila Micanovic,* Brahmananda R. Chitteti,† Pierre C. Dagher,* Edward F. Srour,†

Shehnaz Khan,* Takashi Hato,* Allison Lyle,* Yan Tong, Xue-Ru Wu,§ andTarek M. El-Achkar*|

Divisions of *Nephrology and †Hematology, Microbiology, and Immunology, Indiana University School of Medicine,Indianapolis, Indiana; ‡Department of Biostatistics, Indiana University Schools of Medicine and Public Health,Indianapolis, Indiana; §Departments of Urology and Pathology, New York University School of Medicine and VeteransAffairs New York Harbor Healthcare System Manhattan Campus, New York, New York; and |Roudebush IndianapolisVeterans Affairs Medical Center, Indianapolis, Indiana

ABSTRACTTamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed animportant role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophilinfiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophilhomeostasis. In this study, we show that THP deficiency in mice increases the number of neutrophils, notonly in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the bonemarrow. Using multiplex ELISA, we identified IL-17 as a key granulopoietic cytokine specificallyupregulated in the kidneys but not in the liver of THP2/2 mice. Indeed, neutralization of IL-17 in THP2/2

mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP2/2

kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renalimmune cells and identified the S3 proximal segments, but not renal macrophages, as a major source ofincreased IL-23 synthesis. In conclusion, we show that THP deficiency stimulates proximal epithelial acti-vation of the IL-23/IL-17 axis and systemic neutrophilia. Our findings provide evidence that the kidneyepithelium in the outer medulla can regulate granulopoiesis. When this novel function is added to itsknown role in erythropoiesis, the kidney emerges as an important regulator of the hematopoietic system.

J Am Soc Nephrol 26: 2172–2182, 2015. doi: 10.1681/ASN.2014070664

Tamm-Horsfall protein (THP, also known asUromodulin) is a unique glycoprotein because it isexclusively expressed in the kidney, in tubular cells ofthe thick ascending limbs (TALs).1–3 Within TALcells, THP is targeted predominantly to the apicalmembrane domain, cleaved proteolytically, and se-creted in the urine. However, basolateral release ofTHP in the interstitium and circulation is also ob-served.1,4,5 The association of THP with acute andchronic forms of renal disease, such as familial juvenilehyperuricemic nephropathy, AKI, and CKD, arguesfor important regulatory functions of this glycoproteinin the pathogenesis of kidney disease.1,4,6–10 Interest-ingly, interstitial deposits of THP are frequently asso-ciated with tubulointerstitial diseases, suggesting apotential link between the interstitial presence of

THP and inflammation.8,11–13We recently providedchallenging evidence supporting a role for THP incontrolling neutrophil infiltration during kidneyinjury,2,5,10,14 and promoting recovery.5,14 The pres-ence of THP released fromTALs inhibits the produc-tion of cytokines and chemokines such as CXCL214

in injured neighboring proximal tubules (PTs),

Received July 11, 2014. Accepted October 30, 2014.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Tarek M. El-Achkar, Division of Nephrology,Indiana University School of Medicine, 950 W. Walnut Street,R2 E224, Indianapolis, IN 46202. Email: [email protected]

Copyright © 2015 by the American Society of Nephrology

2172 ISSN : 1046-6673/2609-2172 J Am Soc Nephrol 26: 2172–2182, 2015

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suggesting that THP mediates a regulatory cross-talk betweenTALs and PTs serving to suppress inflammation and neutrophilinfiltration.2

The expression of THP and its level in the circulation aresignificantly decreased in various forms of kidney disease.2,5,15,16

For example, the expression of THP is significantly reduced atthe peak of AKI.5,17,18 Furthermore, several studies have con-firmed that advanced CKD is associated with decreased levels ofTHP in the urine and in the circulation,16,19,20 thereby creating astate of “relative THP deficiency.” Interestingly, in a murineknockout model, THP deficiency in a noninjured state was as-sociated with a systemic increase in proinflammatory cytokinesand chemokines.21 Therefore, these observations prompted usto examine the role of THP in regulating inflammation not onlyin the kidney, but also systemically. The possibility that THPregulates systemic inflammation could explain the general-ized inflammatory phenotype and neutrophilia observed in“THP-deficient states,” such as what is reported with ad-vanced CKD.22–25

This study was designed to investigate the effect of THPdeficiency on systemic neutrophil homeostasis. We hypoth-esized that THP deficiency causes systemic neutrophiliathrough the production of growth factors by the kidney thatstimulate granulopoiesis. We show, for the first time, thatTHP deficiency causes systemic neutrophilia, which is de-pendent on the renal activation of the IL-23/IL-17 axis.Surprisingly, our results underscore the importance of theproximal renal tubular epithelium in regulating systemicneutrophil homeostasis. Our novel findings expand thecross-talk between the kidney and bone marrow beyond theregulation of erythropoiesis, to also include the regulation ofgranulopoiesis.

RESULTS

THP Deficiency Causes Renal and SystemicNeutrophiliaImmunohistochemistry for GR1 (a commonly used marker forneutrophils) in THP+/+ and THP2/2 kidneys shows an increasein the number of GR1+ cells in THP2/2 kidneys (Figure 1). Thiswas verified by flow cytometry for neutrophils in the kidney(Figure 2, A and B), in which neutrophils were defined as CD45+,CD11b+, Ly6G+.26 It is important to note that Ly6G is part ofthe GR1 antigen complex, and is also considered a specificmarker for neutrophils.26,27 The increased number of neutro-phils was present not only in the kidney, but also in the liver ofTHP2/2 mice (Figure 2, C and D) and in the spleen (Supple-mental Figure 1). We subsequently analyzed peripheral bloodcounts in THP+/+ and THP2/2 mice, and demonstrated thatTHP deficiency is associated with a significant increase in cir-culating neutrophil counts (Figure 2, E and F). Taken together,these results suggest that THP deficiency causes not only renalbut also systemic neutrophilia that extends to other organssuch as the liver.

THP Deficiency Causes Enhanced Granulopoiesis in theBone MarrowTo verify whether the generalized systemic neutrophilia in theperiphery and within organs was due to increased granulo-poiesis in the bone marrow, we performed detailed analysis of

Figure 1. Immunohistochemistry of GR1 in THP+/+ and THP2/2

kidney sections. (A–H) Representative images of sections (two sec-tions/kidney, five kidneys per group) encompassing all areas withinthe kidney from THP+/+ and THP2/2mice. Arrows show GR1-stainedcells in various areas within the kidney. (I) Quantitation of GR1+ cellsin each renal zone. Bar graphs are means6SEM. Asterisks repre-sents statistical significance between the two strains (P,0.05).Original magnification, 360 objective.

J Am Soc Nephrol 26: 2172–2182, 2015 THP and Neutrophil Homeostasis 2173

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bone marrow from THP+/+ and THP2/2 mice using flow cy-tometry as described28–30 and shown in Figure 3, SupplementalFigure 2. Figure 3A shows a significant decrease in themarrowofTHP2/2 mice of two classes of granulocyte progenitor cells:common myeloid progenitors and granulocyte-macrophageprogenitors. In parallel, there was a significant increase in the

number of differentiated neutrophils expressing Ly6G (Figure3B). The increased neutrophil count in the bone marrow wasalso verified using an automated hematology analyzer (Supple-mental Figure 3). Taken together, our data provide strong evidenceof enhanced granulopoiesis (decreased progenitors in themarrow,increased circulating differentiated cells), which explains the

Figure 2. Peripheral and organ neutrophil analysis in THP+/+ and THP2/2 mice. (A–D) Flow cytometry analysis of neutrophils in thekidney (A and B) and liver (C and D) from both strains of mice. A and C show representative scatter plots of CD45+ cells in the kidneyand liver, respectively, gated for CD11b and Ly6G. Quantitation of neutrophils (defined as CD45+, CD11b+, Ly6G+) in the kidney andliver are shown in B and D, respectively (n=5 per group). Asterisks denote statistical significance between THP+/+ and THP2/2 (P,0.05).(E and F) Bar graphs are means6SEM of peripheral white blood cell count and its subtypes from THP+/+ and THP2/2 mice (n=8 pergroup). E depicts cell concentration in blood, whereas F shows the percentage of each cell type within the total white blood cell count.A significant increase in neutrophils is noted in THP2/2 versus THP+/+ (*P,0.05). Neut, neutrophil; WBC, white blood cell count;NE, neutrophil; LY, lymphocyte; MO, monocyte; EO, eosinophil; BA, basophil.

2174 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2172–2182, 2015






generalized neutrophilia observed in THP2/2 mice. Because en-hanced granulopoiesis is strongly dependent on the presence ofgranulocyte colony-stimulating factor (G-CSF), we verified thatTHP2/2mice had an increased level of G-CSF in the serum com-pared with THP+/+ mice (Figure 3C).

THP-Deficient Kidneys Are an Important Source ofIncreased IL-17Because THP is uniquely produced in the kidney, we hypoth-esized that its deficiency causes the kidney to secrete growthfactors or cytokines that stimulate granulopoiesis. Therefore, weperformed a cytokine/chemokine multiplex ELISA (32 analytes)on kidney extracts from THP2/2 and THP+/+ mice. We also per-formed the same assayon the liver frombothmice strains. Figure 4shows that kidneys from THP2/2 mice have a significant in-crease in the level of IL-17, CXCL-2, and CXCL-9 comparedwith THP+/+ kidneys. Interestingly, livers extracts fromTHP2/2

mice did not show an increase in any of the progranulocyticfactors tested (including IL-17) compared with THP+/+ livers(Figure 4, lower panel). These data underscore the importance ofTHP in regulating the production of select cytokines/chemokinesin the kidney, notably IL-17.

Increased IL-17 in THP2/2 Kidneys Is a MajorDeterminant of Systemic NeutrophiliaAmong the factors differentially increased in THP2/2 kidneys,IL-17 is known to be an important activator of granulopoiesis,and it is part of the IL-23/IL-17 axis.31–33 In this well recognizedsystem, the cascade is induced by IL-23, which stimulates spe-cialized T cells to produce IL-17.31,34 In turn, IL-17 can stimulategranulopoiesis by causing a systemic increase in G-CSF.31,35 Us-ing ELISA, we showed that THP2/2mice have increased serumIL-17 levels comparedwithTHP+/+mice (Figure5A).Todeterminethe source of increased IL-17 in the kidney, we performed flow

cytometry for IL-17 (Supplemental Figure 4). Although thedistribution of IL-17+, CD3+ cells (T cells) was comparablebetween THP+/+ and THP2/2 kidneys, we did detect a smallincrease in IL-17+ neutrophils in THP2/2, which couldsuggest a potential contribution of these cells to increasedIL-17 levels.

To verify that increased IL-17 is a major determinant ofgranulopoiesis and systemicneutrophilia,weneutralized IL-17in vivo in THP2/2mice with an anti–IL-17 mAb. As shown inFigure 5, IL-17 neutralization significantly reversed the pe-ripheral (Figure 5B) and renal neutrophilia (Figure 5, D andE) in THP2/2 mice (neutrophil levels fell to the range seen inTHP+/+ mice). Furthermore, serum G-CSF levels were signif-icantly decreased by IL-17 neutralization (Figure 5C). Takentogether, these data support the concept that increased IL-17release from THP2/2 kidneys is a major determinant of sys-temic neutrophilia through enhanced granulopoiesis.

THP Regulates the Renal IL-23/IL-17 Axis and theProduction of IL-23 in S3 Epithelial SegmentsTo determine whether the IL-17 surge in THP2/2 kidneys isdue to increased production of IL-23, we measured IL-23mRNA and protein in THP2/2 and THP+/+ kidneys usingreal-time PCR and ELISA, respectively. Figure 6, A and B,shows a significant increase in IL-23 mRNA and protein inTHP2/2 versus THP+/+ kidneys, respectively. These findingssuggest that activation of the IL-23/IL-17 axis in the kidney isregulated by THP. Interestingly, we could not detect IL-23 inthe serum in either strains of mice (Figure 6C), which couldimply that induction of IL-23 is limited to the kidney and doesnot extend systemically.

Wenext sought todetermine the sourceof increased IL-23 inTHP2/2 kidneys. Studies in nonkidney tissues suggested thatinflammatory cells such as activated macrophages and

Figure 3. Bonemarrow analysis by flow cytometry. (A and B) Quantitation of progenitor cells within bonemarrow (A) and quantitation of bonemarrow cells with differentiation markers (B), as described in the Concise Methods, from THP+/+ and THP2/2mice (n=5 each group). The Mw/DC population shown is CD11b+ and Ly6G2. The decrease in granulocyte progenitors and increase in neutrophils strongly support enhancedgranulopoiesis. (C) ELISA assay for G-CSF, performed on sera from THP+/+ and THP2/2 mice (n=5 each group). Asterisk denotes statisticalsignificance between the two strains. LT-HSC, long-term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; MPP, mul-tipotent progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor.




possibly T cells are important producers of IL-23.36,37 There-fore, we performed real-time PCR probing for IL-23 on RNAextracted from glomeruli, S1–S2 and S3 proximal tubular seg-ments, TAL cells, macrophages/dendritic cells (Mws/DCs),and T cells as shown in Figure 6, D and E. Glomeruli, S1–S2, S3, and TAL segments were isolated using laser microdis-section (LMD), shown in Supplemental Figures 5 and 6. FACSanalysis was used to isolate Mws/DCs and T cells using theschema described in Supplemental Figure 7.

Figure 6D shows that in THP+/+ kidneys, IL-23 mRNAwasonly detected in S1–S2 segments, albeit at a very low level. InTHP2/2 kidneys (Figure 6E), IL-23 mRNA was significantlyupregulated, andwas uniquely detected in S3 segments but notin Mws/DCs, T cells, glomeruli, S1–S2 segments, or TAL seg-ments. Therefore, our data show that the proximal tubular

epithelium is a major source of IL-23 synthesis in the kidney.In addition, S3 segments appear to be the key activator of theIL-23/IL-17 axis during THP deficiency.

DISCUSSION

In this study, we investigated the role of THP on systemicneutrophil homeostasis. We show that THP deficiency causessystemic neutrophilia, which is most likely the result of adysregulated increase in renal IL-23/IL-17. We subsequentlyshow that the S3 tubular epithelium is the source of increasedIL-23. To our knowledge, this is the first study showing that thekidney regulates granulopoiesis and neutrophil homeostasis.Therefore, our study expands the key role of the kidney in

Figure 4. Cytokine/chemokine multiplex ELISA of the kidneys and livers in THP2/2 versus THP+/+. Graphs show standardizedmean differencesfor each analyte between THP2/2 and THP+/+ for kidneys (upper, n=5 each group) and liver (lower, n=5 each group). Analytes are groupedbased on their biologic function. Asterisk denotes statistical significance between THP2/2 and THP+/+. GMCSF, granulocyte macrophagecolony stimulating factor; LIF, leukemia inhibitory factor; MCSF, macrophage colony stimulating factor; TNF, tumor necrosis factor-a;VEGF, vascular endothelial growth factor. IL-5 and CXCL5 were undetectable in all the kidneys and livers, respectively.





regulating the hematopoietic system to include not onlyerythropoiesis through the production of erythropoietin,38

but also granulopoiesis through THP-dependent regulationof the IL-23/IL-17 axis. Figure 7 is a summary illustrationoutlying the mechanism of THP-regulated granulopoiesis,on the basis of the current data.

The finding that THP deficiency causes systemic neutro-philia, even without injury is remarkable, and agrees withprevious findings by Liu et al. that THP deficiency causes asystemic proinflammatory phenotype and splenomegaly.21 Todetermine whether the kidney is the source of a progranulo-poetic factor in the setting of THP deficiency, we used an un-biased approachwith multiplex ELISA for 32 preset cytokines/chemokines. Comparing the kidney to the liver enabled us todetermine that the kidney specifically has an increased level ofIL-17, which is a known activator of granulopoiesis.31,32 Thefact that IL-17 is increased in the kidney and the serum, butnot in the liver, in THP2/2 mice strongly supports that the

kidney itself is an important source of IL-17. The key role ofIL-17 in stimulating granulopoiesis and neutrophilia was thenconfirmed by in vivo neutralization. Although IL-1b in con-junction with IL-23 have been reported to stimulate IL-17production,39 there was no differential increase in IL-1b inTHP2/2 kidneys, suggesting that it does not play a significantrole in inducing IL-17 and granulopoiesis. The increase inCXCL9 (in conjunction with IL-17) observed in THP2/2 kid-neys is consistent with recent findings by Paust and colleaguesthat IL-17 stimulates the expression of CXCL9.40 The fact that afew proinflammatory cytokines/chemokines were decreased inTHP2/2 liver (e.g., CXCL2, IL-3, IL-1a, and IL-12p70) could bereactive to the observed neutrophilia, and the systemic inflam-matory environment induced by THP2/2 kidneys.

We attempted to verify the source of IL-17 in THP2/2 kid-neys. Unfortunately, we could not reliably amplify IL-17mRNA in the kidney (using two different primers; Supple-mental Table 1) despite consistent detection of the protein

Figure 5. Effect of IL-17 neutralization on systemic neutrophilia in THP2/2 mice. (A) Bar graphs are means6SEM of the IL-17 level insera from THP+/+ and THP2/2 mice (n=5 per group) measured with ELISA. (B) Neutrophil counts in THP2/2 mice injected with IL-17neutralizing antibody or IgG isotype at various time points during treatment (n=5 each group). (C) Serum G-CSF on day 7 after IL-17neutralization or treatment with IgG isotype. (D and E) Flow cytometry of the kidneys from both groups of mice probing specifically forneutrophils. Representative scatter plots from each group are presented in D, whereas E shows the quantitation of neutrophils shownas bar graphs6SEM. The wild-type bar in E is derived from the data presented in Figure 2, and is shown for comparison. IL-17 neu-tralization in THP2/2 significantly reduced serum G-CSF levels, neutrophilia in the periphery and within the kidney. Asterisk denotesstatistical significance compared to IgG controls, whereas the symbol # denotes significance compared to Day 0 (P,0.05). Neut,neutrophil.





using ELISA. This could be due to the extremely short t1/2 and/or low abundance of IL-17 mRNA. Previous studies showedthat few populations of T cells are major sources of IL-17 inexperimental GN.40,41 This is consistent with findings fromother tissues with chronic inflammation.33 Li et al. also pre-viously showed that neutrophils are a significant source ofIL-17 during kidney injury.42 Our flow cytometry studies sug-gest that increased IL-17+ neutrophils could be a potentialsource for increased IL-17 in THP2/2 kidneys. At this time,we cannot completely rule out additional sources of increasedIL-17 in THP2/2 kidneys such as parenchymal or stromal cells,43

and this is currently the topic ongoing investigations in thelaboratory.

IL-17 is downstream from IL-23 in the well defined IL-23/IL-17 axis.31–34,41,42 We showed an increased level of IL-23mRNA and protein in the THP2/2 kidney, which supportsthat activation of renal IL-23/IL-17 is an important determinantof the observed neutrophilia in THP2/2mice. Interestingly, it isthought that kidney Mws/DCs are the major source of IL-23.41

Furthermore, the site of IL-23 production could depend on thedisease model.41 Using the combination of LMD and FACS toisolate specific epithelial and immune cells in the kidney, weshow that PTs are a major source of IL-23 compared with renalMws/DCs, T cells, glomeruli, or TALs. To our knowledge, this isthe first study to clearly define a major source of IL-23 produc-tion in the kidney using this powerful approach. Our findings

Figure 6. Identification of the source of IL-23 synthesis in kidney using LMD and FACS. (A) IL-23mRNA measurements using real-timePCR in THP+/+ (reference set as 1) and THP2/2 total kidney extracts (n=5 per group). (B and C) IL-23 protein in the kidney and in theserum using ELISA, respectively. Asterisks in A and B denote statistical significance between THP2/2 and THP+/+ (P,0.05). IL-23 couldnot be detected in the serum of either mice strain. (D and E) IL-23 mRNA in specific cell types derived from THP+/+ and THP2/2 kidneys.Glomeruli, S1–S2, S3, and TAL segments were dissected using LMD. Mws/DCs and T cells were obtained using FACS. Total kidneyfrom THP2/2 was used as reference sample (set as 1) in all experiments shown in D and E. The # symbol denotes statistical significanceversus THP+/+ total kidney, whereas the asterisk in E denotes significance compared with THP2/2 total kidney (P,0.05).



underscore the importance of proximal tubular epithelium inregulating the IL-23/IL-17 axis in the kidney. The fact that THPdeficiencymarkedly induces IL-23 in S3 segments emphasizes therole of THP as a major modulator of IL-23 synthesis and fits withour previous findings that THP is an important mediator of tu-bular cross-talk in the outer medulla.2,5,14 Our current data ex-pand the importance of THP-S3 interaction beyond the kidney,and suggest that the kidney outer medulla is an important reg-ulator of systemic neutrophil homeostasis.

The current data also support our previous conclusions thatTHP2/2 kidneys are more prone to upregulation of CXCL2(MIP-2 chemokine, neutrophil chemoattractant) duringAKI.10,14

In fact,we report in thiswork thatTHP2/2kidneys have increasedCXCL2 even at baseline. Collectively, results from this and pre-vious studies will advance our understanding of the pathogenesisof neutrophil infiltration during AKI, especially because a state ofrelative THP deficiency in wild-type kidneys is consistently ob-served in the early stages ofAKI.5,17,18Wepropose that this relativeTHP deficiency at the onset of AKI exquisitely sensitizes the kid-ney to neutrophil infiltration by enhancing granulopoiesis, sys-temic neutrophilia and increasing renal CXCL2. The increasedexpression of THP that we reported during kidney recovery5

could therefore be essential to halt the influx of neutrophils tothe kidney by a dual action on the bone marrow (decreasing pro-duction) and on the kidney (decreased chemotaxis). It is worth

mentioning that previous reports have described a proinflamma-tory role for THP.44,45 These studies were based predominantly onin vitro or ex vivo data with highly aggregated, and potentiallyhighly immunogenic,46 urinary THP.2 To this date, there is noevidence to suggest that interstitial THP exists in the same highlyaggregated form.3 Our in vivo studies with THP2/2 mice dem-onstrate that THP has an anti-inflammatory role, which couldreflect the net outcome of the complex interactions of THP withvarious cells types within the kidney interstitium, especially acounterinflammatory effect on epithelial cells.2,5,14

This study may have clinical implications well beyond AKI.Several studies showed that THP levels in the urine and in theserum decrease with advanced CKD and tubular atro-phy,16,19,20 which is most likely due to decreased expressionby the kidney, as was shown recently by Ledo et al.47 Interest-ingly, recent data from clinical studies also demonstrate thatCKD is associated with development of neutrophilia, an in-creased neutrophil to lymphocyte ratio, and other markers ofsystemic inflammation.22,24,25 We propose that the relativeTHP deficiency occurring in kidneys with CKD activates theIL-23/IL-17 axis, which in turn stimulates granulopoiesisand a systemic inflammatory phenotype. Our study also under-scores the need for large-scale clinical studies to better define thedynamic changes in THP levels during the progression of kidneydisease.

In conclusion, we show that THP defi-ciencystimulatesproximalepithelialactivationof the IL-23/IL-17 axis and systemic neutro-philia. Our findings provide novel insights onhow the kidney, through uniquely producingTHP, can regulate granulopoiesis in the bonemarrowand systemic neutrophil homeostasis.

CONCISE METHODS

MiceAnimal experiments and protocols were ap-

proved by the Indianapolis Veterans Affairs

Animal Care and Use Committee. Age-matched

8- to 12-week-old THP knockout animals (129/

SvEv THP2/2) and wild-type background strain

were used as previously described,5,10,48,49 and

maintained in a specific pathogen-free facility.

Neutralization of IL-17 was done using a rat

mAb (MAB421; R&D Systems) injected daily at

50mg/mouse for 6 days. Isotype IgG2a (14-4321-

85; eBioscience) was used as control. Complete

blood count analysis was performed on 50 ml of

heparinized blood using a Hemavet 950FS ana-

lyzer.

Immunohistochemical AnalysesFor GR-1 staining (ab2557; Abcam, Inc.), 4%

paraformaldehyde perfusion–fixed tissues were

Figure 7. Summary diagram of THP-regulated granulopoiesis. In the kidney, interstitial THPreleased by the TAL exerts an inhibitory effect on the synthesis of IL-23 by S3 proximal tubularsegments in the outer medulla. In the context on THP deficiency, IL-23 is upregulated by S3segments and released within the kidney to act on neutrophils (Neut) and possibly other cellswithin the kidney to induce IL-17. IL-17 is subsequently released into the systemic circulation,where it can stimulate the production of G-CSF. G-CSF acts within the bone marrow tostimulate granulopoiesis and systemic release of excess neutrophils. LT-HSC, long-term he-matopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; MPP, multipotent pro-genitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor.



subsequently embedded in paraffin and processed for standard his-

tochemistry. Negative control without primary antibody was used.

Cells were counted on high (360 objective) power fields, using five

fields from each kidney area per slide (two slides per kidney, and five

kidneys each group).

Flow Cytometry on Kidneys, Livers, and SpleensFlow cytometry was performed on homogenized, digested tissues as

previously described.50 In brief, kidneys or livers were homogenized

and digested with collagenase, and subsequently strained using 70-mm

filters. Spleens were minced between two frosted slides and strained.

Cell counts and viability were done using aCountess (Life Technologies)

automated system. Then, 53106 cells from each sample were used for

staining after blocking nonspecific binding with anti-CD16/CD32

(14-0161-82; eBioscience). Flow cytometry was done using a BD LSRII

flow cytometer. For absolute cell counting, we used a dual platform

approach in which the percentage of cells of interest, out of total live

cells, was determined by flow cytometry. Live cell concentration per

specimen was determined with the Countess automated counter as de-

scribed above. To account for tissueweight variability, we normalized by

the weight of the corresponding tissue.

We used the following anti-mouse fluorochrome-conjugated

mAbs: CD45 (130-091-610; Miltenyi Biotech), Ly6G (560600; BD

Bioscience),CD11b(48-0112-80; eBioscience), andpropidiumiodide

for viability. Of CD45+ cells within the kidney and liver, neutrophils

were defined as CD45+, CD11b+, Ly6G+. FlowJo software (version 10;

FlowJo LLC) was used for flow analysis and plotting.

Flow cytometry for IL-17 was done on kidney cells that underwent

ex vivo stimulation for 4 hours with Cell Stimulation Cocktail (plus

protein transport inhibitors) (00-4975; eBioscience), as described by

others.41,42 Experiments without cell stimulation did not give a reli-

able IL-17 signal (data not shown). Cells were stained for fixable

viability (65-0864-14; eBioscience), CD45, CD11b, Ly6G, and CD3

(48-0032; eBioscience) before undergoing intracellular permeabilization/

fixation (88-8832; eBioscience) and staining for IL-17 (12-7177-81;

eBioscience).

Flow Cytometry on Bone MarrowLow-density bonemarrow cells from both THP+/+ and THP2/2mice

were washed once with stain wash (PBS, 1% bovine calf serum, and

1%penicillin-streptomycin), followedby antibody staining for 15minutes

on ice with fluorochrome-conjugated mAbs against the following mark-

ers: (1) c-Kit (CD117), Sca-1 (CD34), Flk2 (CD135), IL7Ra (CD127), and

Fc-gR (CD16/32) for progenitor cells; and (2) CD3 (T cells), CD11b

(myeloid), CD45R (B cells), Ter119 (erythroid), and Ly6G (neutrophil)

for differentiated cells. All mAbs were obtained from BD Biosciences,

except that CD127 was from eBioscience and anti–Fc-gR was from Biol-

egend. Cells were washed onemore timewith stainwash buffer, and data

were acquired onBDLSRII (BDBiosciences). Progenitor cell contentwas

gated and analyzed (Supplemental Figure 2) as previously described,29,30

according to the following definitions: LSK (Lin2 Sca1+ c-Kit+), long-

termhematopoietic stem cell (Lin2 Sca1+ c-Kit+ CD342CD1352),mul-

tipotent progenitor (Lin2 Sca1+ c-Kit+ CD34+ CD135+), short-term

hematopoietic stem cell (Lin2 Sca1+ c-Kit+ CD34+ CD1352), common

lymphoid progenitor (Lin2Sca1lo CD117lo CD135hi CD127+), common

myeloid progenitor (Lin2 Sca12 CD117+ CD34+ CD16/32lo), and

granulocyte-macrophage progenitor (Lin2 Sca12 CD117+ CD34+

CD16/32hi).

LMDSections from each kidney were snap frozen in optimum cutting

temperature medium using dry ice and kept at 280°C until use. They

were subsequently cut using a microtome at 8-mm sections on Leica

polyphenylene sulfate membrane slides (catalog no. 11505268). Histo-

chemical staining was performed immediately before dissection and

consisted of a series of rapid sequences of the following: (1) 100%

EtOH for 1 minute; (2) 95% EtOH for 30 seconds; (3) 75% EtOH for

30 seconds; (4) 50% EtOH for 30 seconds; (5) water 1, 30 seconds; (6)

water 2, 30 seconds; (7) Histogene Staining Solution (12241-05; Life

Technologies) 100 ml for 40 seconds; (8) water 3, 30 seconds; (9) 75%

EtOH for 30 seconds; (10) 95% EtOH for 30 seconds; (11) 100% EtOH

for 30 seconds; and (12) Xylene 90 seconds. Sections were air dried and

immediately taken to a Leica LMD6000 microscope. Dissection was

performed at 340 magnification. We dissected 40–60 S3 segments

and 80–100 TAL tubules from each section. Three sections were dis-

sected from each kidney/mouse.

For dissection of glomeruli and S1–S2 segments, we used immu-

nofluorescence LMD, using the following sequence of staining: (1) 100%

EtOH for 30 seconds32; (2) 95% EtOH for 20 seconds32; (3) 75%

EtOH for 20 seconds32; (4) 50% EtOH for 20 seconds32; (5) water 1,

30 seconds32; (6) water 2, 30 seconds32; (7) staining with FITC-

phalloidin plus 49,6-diamidino-2-phenylindole (molecular probes)

in PBS plus 2%BSA for 3–5 minutes; (8) phosphate buffered water

wash for 30 seconds33; and (9) air dry for 5 minutes.

RNA was extracted using PicoPure RNA kit (12204-01; Life

Technologies). Because of the finite RNA yield, RNA was pooled for

each group (n=3 mice per group). An additional concentration step

was performed using standard isopropanol precipitation, before RT

and real-time PCR. The purity of RNAwas verified using established

markers for each tubular segment within the nephron (Supplemental

Figures 5 and 6).51,52 RNA from total kidney extracts was used as a

positive control for all markers. To validate our methodology with

additional controls (Supplemental Figure 8), we verified that IL-1b

was exclusively expressed in Mw/DCs,44 whereas macrophage colony

stimulating factor (known to be expressed in myeloid and renal ep-

ithelium26,53) was detected in S3 segments, TALs, and Mws/DCs.

FACS AnalysesFACS was performed using BD FACSAria using the schema shown in

Supplemental Figure 7. Cells were isolated from kidneys similar to

what was described in the flow cytometry section, and were subse-

quently enriched in leukocytes using Lympholyte M (Cedarlane) gra-

dient followed bymagnetic beads directed enrichment of CD45+ cells

using MACS Separation Columns (130-042-201; Miltenyi Biotech).

To maximize yield, kidneys were pooled from eight mice. Mws/DCs

were defined as CD45+, CD11b+, Ly6G2. T cells were defined as

CD45+, CD11b2, CD3+, B2202. The following additional anti-mouse–

conjugated mAbs were used: CD3 (48-0032; eBioscience) and B220

(17-0452; eBioscience). mRNA extraction from recovered cells was per-

formed using the PicoPure RNA kit from Life Technologies.








ELISA AssaysA multiplex cytokine/chemokine assay containing 32 prespecified

analytes (MCYTOMAG-70K-PX32; EMD Millipore) was performed

on kidney and liver lysates from THP+/+ and THP2/2 kidneys (n=5

per group). Values were initially reported in picograms per milligram

protein/tissue after normalization to the protein concentration for

each tissue. Statistical analysis is described below. In separate experiments,

ELISA for IL-17 and G-CSF (MCYTOMAG-70K; EMD Millipore) on

sera from the two strains of mice (n=5 per group) were also performed.

We also performed IL-23 ELISA (MCYTOMAG-74K-01) on kidney tis-

sue extracts and sera from the two strains.

Real-Time PCRReal-time PCR was performed as previously described5 in an Applied

Biosystems ViiA7 system using TaqMan Gene Expression Assays (all

from Applied Biosystems; Supplemental Table 1). All expression was

normalized to gluconate dehydrogenase endogenous control and re-

ported as fold change compared with control using the DD threshold

cycle method, according to the manufacturer’s instructions.

Statistical AnalysesValues of each experimental group are reported as means6SEM. For

most experiments, a two tailed t test was used to examine the differ-

ence in means for continuous data. A nested ANOVAdesign was used

for quantitation of GR1+ cells in immunohistochemistry. Statistical

significance was determined at the 0.05 significance level. For ELISA

multiplex analysis, two-sample t tests were used to compare the value

for each test between the two experimental groups. The mean differ-

ence for each test value between THP2/2 versus THP+/+ and its 95%

confidence interval were then calculated. Because each test was mea-

sured in different metrics, the values were standardized by using the

raw test value divided by its SEM. The standardized test difference

and 95% confidence interval between the two strains for all the tests

are presented in Figure 4 for kidneys and liver separately.

ACKNOWLEDGMENTS

We acknowledge the Bioplex, Pathology, and Flow Cytometry cores at

Indiana University School of Medicine for assistance with this project,

and we thank the Koman Tissue Bank for assistance with LMD.We also

acknowledge Dr. Timothy Sutton for his critical review of this article.

This work was supported by a US Department of Veterans Affairs

merit award to T.M.E.-A.

DISCLOSURESNone.

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