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Tamm-Horsfall Protein Regulates Granulopoiesis and Systemic Homeostasis

† † Radmila Micanovic,* Brahmananda R. Chitteti, Pierre C. Dagher,* Edward F. Srour, Shehnaz Khan,* Takashi Hato,* Allison Lyle,* Yan Tong, Xue-Ru Wu,§ and | Tarek M. El-Achkar*

Divisions of *Nephrology and †Hematology, Microbiology, and , 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 Veterans Affairs New York Harbor Healthcare System Manhattan Campus, New York, New York; and |Roudebush Indianapolis Veterans Affairs Medical Center, Indianapolis, Indiana

ABSTRACT Tamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed an important role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophil infiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophil homeostasis. In this study, we show that THP deficiency in mice increases the number of , not only in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the . Using multiplex ELISA, we identified IL-17 as a key granulopoietic specifically 2 2 2 2 upregulated in the kidneys but not in the liver of THP / mice. Indeed, neutralization of IL-17 in THP / 2 2 mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP / kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renal immune cells and identified the S3 proximal segments, but not renal , as a major source of increased 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 kidney in the outer medulla can regulate granulopoiesis. When this novel function is added to its known role in , 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-Horsfallprotein(THP,alsoknownas THP and inflammation.8,11–13 We recently provided Uromodulin) is a unique glycoprotein because it is challenging evidence supporting a role for THP in exclusively expressed in the kidney, in tubular cells of controlling neutrophil infiltration during kidney the thick ascending limbs (TALs).1–3 Within TAL injury,2,5,10,14 and promoting recovery.5,14 The pres- cells, THP is targeted predominantly to the apical ence of THP released from TALs inhibits the produc- membrane domain, cleaved proteolytically, and se- tion of and chemokines such as CXCL214 creted in the urine. However, basolateral release of in injured neighboring proximal tubules (PTs), THP in the interstitium and circulation is also ob- served.1,4,5 The association of THP with acute and chronic forms of renal disease, such as familial juvenile Received July 11, 2014. Accepted October 30, 2014. hyperuricemic nephropathy, AKI, and CKD, argues Published online ahead of print. Publication date available at for important regulatory functions of this glycoprotein www.jasn.org. 1,4,6–10 in the pathogenesis of kidney disease. Interest- Correspondence: Dr. Tarek M. El-Achkar, Division of Nephrology, ingly, interstitial deposits of THP are frequently asso- Indiana University School of Medicine, 950 W. Walnut Street, ciated with tubulointerstitial diseases, suggesting a R2 E224, Indianapolis, IN 46202. Email: [email protected] potential link between the interstitial presence of Copyright © 2015 by the American Society of Nephrology

2172 ISSN : 1046-6673/2609-2172 JAmSocNephrol26: 2172–2182, 2015 www.jasn.org BASIC RESEARCH suggesting that THP mediates a regulatory cross-talk between TALs and PTs serving to suppress inflammation and neutrophil infiltration.2 The expression of THP and its level in the circulation are significantly decreased in various forms of kidney disease.2,5,15,16 For example, the expression of THP is significantly reduced at the peak of AKI.5,17,18 Furthermore, several studies have con- firmed that advanced CKD is associated with decreased levels of THP in the urine and in the circulation,16,19,20 thereby creating a state of “relative THP deficiency.” Interestingly, in a murine knockout model, THP deficiency in a noninjured state was as- sociated with a systemic increase in proinflammatory cytokines and chemokines.21 Therefore, these observations prompted us to examine the role of THP in regulating inflammation not only in the kidney, but also systemically. The possibility that THP regulates 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 THP deficiency on systemic neutrophil homeostasis. We hypoth- esized that THP deficiency causes systemic neutrophilia through the production of growth factors by the kidney that stimulate granulopoiesis. We show, for the first time, that THP 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 the proximal renal tubular epithelium in regulating systemic neutrophil homeostasis. Our novel findings expand the cross-talk between the kidney and bone marrow beyond the regulation of erythropoiesis, to also include the regulation of granulopoiesis.

RESULTS

THP Deficiency Causes Renal and Systemic Neutrophilia Immunohistochemistry for GR1 (a commonly used marker for 2 2 neutrophils) in THP+/+ and THP / kidneys shows an increase 2 2 in the number of GR1+ cells in THP / kidneys (Figure 1). This 2 2 was verified by flow cytometry for neutrophils in the kidney Figure 1. Immunohistochemistry of GR1 in THP+/+ and THP / (Figure 2, A and B), in which neutrophils were defined as CD45+, kidney sections. (A–H) Representative images of sections (two sec- + + 26 tions/kidney, five kidneys per group) encompassing all areas within CD11b , Ly6G . It is important to note that Ly6G is part of +/+ 2/2 the GR1 antigen complex, and is also considered a specific the kidney from THP and THP mice. Arrows show GR1-stained cells in various areas within the kidney. (I) Quantitation of GR1+ cells marker for neutrophils.26,27 The increased number of neutro- in each renal zone. Bar graphs are means6SEM. Asterisks repre- phils was present not only in the kidney, but also in the liver of fi P, 2/2 sents statistical signi cance between the two strains ( 0.05). THP mice (Figure 2, C and D) and in the spleen (Supple- Original magnification, 360 objective. mental Figure 1). We subsequently analyzed peripheral 2 2 counts in THP+/+ and THP / mice, and demonstrated that THP deficiency is associated with a significant increase in cir- THP Deficiency Causes Enhanced Granulopoiesis in the culating neutrophil counts (Figure 2, E and F). Taken together, Bone Marrow these results suggest that THP deficiency causes not only renal To verify whether the generalized systemic neutrophilia in the but also systemic neutrophilia that extends to other organs periphery and within organs was due to increased granulo- such as the liver. poiesis in the bone marrow, we performed detailed analysis of

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

2 2 bone marrow from THP+/+ and THP / mice using flow cy- number of differentiated neutrophils expressing Ly6G (Figure tometry as described28–30 and shown in Figure 3, Supplemental 3B). The increased neutrophil count in the bone marrow was Figure 2. Figure 3A shows a significant decrease in the marrow of also verified using an automated hematology analyzer (Supple- 2 2 THP / mice of two classes of progenitor cells: mental Figure 3). Taken together, our data provide strong evidence common myeloid progenitors and granulocyte- of enhanced granulopoiesis (decreased progenitors in the marrow, progenitors. In parallel, there was a significant increase in the increased circulating differentiated cells), which explains the

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Figure 3. Bone marrow analysis by flow cytometry. (A and B) Quantitation of progenitor cells within bone marrow (A) and quantitation of bone 2 2 marrow cells with differentiation markers (B), as described in the Concise Methods, from THP+/+ and THP / mice (n=5 each group). The Mw/ 2 DC population shown is CD11b+ and Ly6G . The decrease in granulocyte progenitors and increase in neutrophils strongly support enhanced 2 2 granulopoiesis. (C) ELISA assay for G-CSF, performed on sera from THP+/+ and THP / mice (n=5 each group). Asterisk denotes statistical significance between the two strains. LT-HSC, long-term ; ST-HSC, short-term hematopoietic stem cell; MPP, mul- tipotent progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor.

2 2 generalized neutrophilia observed in THP / mice. Because en- cytometry for IL-17 (Supplemental Figure 4). Although the hanced granulopoiesis is strongly dependent on the presence of distribution of IL-17+,CD3+ cells (T cells) was comparable 2 2 granulocyte colony-stimulating factor (G-CSF), we verified that between THP+/+ and THP / kidneys, we did detect a small 2 2 2 2 THP / mice had an increased level of G-CSF in the serum com- increase in IL-17+ neutrophils in THP / , which could pared with THP+/+ mice (Figure 3C). suggest a potential contribution of these cells to increased IL-17 levels. THP-Deficient Kidneys Are an Important Source of To verify that increased IL-17 is a major determinant of Increased IL-17 granulopoiesis and systemic neutrophilia, we neutralized IL-17 2/2 Because THP is uniquely produced in the kidney, we hypoth- in vivo in THP mice with an anti–IL-17 mAb. As shown in esized that its deficiency causes the kidney to secrete growth Figure 5, IL-17 neutralization significantly reversed the pe- factors or cytokines that stimulate granulopoiesis. Therefore, we ripheral (Figure 5B) and renal neutrophilia (Figure 5, D and 2 2 performed a cytokine/chemokine multiplex ELISA (32 analytes) E) in THP / mice (neutrophil levels fell to the range seen in 2 2 on kidney extracts from THP / and THP+/+ mice. We also per- THP+/+ mice). Furthermore, serum G-CSF levels were signif- formedthesameassayontheliverfrom both mice strains. Figure 4 icantly decreased by IL-17 neutralization (Figure 5C). Taken 2 2 shows that kidneys from THP / mice have a significant in- together, these data support the concept that increased IL-17 2 2 crease in the level of IL-17, CXCL-2, and CXCL-9 compared release from THP / kidneys is a major determinant of sys- 2 2 with THP+/+ kidneys. Interestingly, livers extracts from THP / temic neutrophilia through enhanced granulopoiesis. mice did not show an increase in any of the progranulocytic +/+ factors tested (including IL-17) compared with THP livers THP Regulates the Renal IL-23/IL-17 Axis and the (Figure 4, lower panel). These data underscore the importance of Production of IL-23 in S3 Epithelial Segments 2 2 THP in regulating the production of select cytokines/chemokines To determine whether the IL-17 surge in THP / kidneys is in the kidney, notably IL-17. due to increased production of IL-23, we measured IL-23 2 2 mRNA and protein in THP / and THP+/+ kidneys using 2 2 Increased IL-17 in THP / Kidneys Is a Major real-time PCR and ELISA, respectively. Figure 6, A and B, Determinant of Systemic Neutrophilia shows a significant increase in IL-23 mRNA and protein in 2 2 2 2 Among the factors differentially increased in THP / kidneys, THP / versus THP+/+ kidneys, respectively. These findings IL-17 is known to be an important activator of granulopoiesis, suggest that activation of the IL-23/IL-17 axis in the kidney is and it is part of the IL-23/IL-17 axis.31–33 In this well recognized regulated by THP. Interestingly, we could not detect IL-23 in system, the cascade is induced by IL-23, which stimulates spe- the serum in either strains of mice (Figure 6C), which could cialized T cells to produce IL-17.31,34 In turn, IL-17 can stimulate imply that induction of IL-23 is limited to the kidney and does granulopoiesis by causing a systemic increase in G-CSF.31,35 Us- not extend systemically. 2 2 ing ELISA, we showed that THP / mice have increased serum Wenextsoughtto determine the sourceofincreasedIL-23in 2 2 IL-17 levels compared with THP+/+ mice (Figure 5A). Todetermine THP / kidneys. Studies in nonkidney tissues suggested that the source of increased IL-17 in the kidney, we performed flow inflammatory cells such as activated macrophages and

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2 2 Figure 4. Cytokine/chemokine multiplex ELISA of the kidneys and livers in THP / versus THP+/+. Graphs show standardized mean differences 2 2 for each analyte between THP / and THP+/+ for kidneys (upper, n=5 each group) and liver (lower, n=5 each group). Analytes are grouped 2 2 based on their biologic function. Asterisk denotes statistical significance between THP / and THP+/+. GMCSF, granulocyte macrophage colony 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. possibly T cells are important producers of IL-23.36,37 There- epithelium is a major source of IL-23 synthesis in the kidney. fore, we performed real-time PCR probing for IL-23 on RNA In addition, S3 segments appear to be the key activator of the extracted from glomeruli, S1–S2 and S3 proximal tubular seg- IL-23/IL-17 axis during THP deficiency. 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- DISCUSSION section (LMD), shown in Supplemental Figures 5 and 6. FACS analysis was used to isolate Mws/DCs and T cells using the In this study, we investigated the role of THP on systemic schema described in Supplemental Figure 7. neutrophil homeostasis. We show that THP deficiency causes Figure 6D shows that in THP+/+ kidneys, IL-23 mRNA was systemic neutrophilia, which is most likely the result of a only detected in S1–S2 segments, albeit at a very low level. In dysregulated increase in renal IL-23/IL-17. We subsequently 2 2 THP / kidneys (Figure 6E), IL-23 mRNA was significantly show that the S3 tubular epithelium is the source of increased upregulated, and was uniquely detected in S3 segments but not IL-23. Toour knowledge, this is the first study showing that the in Mws/DCs, T cells, glomeruli, S1–S2 segments, or TAL seg- kidney regulates granulopoiesis and neutrophil homeostasis. ments. Therefore, our data show that the proximal tubular Therefore, our study expands the key role of the kidney in

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2 2 Figure 5. Effect of IL-17 neutralization on systemic neutrophilia in THP / mice. (A) Bar graphs are means6SEM of the IL-17 level in 2 2 2 2 sera from THP+/+ and THP / mice (n=5 per group) measured with ELISA. (B) Neutrophil counts in THP / mice injected with IL-17 neutralizing antibody or IgG isotype at various time points during treatment (n=5 each group). (C) Serum G-CSF on day 7 after IL-17 neutralization or treatment with IgG isotype. (D and E) Flow cytometry of the kidneys from both groups of mice probing specifically for neutrophils. Representative scatter plots from each group are presented in D, whereas E shows the quantitation of neutrophils shown as 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- 2 2 tralization in THP / significantly reduced serum G-CSF levels, neutrophilia in the periphery and within the kidney. Asterisk denotes statistical significance compared to IgG controls, whereas the symbol # denotes significance compared to Day 0 (P,0.05). Neut, neutrophil. regulating the hematopoietic system to include not only kidney itself is an important source of IL-17. The key role of erythropoiesis through the production of erythropoietin,38 IL-17 in stimulating granulopoiesis and neutrophilia was then but also granulopoiesis through THP-dependent regulation confirmed by in vivo neutralization. Although IL-1b in con- of the IL-23/IL-17 axis. Figure 7 is a summary illustration junction with IL-23 have been reported to stimulate IL-17 outlying the mechanism of THP-regulated granulopoiesis, production,39 there was no differential increase in IL-1b in 2 2 on the basis of the current data. THP / kidneys, suggesting that it does not play a significant The finding that THP deficiency causes systemic neutro- role in inducing IL-17 and granulopoiesis. The increase in 2 2 philia, even without injury is remarkable, and agrees with CXCL9 (in conjunction with IL-17) observed in THP / kid- previous findings by Liu et al. that THP deficiency causes a neys is consistent with recent findings by Paust and colleagues systemic proinflammatory phenotype and splenomegaly.21 To that IL-17 stimulates the expression of CXCL9.40 The fact that a determine whether the kidney is the source of a progranulo- few proinflammatory cytokines/chemokines were decreased in 2 2 poetic factor in the setting of THP deficiency, we used an un- THP / liver (e.g., CXCL2, IL-3, IL-1a, and IL-12p70) could be biased approach with multiplex ELISA for 32 preset cytokines/ reactive to the observed neutrophilia, and the systemic inflam- 2 2 chemokines. Comparing the kidney to the liver enabled us to matory environment induced by THP / kidneys. 2 2 determine that the kidney specifically has an increased level of We attempted to verify the source of IL-17 in THP / kid- IL-17, which is a known activator of granulopoiesis.31,32 The neys. Unfortunately, we could not reliably amplify IL-17 fact that IL-17 is increased in the kidney and the serum, but mRNA in the kidney (using two different primers; Supple- 2 2 not in the liver, in THP / mice strongly supports that the mental Table 1) despite consistent detection of the protein

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Figure 6. Identification of the source of IL-23 synthesis in kidney using LMD and FACS. (A) IL-23mRNA measurements using real-time 2 2 PCR in THP+/+ (reference set as 1) and THP / total kidney extracts (n=5 per group). (B and C) IL-23 protein in the kidney and in the 2 2 serum using ELISA, respectively. Asterisks in A and B denote statistical significance between THP / and THP+/+ (P,0.05). IL-23 could 2 2 not be detected in the serum of either mice strain. (D and E) IL-23 mRNA in specific cell types derived from THP+/+ and THP / kidneys. Glomeruli, S1–S2, S3, and TAL segments were dissected using LMD. Mws/DCs and T cells were obtained using FACS. Total kidney 2 2 from THP / was used as reference sample (set as 1) in all experiments shown in D and E. The # symbol denotes statistical significance 2 2 versus THP+/+ total kidney, whereas the asterisk in E denotes significance compared with THP / total kidney (P,0.05).

using ELISA. This could be due to the extremely short t1/2 and/ IL-17 is downstream from IL-23 in the well defined IL-23/ or low abundance of IL-17 mRNA. Previous studies showed IL-17 axis.31–34,41,42 We showed an increased level of IL-23 2 2 that few populations of T cells are major sources of IL-17 in mRNA and protein in the THP / kidney, which supports experimental GN.40,41 This is consistent with findings from that activation of renal IL-23/IL-17 is an important determinant 2 2 other tissues with chronic inflammation.33 Li et al. also pre- of the observed neutrophilia in THP / mice. Interestingly, it is viously showed that neutrophils are a significant source of thought that kidney Mws/DCs are the major source of IL-23.41 IL-17 during kidney injury.42 Our flow cytometry studies sug- Furthermore, the site of IL-23 production could depend on the gest that increased IL-17+ neutrophils could be a potential disease model.41 Using the combination of LMD and FACS to 2 2 source for increased IL-17 in THP / kidneys. At this time, isolate specific epithelial and immune cells in the kidney, we we cannot completely rule out additional sources of increased show that PTs are a major source of IL-23 compared with renal 2 2 IL-17 in THP / kidneys such as parenchymal or stromal cells,43 Mws/DCs, T cells, glomeruli, or TALs. To our knowledge, this is and this is currently the topic ongoing investigations in the the first study to clearly define a major source of IL-23 produc- laboratory. tion in the kidney using this powerful approach. Our findings

2178 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2172–2182, 2015 www.jasn.org BASIC RESEARCH underscore the importance of proximal tubular epithelium in mentioning that previous reports have described a proinflamma- regulating the IL-23/IL-17 axis in the kidney. The fact that THP tory role for THP.44,45 These studies were based predominantly on deficiency markedly induces IL-23 in S3 segments emphasizes the in vitro or ex vivo data with highly aggregated, and potentially role of THP as a major modulator of IL-23 synthesis and fits with highly immunogenic,46 urinary THP.2 To this date, there is no our previous findings that THP is an important mediator of tu- evidence to suggest that interstitial THP exists in the same highly 2 2 bular cross-talk in the outer medulla.2,5,14 Our current data ex- aggregated form.3 Our in vivo studies with THP / mice dem- pand the importance of THP-S3 interaction beyond the kidney, onstrate that THP has an anti-inflammatory role, which could and suggest that the kidney outer medulla is an important reg- reflect the net outcome of the complex interactions of THP with ulator of systemic neutrophil homeostasis. various cells types within the kidney interstitium, especially a The current data also support our previous conclusions that counterinflammatory effect on epithelial cells.2,5,14 2 2 THP / kidneys are more prone to upregulation of CXCL2 This study may have clinical implications well beyond AKI. (MIP-2 chemokine, neutrophil chemoattractant) during AKI.10,14 Several studies showed that THP levels in the urine and in the 2 2 In fact, we report in this work that THP / kidneys have increased serum decrease with advanced CKD and tubular atro- CXCL2 even at baseline. Collectively, results from this and pre- phy,16,19,20 which is most likely due to decreased expression vious studies will advance our understanding of the pathogenesis by the kidney, as was shown recently by Ledo et al.47 Interest- of neutrophil infiltration during AKI, especially because a state of ingly, recent data from clinical studies also demonstrate that relative THP deficiency in wild-type kidneys is consistently ob- CKD is associated with development of neutrophilia, an in- served in the early stages of AKI.5,17,18 Weproposethatthisrelative creased neutrophil to lymphocyte ratio, and other markers of THP deficiency at the onset of AKI exquisitely sensitizes the kid- systemic inflammation.22,24,25 We propose that the relative ney to neutrophil infiltration by enhancing granulopoiesis, sys- THP deficiency occurring in kidneys with CKD activates the temic neutrophilia and increasing renal CXCL2. The increased IL-23/IL-17 axis, which in turn stimulates granulopoiesis expression of THP that we reported during kidney recovery5 and a systemic inflammatory phenotype. Our study also under- could therefore be essential to halt the influx of neutrophils to scores the need for large-scale clinical studies to better define the the kidney by a dual action on the bone marrow (decreasing pro- dynamic changes in THP levels during the progression of kidney duction) and on the kidney (decreased chemotaxis). It is worth disease. In conclusion, we show that THP defi- ciencystimulates proximalepithelialactivation of the IL-23/IL-17 axis and systemic neutro- philia. Our findings provide novel insights on how the kidney, through uniquely producing THP, can regulate granulopoiesis in the bone marrow and systemic neutrophil homeostasis.

CONCISE METHODS

Mice Animal 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/ 2 2 SvEv THP / ) 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 50 mg/mouse for 6 days. Isotype IgG2a (14-4321- Figure 7. Summary diagram of THP-regulated granulopoiesis. In the kidney, interstitial THP 85; eBioscience) was used as control. Complete released by the TAL exerts an inhibitory effect on the synthesis of IL-23 by S3 proximal tubular blood count analysis was performed on 50 mlof segments in the outer medulla. In the context on THP deficiency, IL-23 is upregulated by S3 heparinized blood using a Hemavet 950FS ana- segments and released within the kidney to act on neutrophils (Neut) and possibly other cells within the kidney to induce IL-17. IL-17 is subsequently released into the systemic circulation, lyzer. where it can stimulate the production of G-CSF. G-CSF acts within the bone marrow to stimulate granulopoiesis and systemic release of excess neutrophils. LT-HSC, long-term he- Immunohistochemical Analyses matopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; MPP, multipotent pro- For GR-1 staining (ab2557; Abcam, Inc.), 4% genitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor. paraformaldehyde perfusion–fixed tissues were

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2 2 subsequently embedded in paraffin and processed for standard his- myeloid progenitor (Lin Sca1 CD117+ CD34+ CD16/32lo), and 2 2 tochemistry. Negative control without primary antibody was used. granulocyte-macrophage progenitor (Lin Sca1 CD117+ CD34+ Cells were counted on high (360 objective) power fields, using five CD16/32hi). fields from each kidney area per slide (two slides per kidney, and five kidneys each group). LMD Sections from each kidney were snap frozen in optimum cutting Flow Cytometry on Kidneys, Livers, and Spleens temperature medium using dry ice and kept at 280°C until use. They Flow cytometry was performed on homogenized, digested tissues as were subsequently cut using a microtome at 8-mm sections on Leica previously described.50 In brief, kidneys or livers were homogenized polyphenylene sulfate membrane slides (catalog no. 11505268). Histo- and digested with collagenase, and subsequently strained using 70-mm chemical staining was performed immediately before dissection and filters. Spleens were minced between two frosted slides and strained. consisted of a series of rapid sequences of the following: (1) 100% Cell counts and viability were done using a Countess (Life Technologies) EtOH for 1 minute; (2) 95% EtOH for 30 seconds; (3) 75% EtOH for automated system. Then, 53106 cells from each sample were used for 30 seconds; (4) 50% EtOH for 30 seconds; (5) water 1, 30 seconds; (6) staining after blocking nonspecific binding with anti-CD16/CD32 water 2, 30 seconds; (7) Histogene Staining Solution (12241-05; Life (14-0161-82; eBioscience). Flow cytometry was done using a BD LSRII Technologies) 100 ml for 40 seconds; (8) water 3, 30 seconds; (9)75% flow cytometer. For absolute cell counting, we used a dual platform EtOH for 30 seconds; (10) 95% EtOH for 30 seconds; (11)100%EtOH approach in which the percentage of cells of interest, out of total live for 30 seconds; and (12) Xylene 90 seconds. Sections were air dried and cells, was determined by flow cytometry. Live cell concentration per immediately taken to a Leica LMD6000 microscope. Dissection was specimen was determined with the Countess automated counter as de- performed at 340 magnification. We dissected 40–60 S3 segments scribed above. Toaccount for tissue weight variability, we normalized by and 80–100 TAL tubules from each section. Three sections were dis- the weight of the corresponding tissue. sected from each kidney/mouse. We used the following anti-mouse fluorochrome-conjugated For dissection of glomeruli and S1–S2 segments, we used immu- mAbs: CD45 (130-091-610; Miltenyi Biotech), Ly6G (560600; BD nofluorescence LMD, using the following sequence of staining: (1)100% Bioscience),CD11b(48-0112-80;eBioscience),and propidiumiodide EtOH for 30 seconds32; (2) 95% EtOH for 20 seconds32; (3) 75% for viability. Of CD45+ cells within the kidney and liver, neutrophils EtOH for 20 seconds32; (4)50%EtOHfor20seconds32; (5)water1, were defined as CD45+,CD11b+,Ly6G+. FlowJo software (version 10; 30 seconds32; (6) water 2, 30 seconds32; (7) staining with FITC- FlowJo LLC) was used for flow analysis and plotting. phalloidin plus 49,6-diamidino-2-phenylindole (molecular probes) Flow cytometry for IL-17 was done on kidney cells that underwent in PBS plus 2%BSA for 3–5minutes;(8) phosphate buffered water ex vivo stimulation for 4 hours with Cell Stimulation Cocktail (plus wash for 30 seconds33; and (9) air dry for 5 minutes. protein transport inhibitors) (00-4975; eBioscience), as described by RNA was extracted using PicoPure RNA kit (12204-01; Life others.41,42 Experiments without cell stimulation did not give a reli- Technologies). Because of the finite RNA yield, RNA was pooled for able IL-17 signal (data not shown). Cells were stained for fixable each group (n=3 mice per group). An additional concentration step viability (65-0864-14; eBioscience), CD45, CD11b, Ly6G, and CD3 was performed using standard isopropanol precipitation, before RT (48-0032; eBioscience) before undergoing intracellular permeabilization/ and real-time PCR. The purity of RNA was verified using established fixation (88-8832; eBioscience) and staining for IL-17 (12-7177-81; markers for each tubular segment within the nephron (Supplemental eBioscience). 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 Flow Cytometry on Bone Marrow additional controls (Supplemental Figure 8), we verified that IL-1b 2 2 Low-density bone marrow cells from both THP+/+ and THP / mice was exclusively expressed in Mw/DCs,44 whereas macrophage colony were washed once with stain wash (PBS, 1% bovine calf serum, and stimulating factor (known to be expressed in myeloid and renal ep- 1% penicillin-streptomycin), followed by antibody staining for 15 minutes ithelium26,53) was detected in S3 segments, TALs, and Mws/DCs. on ice with fluorochrome-conjugated mAbs against the following mark- ers: (1) c-Kit (CD117), Sca-1 (CD34), Flk2 (CD135), IL7Ra (CD127), and FACS Analyses Fc-gR (CD16/32) for progenitor cells; and (2) CD3 (T cells), CD11b FACS was performed using BD FACSAria using the schema shown in (myeloid), CD45R (B cells), Ter119 (erythroid), and Ly6G (neutrophil) Supplemental Figure 7. Cells were isolated from kidneys similar to for differentiated cells. All mAbs were obtained from BD Biosciences, what was described in the flow cytometry section, and were subse- except that CD127 was from eBioscience and anti–Fc-gRwasfromBiol- quently enriched in leukocytes using Lympholyte M (Cedarlane) gra- egend. Cells were washed one more time with stain wash buffer, and data dient followed by magnetic beads directed enrichment of CD45+ cells were acquired on BD LSRII (BD Biosciences). Progenitor cell content was using MACS Separation Columns (130-042-201; Miltenyi Biotech). gated and analyzed (Supplemental Figure 2) as previously described,29,30 To maximize yield, kidneys were pooled from eight mice. Mws/DCs 2 2 accordingtothefollowingdefinitions: LSK (Lin Sca1+ c-Kit+), long- were defined as CD45+, CD11b+,Ly6G .Tcellsweredefined as 2 2 2 2 2 term hematopoietic stem cell (Lin Sca1+ c-Kit+ CD34 CD135 ), mul- CD45+, CD11b ,CD3+,B220 . The following additional anti-mouse– 2 tipotent progenitor (Lin Sca1+ c-Kit+ CD34+ CD135+), short-term conjugated mAbs were used: CD3 (48-0032; eBioscience) and B220 2 2 hematopoietic stem cell (Lin Sca1+ c-Kit+ CD34+ CD135 ), common (17-0452; eBioscience). mRNA extraction from recovered cells was per- 2 lymphoid progenitor (Lin Sca1lo CD117lo CD135hi CD127+), common formed using the PicoPure RNA kit from Life Technologies.

2180 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2172–2182, 2015 www.jasn.org BASIC RESEARCH

ELISA Assays 2. El-Achkar TM, Wu XR: Uromodulin in kidney injury: An instigator, by- A multiplex cytokine/chemokine assay containing 32 prespecified stander, or protector? Am J Kidney Dis 59: 452–461, 2012 analytes (MCYTOMAG-70K-PX32; EMD Millipore) was performed 3. Rampoldi L, Scolari F, Amoroso A, Ghiggeri G, Devuyst O: The redis- 2 2 on kidney and liver lysates from THP+/+ and THP / kidneys (n=5 covery of uromodulin (Tamm-Horsfall protein): From tubulointerstitial nephropathy to chronic kidney disease. Kidney Int 80: 338–347, 2011 per group). Values were initially reported in picograms per milligram 4. Jennings P, Aydin S, Kotanko P, Lechner J, Lhotta K, Williams S, Thakker protein/tissue after normalization to the protein concentration for RV, Pfaller W: Membrane targeting and secretion of mutant uromodulin each tissue. Statistical analysis is described below. In separate experiments, in familial juvenile hyperuricemic nephropathy. J Am Soc Nephrol 18: ELISA for IL-17 and G-CSF (MCYTOMAG-70K; EMD Millipore) on 264–273, 2007 sera from the two strains of mice (n=5 per group) were also performed. 5. El-Achkar TM, McCracken R, Liu Y, Heitmeier MR, Bourgeois S, Ryerse J, Wu XR: Tamm-Horsfall protein translocates to the basolateral domain of We also performed IL-23 ELISA (MCYTOMAG-74K-01) on kidney tis- thick ascending limbs, interstitium, and circulation during recovery from sue extracts and sera from the two strains. acute kidney injury. Am J Physiol Renal Physiol 304: F1066–F1075, 2013 6. Howie AJ, Brewer DB: Extra-tubular deposits of Tamm-Horsfall protein Real-Time PCR in renal allografts. J Pathol 139: 193–206, 1983 Real-time PCR was performed as previously described5 in an Applied 7. Serafini-Cessi F, Malagolini N, Cavallone D: Tamm-Horsfall glycopro- Am J Kidney Dis – Biosystems ViiA7 system using TaqMan Gene Expression Assays (all tein: Biology and clinical relevance. 42: 658 676, 2003 8. Zager RA, Cotran RS, Hoyer JR: Pathologic localization of Tamm- from Applied Biosystems; Supplemental Table 1). All expression was Horsfall protein in interstitial deposits in renal disease. Lab Invest 38: normalized to gluconate dehydrogenase endogenous control and re- 52–57, 1978 ported as fold change compared with control using the DD threshold 9. Köttgen A, Glazer NL, Dehghan A, Hwang SJ, Katz R, Li M, Yang Q, cycle method, according to the manufacturer’s instructions. Gudnason V, Launer LJ, Harris TB, Smith AV, Arking DE, Astor BC, Boerwinkle E, Ehret GB, Ruczinski I, Scharpf RB, Chen YD, de Boer IH, Haritunians T, Lumley T, Sarnak M, Siscovick D, Benjamin EJ, Statistical Analyses Levy D, Upadhyay A, Aulchenko YS, Hofman A, Rivadeneira F, 6 Values of each experimental group are reported as means SEM. For UitterlindenAG,vanDuijnCM,ChasmanDI,ParéG,RidkerPM,Kao most experiments, a two tailed t test was used to examine the differ- WH, Witteman JC, Coresh J, Shlipak MG, Fox CS: Multiple loci associated ence in means for continuous data. A nested ANOVAdesign was used with indices of renal function and chronic kidney disease. Nat Genet 41: for quantitation of GR1+ cells in immunohistochemistry. Statistical 712–717, 2009 significance was determined at the 0.05 significance level. For ELISA 10. El-Achkar TM, Wu XR, Rauchman M, McCracken R, Kiefer S, Dagher PC: Tamm-Horsfall protein protects the kidney from ischemic injury by multiplex analysis, two-sample t tests were used to compare the value decreasing inflammation and altering TLR4 expression. Am J Physiol for each test between the two experimental groups. The mean differ- Renal Physiol 295: F534–F544, 2008 2 2 ence for each test value between THP / versus THP+/+ and its 95% 11. Patel R, McKenzie JK, McQueen EG: Tamm-Horsfall urinary mucopro- confidence interval were then calculated. Because each test was mea- tein and tubular obstruction by casts in acute renal failure. Lancet 1: – sured in different metrics, the values were standardized by using the 457 461, 1964 12. Resnick JS, Sisson S, Vernier RL: Tamm-Horsfall protein. Abnormal lo- raw test value divided by its SEM. The standardized test difference calization in renal disease. Lab Invest 38: 550–555, 1978 fi and 95% con dence interval between the two strains for all the tests 13. Cotran RS, Galvanek E: Immunopathology of human tubulo-interstitial are presented in Figure 4 for kidneys and liver separately. diseases: Localization of immunoglobulins complement and Tamm- Horsfall protein. Contrib Nephrol 16: 126–131, 1979 14. El-Achkar TM, McCracken R, Rauchman M, Heitmeier MR, Al-Aly Z, Dagher PC, Wu XR: Tamm-Horsfall protein-deficient thick ascending ACKNOWLEDGMENTS limbs promote injury to neighboring S3 segments in an MIP-2- dependent mechanism. Am J Physiol Renal Physiol 300: F999–F1007, 2011 We acknowledge the Bioplex, Pathology, and Flow Cytometry cores at 15. Köttgen A, Hwang SJ, Larson MG, Van Eyk JE, Fu Q, Benjamin EJ, Indiana University School of Medicine for assistance with this project, Dehghan A, Glazer NL, Kao WH, Harris TB, Gudnason V, Shlipak MG, and we thank the Koman Tissue Bank for assistance with LMD. We also Yang Q, Coresh J, Levy D, Fox CS: Uromodulin levels associate with a acknowledge Dr. Timothy Sutton for his critical review of this article. common UMOD variant and risk for incident CKD. J Am Soc Nephrol This work was supported by a US Department of Veterans Affairs 21: 337–344, 2010 merit award to T.M.E.-A. 16. 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JClinInvest122: 4519–4532, 2012 Waal-Malefyt R, Hannum C, Bazan JF, Kastelein RA: Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13: 715–725, 2000 38. Fried W: Erythropoietin and erythropoiesis. Exp Hematol 37: 1007– This article contains supplemental material online at http://jasn.asnjournals. 1015, 2009 org/lookup/suppl/doi:10.1681/ASN.2014070664/-/DCSupplemental.

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

IL23 Mm00518984_m1 IL17 (primer 1) Mm00439618-m1 IL17 (primer 2) Mm00439619-m1 SGLT2 Mm00453831_m1 rBAT Mm00486218_m1 NKCC2 Mm00441424_m1 NCC Mm00490213_m1 Nphs2 (Podocin) Mm01292252-m1 AQP2 Mm00437575_m1 MCSF Mm00432686-m1 IL-1β Mm00434228-m1 GAPDH Mm99999915_g1

Supplemental Table 1: TaqMan gene expression assays used in real-time PCR (all from Applied Biosystems) Supplemental Figure legends

Supplemental Figure 1: Flow cytometry for neutrophils in the spleen

Panel A shows a representative scatter plot of flow cytometry analysis of CD45+ cells from THP+/+ and THP-/- spleens (n=5 per group), gated for CD11b and Ly6G.

Quantitation of neutrophils (defined as CD45+, CD11b+, Ly6G+) is shown in panel B.

Asterisk denotes statistical significance between THP+/+ and THP-/- (p<0.05).

Supplemental Figure 2: Gating strategy for bone marrow flow cytometry and analysis

Representative flow cytometry dot plots of bone marrow from THP+/+ and THP-/- mice are shown, along with the corresponding gating controls. The definitions of progenitor cells were discussed in details in the methods sections.

Supplemental Figure 3: Analysis of bone marrow cells using an automated hematology analyzer

Bar graphs are mean± standard error of various types of differentiated leukocytes in the bone marrow of THP+/+ and THP-/- mice, measured using a Hemavet analyzer (N=6 per group). Asterisk denotes statistical significance p<0.05 between groups.

Supplemental Figure 4: Flow cytometry for IL17 producing cells in the kidney

Panel A shows a representative flow cytometry analysis of CD45+ cells from THP+/+

and THP-/- kidneys (n=5 each group), gated for CD3 and IL-17. The percentages shown

within the quadrants (orange, CD3+, IL-17+; Blue, CD3-, IL-17+) represent the group

average ± standard error. Asterisk denotes statistical significance between THP+/+ and

THP-/- (p<0.05). Panel B shows the percentage of neutrophils (defined as CD11b hi,

Ly6G+) within CD3-, IL17+ cells.

Supplemental Figure 5: Laser Micro-dissection (LMD) of S3 segments and Thick

ascending limbs (TAL)

Panels A and B show LMD of S3 and TAL segments in the kidney, respectively.

Verification of the purity of the RNA extracted from each segments was performed using

real-time PCR probing for tubular markers expressed only in specific nephron

segments: SGLT2 for S1 and S2 segments; rBAT for S3 segments, NKCC2 for TAL,

NCC for distal tubules, and Aquaporin 2 (AQP2) for collecting ducts. Total kidney RNA

was used as positive control and set as reference Asterisk denotes statistical

significance from total kidney (p<0.05). The representative data shown is from THP-/-

kidneys, and identical results were obtained in THP+/+.

Supplemental Figure 6: Immuno-Fluoresence LMD (I-LMD) of glomeruli and S1-S2

segments.

Panels A-C and E-G show I-LMD of a glomerulus and S1-S2 segments from the kidney

cortex, respectively. Nuclei were stained blue with DAPI; S1-S2 segments were

identified using Oregon green-phalloidin (green brush border stain, E and F). As a

positive control, we performed real time PCR on the extracted RNA for podocin

(glomerulus specific, panel D) and SGLT2 (S1-S2 specific, panel H). Asterisk denotes

statistical significance compared to total kidney used as reference. The representative

data shown is from THP-/- kidneys, and comparable results were obtained in THP+/+.

Supplemental Figure 7: Gating strategy for FACS

Representative dot plots for the gating strategy used to sort macrophages/dendritic cells

(Mφ/DC) and T cells are shown. Mφ/DC were defined as CD45+, CD11b+, Ly6G-. T cells were defined as CD45+, CD11b-, CD3+, B220-

Supplemental Figure 8: Controls for the LMD-FACS procedures

Panels A and B show real-time PCR for IL1β and MCSF (respectively) performed on

RNA extracted from S3 and TAL (obtained using LMD); Mφ/DC and T cells (obtained

using FACS). Total kidney was used as positive control and set as reference. Asterisk

denotes P<0.05 compared to total kidney, suggesting that the particular cell type is a

major source of the corresponding cytokine or growth factor.