KLF11 deficiency enhances chemokine generation and fibrosis in murine unilateral ureteral obstruction.

Progression of virtually all forms of chronic kidney disease (CKD) is associated with activation of pro-inflammatory and pro-fibrotic signaling pathways. Despite extensive research, progress in identifying therapeutic targets to arrest or slow progression of CKD has been limited by incomplete understanding of basic mechanisms underlying renal inflammation and fibrosis in CKD. Recent studies have identified Kruppel-like transcription factors that have been shown to play critical roles in renal development, homeostasis, and response to injury. Although KLF11 deficiency has been shown to increase collagen production in vitro and tissue fibrosis in other organs, no previous study has linked KLF11 to the development of CKD. We sought to test the hypothesis that KLF11 deficiency promotes CKD through upregulation of pro-inflammatory and pro-fibrogenic signaling pathways in murine unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis. We found that KLF11-deficiency exacerbates renal injury in the UUO model through activation of the TGF-β/SMAD signaling pathway and through activation of several pro-inflammatory chemokine signaling pathways. Based on these considerations, we conclude that agents increase KLF11 expression may provide novel therapeutic targets to slow the progression of CKD.

Introduction

Kruppel-like factors (KLFs) are a family of zinc-finger transcription factors that play important roles in regulation of growth and development, proliferation, and regeneration following injury [1-4]. There are over 17 members of the KLF family which can be divided into three phylogenetic groups: Group 1 (KLF 3, 8, and 12) which serve as transcriptional repressors through interaction with C terminal binding protein; Group 2 (KLF 1, 2, 4, 5, 6, 7) which are, in general, transcriptional activators; and Group 3 (KLF 9, 10, 11, 13, 14, and 16) possess an alpha helical motif that mediates binding to Sin3A and function as transcriptional repressors [5, 6]. Several KLF family members have been shown to mediate fibrosis through interaction with the TGF-β/SMAD3 signaling pathway [7, 8].

Several recent studies have implicated KLF family members in renal pathophysiology. KLFs which have been shown to mediate kidney injury include KLF 2, 4, 5, 6, and 15. Members of the KLF family have been linked to podocyte dysfunction (KLF 6, 15) and renal fibrosis (KLF 4, 5, 6, 15), at least in part through modulation of TGF-β/SMAD3 signaling [5, 9]. These KLFs are involved in a variety of critical physiologic functions, including preservation of the endothelial capillary barrier, prevention of apoptosis, regulation of mitochondrial function, and maintenance of podocyte differentiation [5].

In vitro studies have shown that KLF11 represses gene expression via Sp1-like binding sites and binds either GC-rich or CACC sequences. Although KLF11 downregulates collagen I production [10] and KLF11 deficiency enhances hepatic fibrosis [7], there have been no previous studies linking KLF11 signaling to fibrosis or inflammation in chronic renal injury, an issue that has been identified as an important area for future investigation [5]. Based on observations that KLF11 deficiency may increase inflammation in non-renal experimental systems [11], we sought to test the hypothesis that KLF11 deficiency exacerbates renal damage in the murine Unilateral Ureteral Obstruction (UUO) model of renal fibrosis [12] by promoting a pro-inflammatory and pro-fibrotic phenotype.

Materials and methods

Animals

Mice KLF11 knockout (KLF11 KO) [10, 13] were crossed back into a pure C57BL/6 background for more than 20 generations in our laboratory. Wild type (C57BL6/J) (WT) mice for backcrossing were purchased from the Jackson Laboratory (Bar Harbor, ME). WT mice (n = 19 UUO, n = 11 Sham) and KLF11 KO (n = 18 UUO, n = 12 Sham) mice underwent UUO or Sham surgery at 8–12 weeks of age. UUO surgery was performed by a double ligation in the right ureter at approximately halfway between the kidney and the bladder. For Sham surgery (Control) the right ureter was identified and manipulated without generating any ureteric obstruction (no ligation and no cut in the ureter). Mice were sacrificed at day 9 after ureteral ligation. All the animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) prior to conducting any experiments. These animal procedures were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Histologic and immunohistochemical analysis

Histologic and immunohistochemical analysis was performed on right kidney tissue from mice that underwent UUO or Sham surgery. The kidney tissues were fixed in 10% neutral buffered formalin and processed using standard techniques. 5 μm histological sections were prepared and stained with hematoxylin-eosin (H&E). Renal atrophy was semi quantitatively assessed as the percentage of cortical surface area occupied by atrophic tubules, compared to the entire cortical surface area, according to methods previously established in our laboratory [14, 15]. Immunohistochemical staining was done for anti-F4/80 (1:200, BIO-RAD, Cat. No. MCA497RT), anti-KLF11 (1:1600, Novus Biological, Cat# H00008462-M03), anti-CD3 (1:100, Agilent Dako, Code Number A045), anti-CD68 (1:200, Abcam, Cat# ab125212), anti-CD163 (1:400, Abcam, Cat# ab182422), and anti-CD206 (1:800, Abcam, Cat# ab64693). Sections were stained with Sirius Red to quantitate matrix deposition. Ten random fields per kidney section were examined for each marker (F4/80, CD3, CD68, CD163, CD206 and Sirius Red) and the average of positive areas was expressed as percentage of total analyzed area. All measurements and quantifications were performed in a blinded fashion using NIS elements BR 4.13.00 64-bit image analysis system (Nikon Instruments INC., Melville, NY) at 200 X magnification.

Renal function assay

For exanimating renal function, blood and terminal organ harvest was performed at day 9 after the surgery. The blood levels of Albumin (Photometric, Dye Binding-Bromcresol Green), serum BUN (Urease with GLDH (coupled Enzymes)), Creatinine (CREA) (Enzymatic) and Glucose (Hexokinase G6PDG/UV (enzymatic colorimetric assay) were examined as markers of renal dysfunction.

Real-time PCR based array analysis

Total RNA was extracted from kidney tissues (RNeasy plus Mini kit, Qiagen, Valencia, CA) and quantified by spectrophotometry (NanoDrop Technologies, Wilmington, DE). After the RNA quality was evaluated the RNAs were reverse transcribed and the first-strand cDNA was prepared from total RNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The gene expression was analyzed with RT2 Profiler Inflammatory Response and Autoimmunity PCR Array (Qiagen cat. PAMM-077Z), TGF-β/BMP signaling pathway PCR Array (Qiagen cat. PAMM-035Z) and Fibrosis PCR Array (Qiagen cat. PAMM-120Z) as per manufacturer’s protocol. The gene expression of the UUO samples was calculated comparing each condition with WT-Sham. Data from all conditions were normalized to mean Ct value of Gapdh and Hsp90ab1 genes [16].

RNAseq

The raw RNA sequencing paired-end reads for the samples was processed through the Mayo Clinic RNA-Seq bioinformatics pipeline, MAP-RSeq version 3.1.4 [17]. MAP-RSeq employs the very fast, accurate and splice-aware aligner, STAR [18], to align reads to the reference human genome build hg38. Gene and exon expression quantification was performed using the Subread [19] package to obtain both raw and normalized reads (RPKM–Reads Per Kilobase per Million mapped reads. Finally, comprehensive analyses were run on the aligned reads to assess quality of the sequenced libraries. Using the raw gene counts report from MAP-RSeq, genes that are differentially expressed between the groups was assessed using the bioinformatics package DEseq. Genes found different between the groups will be reported with their magnitude of change (log2 scale).

Statistical analysis

Data are presented as means ± SEM. One-way analysis of variance (ANOVA) or t-test were performed for comparison between groups using ANOVA followed by Student’s t-test. Differences between the groups were considered statistically significant when p ≤0.05. Statistical analyses and heatmaps were performed with GraphPad Prism 8 XML Project version 8.2.1 (GraphPad Software, La Jolla, CA). The Principal Component Analysis (PCA) was calculated using ClustVis 2.0 (https://biit.cs.ut.ee/clustvis/).

Results

Effect of KLF11 deficiency on basal gene expression of markers of inflammation, fibrosis, and TGF-β/BMP pathway

There were no significant morphologic differences identified between the WT and KLF11 KO-Sham kidneys. In particular, the glomeruli and tubules were of normal size, with no significant interstitial fibrosis or tubular atrophy. Nevertheless, Profiler PCR Array identified significant differences in basal expression of several members of the TGF-β and BMP superfamilies. A summary of genes differentially expressed in KLF11 KO-Sham and WT-Sham is provided in Tables 1 and 2, S1 and S2 Tables (significant differences designated by (a)).

Table 1

Differentially expressed genes in the TGF-β/BMP pathway and fibrotic response in Sham mice compared to UUO mice.

	WT-Sham	KLF11 KO-Sham	WT-UUO	KLF11 KO-UUO
BMP Family (Bone Morphogenetic Proteins)
Bmp1	1±0.15	1.9±0.49 (a)ns	7.9±0.99 (b) ****	13±1.2 (c) ****	(d) **
Bmp3	1±0.094	1.4±0.3 (a)ns	3.1±0.43 (b) *	5±0.67 (c) ****	(d) *
Bmp5	1±0.16	9.3±2.7 (a) *	1.5±0.21 (b)ns	3.7±1 (c) *	(d) *
Bmp7	1±0.074	1.1±0.061 (a)ns	0.44±0.039 (b) ****	0.58±0.05 (c) ****	(d) *
Bmpr1a	1±0.078	1.6±0.2 (a) *	2±0.2 (b) *	3.7±0.33 (c) ****	(d) ***
Bmpr1b	1±0.18	1.9±0.35 (a) *	1.8±0.25 (b)ns	3.4±0.32 (c) **	(d) **
Bmpr2	1±0.091	2.1±0.27 (a) **	2.3±0.29 (b)ns	4.5±0.55 (c) ***	(d) **
Cell Adhesion Molecules
Itga1	1±0.12	0.94±0.082 (a)ns	2±0.15 (b) ****	2.5±0.092 (c) ****	(d) *
Itga3	1±0.098	1.4±0.35 (a)ns	3.4±0.3 (b) ****	5±0.46 (c) ****	(d) **
Itgav	1±0.081	1.1±0.079 (a)ns	2.8±0.2 (b) ****	3.4±0.2 (c) ****	(d) *
Cell cycle progression
Cdkn1b	1±0.092	1.5±0.14 (a) **	1.8±0.16 (b)ns	3±0.34 (c) ***	(d) **
ECM Remodeling Enzymes
Plau	1±0.11	1.4±0.22 (a)ns	1.3±0.089 (b)ns	2.3±0.37 (c) *	(d) *
Plg	1±0.27	1.8±0.57 (a)ns	0.78±0.11 (b)ns	2.7±0.77 (c)ns	(d) *
Serpina1a	1±0.3	0.031±0.0056 (a) *	1.1±0.2 (b)ns	0.12±0.025 (c)ns	(d) ***
Timp3	1±0.053	1.4±0.098 (a) **	0.59±0.05 (b) ***	0.85±0.033 (c) ****	(d) ***
Timp4	1±0.098	1.6±0.5 (a)ns	0.68±0.071 (b)ns	1.2±0.2 (c)ns	(d) *
Growth Factors
Hgf	1±0.082	0.82±0.13 (a)ns	3.9±0.27 (b) ****	4.7±0.27 (c) ****	(d) *
Pdgfa	1±0.1	1.2±0.18 (a)ns	3.5±0.32 (b) ****	4.7±0.29 (c) ****	(d) *
Pdgfb	1±0.078	1.3±0.13 (a)ns	6±0.49 (b) ***	10±1.2 (c) ****	(d) **
SMAD Family
SMAD1	1±0.096	1.2±0.16 (a)ns	3.6±0.35 (b) ****	5.5±0.45 (c) ****	(d) **
SMAD2	1±0.03	1.4±0.11 (a) **	2.4±0.16 (b) ***	4.3±0.34 (c) ****	(d) ****
SMAD3	1±0.088	1.3±0.16 (a)ns	2.6±0.2 (b) **	4±0.52 (c) ****	(d) *
SMAD4	1±0.062	1.4±0.2 (a)ns	1.8±0.12 (b) *	2.6±0.24 (c) ****	(d) **
SMAD5	1±0.088	1.3±0.16 (a)ns	2.6±0.2 (b) ***	3.6±0.38 (c) ****	(d) *
Smurf1	1±0.064	1.5±0.27 (a)ns	3.4±0.37 (b) ***	5±0.52 (c) ****	(d) *
TGF-β Superfamily Members
Acvr1	1±0.099	1.5±0.22 (a)ns	3.1±0.3 (b) **	4±0.28 (c) ****	(d) *
Acvr2a	1±0.087	1.7±0.3 (a) *	1.6±0.19 (b)ns	2.8±0.29 (c) *	(d) **
Acvrl1	1±0.075	1.2±0.17 (a)ns	2.6±0.33 (b) **	3.8±0.45 (c) ****	(d) *
Lefty1	1±0.12	1.4±0.13 (a) *	3.6±0.37 (b)**	7.2±0.88 (c) ****	(d) **
Ltbp1	1±0.12	1.2±0.21 (a)ns	2.3±0.28 (b) *	3.8±0.41 (c) ****	(d) **
Ltbp2	1±0.17	18±11 (a)ns	23±4.9 (b)ns	42±6 (c) *	(d) *
Ltbp4	1±0.085	0.88±0.15 (a)ns	2.3±0.28 (b) *	3.3±0.39 (c) ****	(d) *
TGF-β1	1±0.06	1.1±0.098 (a)ns	7.1±0.65 (b) ****	9.3±0.58 (c) ****	(d) *
TGF-β2	1±0.26	2.3±0.71 (a)ns	5.2±0.63 (b) **	12±1 (c) ****	(d) ****
TGF-β3	1±0.12	3.4±1.6 (a)ns	13±2.1 (b) ***	20±2.2 (c) ****	(d) *
TGF-βi	1±0.06	1±0.088 (a)ns	8.1±0.82 (b) ****	10±0.44 (c) ****	(d) *
TGF-βr1	1±0.051	1.6±0.19 (a) **	3.4±0.3 (b) **	6.7±0.71 (c) ****	(d) ***
TGF-βr2	1±0.079	1.6±0.18 (a) **	5.7±0.55 (b) ***	8.7±1.1 (c) ****	(d) *
TNF receptor superfamily
Tnf	1±0.22	1.8±0.69 (a)ns	23±2.5 (b) ****	33±3.6 (c) ****	(d) *
Tnfsf10	1±0.065	2±0.4 (a)ns	1.5±0.21 (b)ns	3.1±0.33 (c)ns	(d) ***
Transcription Factors
Akt1	1±0.086	1.2±0.097 (a)ns	2.7±0.2 (b) ****	3.4±0.17 (c) ****	(d) *
Junb	1±0.12	1.1±0.36 (a)ns	13±1.1 (b) ****	2.7±0.75 (c)ns	(d) ****
Nfkb1	1±0.064	1±0.098 (a)ns	4.2±0.33 (b) ****	5.3±0.24 (c) ****	(d) *
Runx1	1±0.11	1.3±0.3 (a)ns	29±4.1 (b) ****	54±4.9 (c) ****	(d) **
Sp1	1±0.064	1.1±0.067 (a)ns	1.5±0.11 (b) *	2.3±0.17 (c) ****	(d) **
Stat1	1±0.22	0.77±0.063 (a)ns	2.3±0.2 (b)ns	3.7±0.6 (c) ****	(d) *
Stat6	1±0.1	1.2±0.11 (a)ns	2.2±0.21 (b) ***	3.5±0.29 (c) ****	(d) **
Tsc22d1	1±0.099	2.4±0.47 (a) *	3.8±0.35 (b) *	5.9±0.91 (c) ***	(d) *
ECM Structural Constituents
Col1a2	1±0.057	1.1±0.23 (a)ns	17±2.4 (b) ***	26±3.6 (c) ****	(d) *
Other Genes
Emp1	1±0.13	2.7±0.79 (a) *	9.2±1.4 (b) ***	15±1.7 (c) ****	(d) *

Gene expression analysis was performed employing the pathway Detect RNA array. The table showed the differentially expressed genes by RTPCR after 9 days of Surgery Sham/UUO. Statistical significance was determined by Student’s t-test. (a) KLF11 KO-Sham compared with WT-Sham, (b) WT-UUO compared with WT-Sham, (c) KLF11 KO-UUO compared with KLF11 KO-Sham, (d) KLF11 KO-UUO compared with WT-UUO. Values are means ± SEM. p values ≤0.05 were considered as significant (GraphPad Software, La Jolla, CA). Statistically significant values are highlighted in bold

*p ≤ 0.05

**p ≤ 0.01

*** p ≤ 0.001

****p ≤ 0.0001, ns: not significant.

Table 2

Differentially expressed genes of inflammatory response in KLF11 KO mice compared to WT.

	WT-Sham	KLF11 KO-Sham	WT-UUO	KLF11 KO-UUO
Complement components/regulation
C3	1±0.14	0.77±0.24 (a)ns	44±6.1 (b) ****	70±7 (c) ****	(d) *
C4b	1±0.4	0.33±0.068 (a)ns	3.6±0.67 (b)ns	7.7±1 (c) ****	(d) **
Chemokine (CC)
Ccl2	1±0.14	0.67±0.15 (a)ns	67±9.7 (b) ****	103±11 (c) ****	(d) *
Ccl5	1±0.21	0.71±0.17 (a)ns	9.8±1.5 (b)ns	30±9.6 (c) **	(d) *
Ccl7	1±0.27	0.71±0.24 (a)ns	156±26 (b) **	259±39 (c) ****	(d) *
Ccl8	1±0.42	0.21±0.064 (a)ns	21±3.7 (b) *	44±7.4 (c) ****	(d) *
Ccl12	1±0.34	0.32±0.038 (a)ns	32±3.5 (b) ****	49±7 (c) ****	(d) *
Ccl17	1±0.25	0.74±0.17 (a)ns	34±5.3 (b) ****	49±4.3 (c) ****	(d) *
Chemokine Receptors (CC)
Ccr2	1±0.22	0.6±0.064 (a)ns	20±2.4 (b) ****	28±1.3 (c) ****	(d) *
Chemokine (CXC) ligands and receptors
Cxcl1	1±0.24	2.3±0.45 (a) *	117±18 (b) ****	183±14 (c) ****	(d) *
Cxcl2	1±0.43	0.58±0.093 (a)ns	634±157 (b) ***	246±31 (c)ns	(d) *
Interleukin
Il13	1±0.15	2.3±0.69 (a)ns	3.3±0.6 (b) *	5.4±0.57 (c) **	(d) *
Il18	1±0.11	1.4±0.099 (a) *	2.5±0.28 (b) ***	3.7±0.33 (c) ****	(d) *
Il6	1±0.26	0.58±0.34 (a)ns	97±34 (b)ns	15±2.8 (c)ns	(d) *
Il23a	1±0.22	1.5±0.32 (a)ns	3.8±0.62 (b)ns	6.6±1.2 (c) ***	(d) *
Interleukin Receptor
Il10rb	1±0.096	1.2±0.14 (a)ns	1.9±0.13 (b) ****	2.2±0.088 (c) ****	(d) *
Toll-like receptor
Tlr1	1±0.13	0.95±0.18 (a)ns	12±1.5 (b) ****	17±1.7 (c) ****	(d) *
Tlr9	1±0.15	0.69±0.13 (a)ns	9±0.99 (b) ****	13±1.5 (c) ****	(d) *
Other Immune response members
Cd14	1±0.069	1.8±0.28 (a) *	24±2.9 (b) **	59±8 (c) ****	(d) ***
Cd40	1±0.067	1±0.17 (a)ns	7±0.82 (b) ****	12±1.1 (c) ****	(d) **
FasL	1±0.15	0.86±0.2 (a)ns	4.2±0.51 (b) **	7.9±1.1 (c) ****	(d) **
Lta	1±0.41	0.34±0.078 (a)ns	1.6±0.27 (b)ns	2.7±0.47 (c) **	(d) *
Ltb	1±0.089	0.76±0.21 (a)ns	9.6±1.4 (b) **	17±2.6 (c) ****	(d) *
Ly96	1±0.12	0.81±0.069 (a)ns	1.6±0.11 (b) **	1.9±0.039 (c) ****	(d) **
Ripk2	1±0.052	1.4±0.18 (a)ns	2±0.13 (b) **	2.6±0.32 (c) **	(d) *

Gene expression analysis was performed employing the pathway Detect RNA array. The table showed the differentially expressed genes by RTPCR after 9 days of Surgery UUO. Statistical significance was determined by Student’s t-test. (A) KLF11 KO-UUO compared with WT-UUO. p values ≤0.05 were considered as significant (GraphPad Software, La Jolla, CA). (B) List of genes differentially expressed between WT-Sham vs WT-UUO and KLF11 KO-Sham vs KLF11 KO-UUO but not differently expressed between WT-UUO vs KLF11 KO-UUO. Statistically significant values are highlighted in bold

*p ≤ 0.05

**p ≤ 0.01

*** p ≤ 0.001

****p ≤ 0.0001, ns: not significant.

Significantly upregulated genes in KLF11 KO-Sham mice included members of the BMP Family (Bmp5, Bmpr1a, Bmpr1b, Bmpr2), Cell cycle progression (Cdkn1b), ECM Remodeling Enzymes (Mmp1a, Timp3), Growth Factors (Gdf7, Igf1), SMAD Family (SMAD2, SMAD7), TGF-β Superfamily Members (Acvr2a, Chrd, Inha, Lefty1, TGF-βr1, TGF-βr2), and Transcription Factors (Atf4, Id2, Tsc22d1), Bcl2, Emp1, Fst. Conversely, expression of Serpina1a (ECM Remodeling Enzyme) was significantly decreased in KLF11 KO-Sham mice, compared to WT-Sham mice (Table 1 and S1 Table). Expression of several members of the immune response family were downregulated in KLF11 KO-Sham mice, compared to WT-Sham mice, including the Chemokine (CC) (Ccl3, Ccl4, Ccl19, Ccl22), Chemokine Receptors (CC) (Ccr7) and Itgb2, whereas several members of the Chemokine (CXC) ligands (Cxcl1, Cxcl11), Interleukin (Il13, Il18) and Cd14 were upregulated (Table 2 and S2 Table).

Genetic inactivation of KLF11 increases renal injury in UUO model

Other KLF family members have been linked to podocyte dysfunction and renal fibrosis but the role of KLF11 in mediating renal inflammation and fibrosis has not been established. To assess the role of KLF11 in renal injury, Unilateral Ureteral Obstruction (UUO) was performed in WT (n = 19) and KLF11 KO mice (n = 18), and kidneys were harvested 9 days after the surgery. In Sham mice, the ureter was localized and manipulated without ureteral ligation. Immunohistochemical localization of KLF11 in Sham and UUO kidney is shown in Fig 1. There was minimal staining for KLF11 in Sham mice. In WT mice subjected to UUO, there was strong nuclear staining predominantly in tubular epithelial cells. Focal positive staining of glomerular podocytes was observed (Fig 1A). As expected, there was minimal staining for KLF11 in the KLF11 KO mice. A heatmap showing relative expression of other KLF family members in Sham and UUO mice is shown in Fig 1B. KLF14 and, to a lesser extent, KLF16 expression was increased in KLF11 KO-UUO mice compared to WT-UUO mice. Several other KLF family members (KLF 2, 4, 5, 6, and 11) showed lower expression in KLF11 KO-UUO mice compared to WT-UUO mice (Fig 1B).

Fig 1

Gene expression of KLF Family members in the murine unilateral ureteral obstruction (UUO) model.

(A) Representative histological images of KLF11-stained kidney sections at 400 X magnification, showing WT and KLF11 KO at 9 days after the UUO/Sham surgery. Tubular epithelial cell nuclei show strong positive staining (brown). Glomerulus (arrow, inset) shows focal positive staining within visceral epithelial cells. Scale bar represents 100 microns. (B) Heatmap. Color scale shows high and low expressions as red and green, respectively.

Tubular atrophy was semi-quantitatively assessed, in a blinded fashion, as the percentage of atrophic tubules over the entire cortical surface area, as previously described by us [14, 15]. Tubular atrophy was significantly higher in KLF11 KO mice subjected to UUO (KLF11 KO-UUO) compared to wild type mice subjected to UUO (WT-UUO) (68% vs 53%, p ≤ 0.001) (Fig 2B). No significant differences were observed in renal function parameters (Albumin, Blood Urea Nitrogen, Creatinine, Glucose) between WT-UUO and KLF11 KO-UUO (S3 Table).

Fig 2

KLF11 deficiency exacerbates renal damage in the murine unilateral ureteral obstruction (UUO) model.

(A) Representative histological images of H&E-stained kidney sections at 200 X magnification, showing WT and KLF11 KO at 9 days after the UUO/Sham surgery. Scale bar represents 100 microns. (B) Semiquantitative analysis of tubular atrophy. ***p ≤ 0.001. Values are means ± SEM.

KLF11 deficiency increases renal fibrosis following UUO

We performed quantitative histopathologic analysis of fibrosis in KLF11 KO and WT mice subjected to UUO in Sirius Red stained slides. There were no differences in the percentage of cortical surface area staining positively for Sirius Red in the KLF11 KO-Sham (0.81±0.28) vs WT-Sham controls (0.8±0.26) (Fig 3A and 3B). Interstitial fibrosis was significantly greater in KLF11 KO-UUO (2.4±0.32) mice, compared to WT-UUO mice (1.3±0.17, p = 0.0041) (Fig 3B).

Fig 3

Collagen deposition and expression is higher in the obstructed kidney of KLF11 KO-UUO mice compared to WT-UUO mice.

(A) representative histological images from UUO mice stained with Sirius Red (SR) at 200 X magnification, showing increased fibrosis in the obstructed kidneys of KLF11 KO-UUO comparing with WT-UUO. Scale bar represents 100 microns. (B) Quantitative analysis of the percent cortical surface area staining positively for Sirius Red (extracellular matrix). **p ≤ 0.01. Values are means ± SEM.

KLF11 deletion leads to increased expression of TGF-β/BMP and other pro-fibrotic genes after UUO

We sought to identify differentially regulated genes related to the TGF-β/BMP signaling pathway and fibrosis in KLF11 KO-UUO mice compared to WT-UUO mice. Heatmaps providing a summary of differentially regulated genes in WT-Sham, KLF11 KO-Sham, WT-UUO, and KLF11 KO-UUO mice are shown in Fig 4A and 4B. Table 1 shows the classification of gene expression between: UUO groups where the expression of KLF11 KO-UUO genes were statistically different than WT-UUO (designated (d)); between UUO and Sham groups, designated (b) for a significant change in the expression in WT-UUO vs WT-Sham and designated (c) where the gene expression in KLF11 KO-UUO was significantly different than KLF11 KO-Sham; and between Sham groups for significant differences between the KLF11 KO-Sham and WT-Sham (designated (a)).

Fig 4

Analysis of differentially expressed genes of TGF-β/BMP/Fibrotic pathways between the KLF11 KO vs WT (Sham and UUO).

Heatmap for (A) TGF-β/BMP pathway (B) Fibrotic response were generated using GraphPad Prism version 8.2.1. Color scale shows high and low expressions as red and green, respectively.

As expected from the Sirius Red results (Fig 3B), the expression of Col1a2 was significantly higher in KLF11 KO-UUO mice compared to WT-UUO mice (Table 1). Several transcription factors that regulate TGF-β signaling and fibrosis, including Akt1, Nfkb1, Runx1, Sp1, Stat1, Stat6 and Tsc22d1 were significantly upregulated in the KLF11 KO-UUO mice. Most members of the BMP Family (Bmp1, Bmp3, Bmp5, Bmp7, Bmpr1a, Bmpr1b, Bmpr2), members of the TGF-β Superfamily Members (TGF-β1, TGF-β2, TGF-β3, TGF-βi), TGF-β receptors (Acvr1, Acvr2a, Acvrl1, TGF-βr1, TGF-βr2), SMAD Family (SMADs 1–5 and Smurf1), Lefty1, Ltbp1, Ltbp2, Ltbp4, Cell Adhesion Molecules (Itga1, Itga3, Itgav), Cell cycle progression (Cdkn1b), ECM Remodeling Enzymes (Plau, Plg), Growth Factors (Hgf, Pdgfa, Pdgfb), TNF receptor superfamily (Tnf, Tnfsf10), and Emp1 were more highly expressed in KLF11 KO-UUO mice compared to WT-UUO mice. Conversely, Junb, Serpina1a and Timp3 expression was significantly lower in KLF11 KO-UUO mice than WT-UUO mice (Table 1).

The expression of BMP Family (Bmp2), Cell Adhesion Molecules (Itgb1, Itga2, Itgb3, Itgb5, Itgb6), Cell cycle progression (Cdkn1a, Gadd45b), ECM Remodeling Enzymes (Lox, Mmp2, Mmp3, Mmp14, Plat, Serpine1, Serpinh1, Timp1, Timp2), Growth Factors (Edn1, Tdgf1), Pro-fibrotic genes (Acta2, Ctgf), SMAD Family (SMAD7), TGF-β Superfamily Members (Cav1, Chrd, Eng, Grem1, Inhba, TGF-β1i1, Tgif1, Thbs1, Thbs2), Transcription Factors (Cebpb, Fos, Jun, Myc, Snai1, Sox4), ECM Structural Constituents (Col1a1, Col3a1) and Bcl2, were significantly higher in WT-UUO and KLF11 KO-UUO compared with the respective Shams, but there were no significant differences between KLF11 KO-UUO and WT-UUO mice (S1 Table).

Conversely, expression of Bmp7, Egf, Herpud, Mmp1a, Vegfa, Timp3 was significantly lower in the UUO (WT and KLF11 KO) compared to Sham (WT and KLF11 KO) (Table 1 and S1 Table).

Macrophage influx increased in KLF11 KO mice compared to WT mice following UUO

F4/80 Macrophages

Macrophage influx was estimated as the percentage of cortical surface area staining positively for the macrophage marker F4/80 (F4/80+). F4/80+ macrophages were seen in low level in WT-Sham and KLF11 KO-Sham renal cortex. The average expression at day 9 of F4/80+ increased in the renal cortex of UUO compared with Sham. The infiltration of F4/80 increased 35 times in the WT-UUO (2.7±0.36) compared with WT-Sham (0.078±0.024) and more than 47 times in the KLF11 KO-UUO (4.7±0.71) compared with KLF11 KO-Sham (0.099±0.018) (Fig 5B). On day 9, the expression of F4/80+ macrophages were significantly increased in the KLF11 KO-UUO compared with WT-UUO (Fig 5B).

Fig 5

KLF11 deficiency is associated with an increase in F4/80+ Macrophage influx after UUO.

Percent of cortical surface area staining positively for anti-F4/80 antibody. (A) representative histological images from Sham and UUO mice stained with anti-F4/80 antibody (F4/80) at 200 X magnification, showing increased staining in the obstructed kidneys of KLF11 KO-UUO compared toWT-UUO. Scale bar represents 100 microns. (B) Quantitative analysis of the percent cortical surface area staining positively for F4/80. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Values are means ± SEM.

CD206 macrophages

It is recognized that “alternatively activated” M2 macrophages can mediate tissue repair as well as fibrosis. We therefore sought to determine whether UUO was associated with increased M2 macrophage influx, employing the immunohistochemical marker CD206. Only a few CD206+ macrophages were seen in the Sham controls of WT and KLF11 KO (Fig 6A). The number of CD206+ macrophages in the renal cortex was significantly increased in KLF11 KO-UUO (1.2±0.18) compared with the WT-UUO (0.69±0.099, p = 0.0302) (Fig 6B). The levels of CD206 also increased in the renal medulla but not differences were observed between WT-UUO and KLF11 KO-UUO.

Fig 6

KLF11 deficiency is associated with increased CD206+ macrophage influx after UUO.

Percent of cortical surface area staining positively for anti-CD206 antibody (A) representative histological images from Sham and UUO mice stained with anti-CD206 antibody (CD206) at 200 X magnification, showing increased staining in the obstructed kidneys of KLF11 KO-UUO comparedto WT-UUO. Scale bar represents 100 microns. (B) Quantitative analysis of the percent cortical surface area staining positively for CD206. *p ≤ 0.05; Values are means ± SEM.

We found no significant differences in macrophages staining positively for the M1 marker CD163 between WT-UUO and KLF11 KO-UUO mice. Furthermore, we found no significant differences in CD3+ T cell infiltration between the WT-UUO and

KLF11 deletion leads to increased production of pro-Inflammatory cytokines after UUO surgery

Given that KLF11 KO-UUO mice showed increased macrophage influx compared with WT-UUO mice (Figs 5 and 6), we analyzed differentially regulated pro-inflammatory genes employing the Profile PCR array. A heatmap of differentially regulated genes in WT-Sham, KLF11 KO-Sham, WT-UUO, and KLF11 KO-UUO is shown in Fig 7A. Table 2 shows the classification of gene expression between: UUO groups where the expression of KLF11 KO-UUO genes were statistically different than WT-UUO (designated (d)); between UUO and Sham groups, designated (b) for a significant change in the expression in WT-UUO vs WT-Sham and designated (c) where the gene expression in KLF11 KO-UUO was significantly different than KLF11 KO-Sham; and between Sham groups for significant differences between the KLF11 KO-Sham and WT-Sham (designated (a)). We found that expression of Complement components/regulation (C3, C4b), Chemokine (CC) (Ccl2, Ccl5, Ccl7, Ccl8, Ccl12, Ccl17), Chemokine Receptors (CC) (Ccr2), Chemokine (CXC) ligands (Cxcl1, Cxcl2), Interleukin (Il13, Il6, Il18, Il23a), Interleukin Receptors (Il10rb), Toll-like receptor (Tlr1, Tlr9), Cd14, Cd40, FasL, Lta, Ltb, Ly96 and Ripk2 was significantly upregulated in KLF11 KO-UUO compared to WT-UUO mice. We also found that the expression of Complement components/regulation (C3ar1), Chemokine (CC) (Ccl4, Ccl11, Ccl19, Ccl20, Ccl22), Chemokine Receptors (CC) (Ccr1, Ccr3, Ccr7), Chemokine (CXC) ligands (Cxcr4, Cxcl5), Interleukin (Il1b, Il7), Interleukin Receptors (Il1r1, Il1rap, Il1rn, Il6ra, Il23r), Toll-like receptor (Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tirap), Bcl6, Csf1, Itgb2, Kng1, Myd88, and Nos2 were upregulated in WT-UUO and KLF11 KO-UUO compared with WT-Sham and KLF11 KO-Sham respectively. The Principal Component Analysis of the expression of genes involved in the TGF-β/BMP/Fibrotic/Inflammatory pathways indicates there are differences between the genotypes KLF11 KO-UUO and WT-UUO but not between KLF11 KO-Sham and WT-Sham (Fig 7B).

Fig 7

Heatmap of the differential gene expression of the inflammatory response.

Gene expression of the Inflammatory response for WT-Sham, WT-UUO, KLF11 KO-Sham, KLF11 KO-UUO was measured, compared, and plotted in a Heatmap (A). Color scale shows high and low expressions as red and green, respectively. (B) PCA plot. 55.5% (PC1) and 8.6% (PC2) of the total variance (n = 33 data points). SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 (PC1) and principal component 2 (PC2). Prediction ellipses are such that with probability 0.95, a new observation from the same group will fall inside the ellipse. Ellipses and shapes show clustering of the samples. Sham WT (n = 8), UUO WT (n = 9), Sham KLF11 KO (n = 6), UUO KLF11 KO (n = 9).

Discussion

KLF proteins have been recognized as critical mediators of physiologic and pathophysiologic functions in many organ systems. Members of the KLF family have been linked to the pathogenesis of diabetes, obesity, inflammation, cancer, and cardiovascular disease [8, 20–22]. Although several KLF proteins have been implicated in the pathogenesis of renal disease, a potential role for KLF11 has not previously been established.

KLF KO mice are phenotypically normal, are fertile, and have normal lifespans [13]. In humans, amino acid changes in KLF11 are associated with maturity onset diabetes of the young type VII, whereas complete inactivation of this pathway by the -331-insulin mutation causes neonatal diabetes mellitus. KLF11 regulates expression of metabolic genes via an evolutionarily conserved protein interaction domain functionally disrupted in maturity onset diabetes of the young [23]. Although mutations in the KLF11 gene have been linked to maturity onset diabetes of the young type VII [9], KLF11 KO mice have decreased circulating insulin levels and increased insulin sensitivity, but do not develop overt diabetes [24, 25].

In the UUO model, ureteral obstruction leads to pressure-induced atrophy of tubular epithelial cells, leading to interstitial inflammation and fibrosis. Immunohistochemical stains for KLF11 showed relatively weak staining in WT sham mice. There was strong immunostaining for KLF11 in WT mice subjected to UUO, predominantly within tubular epithelial cells, the primary target in UUO.

We found no significant renal histopathologic alterations in the KLF11 KO Sham mice. In particular, there was no significant tubular atrophy, interstitial fibrosis, or interstitial inflammation noted. Despite the normal morphology, expression of several TGF-β/BMP family members, including TGF-βr1, TGF-βr2, and SMAD3 were higher in the KLF11 KO-Shams than WT-Shams.

We sought to test the hypothesis that KLF11 regulates inflammation and fibrosis in unilateral ureteral obstruction, a well-established model of renal fibrosis. We found that renal atrophy was more severe in KLF11 KO mice subjected to UUO, comparted to WT mice with UUO. Renal atrophy was associated with a significant influx of F4/80+ macrophages and CD206+ macrophages (a marker for M2 macrophages). There were no significant differences in the extent of CD163+ cells or CD3+ T cells between KLF11 KO-UUO mice and WT-UUO mice. Expression of pro-inflammatory cytokines was higher in KLF11 KO-UUO mice compared to WT-UUO mice (Table 2). Deposition of extracellular matrix was 1.7-fold higher in KLF11 KO-UUO mice, compared to WT-UUO mice, as assessed by Sirius Red staining. Expression of pro-fibrotic genes and genes associated with the TGF-β/BMP pathway were significantly higher in KLF11 KO-UUO mice compared to WT-UUO mice (Table 1). Based on the strong and diffuse staining of tubular epithelial cells in WT mice subjected to UUO, we hypothesize that KLF11 deficiency may exacerbate tubular injury in response to ureteric obstruction. Support for the notion that tubular epithelial cells can initiate pro-inflammatory and pro-fibrotic signaling pathways has been obtained in other models of renal injury. In a murine model of renovascular hypertension, we found that tubular epithelial cells in the stenotic kidney strongly expressed Ccl2, a potent monocyte chemoattractant factor, within 24 hours of surgery to establish renal artery stenosis [26]. At this time point, no significant interstitial fibrosis, tubular atrophy, or interstitial inflammation was observed, indicating that, similar to ureteric obstruction, injured tubular epithelial cells can initiate proinflammatory and profibrotic pathways. We propose that KLF11 serves as a negative regulator of such pathways in injured tubular epithelial cells.

Although a role for KLF11 in the kidney has not previously been established, there is ample evidence that KLF11 plays a critical role in regulation of inflammation and fibrosis in other organs. For example, KLF11 overexpression inhibits cardiac hypertrophy and fibrosis in mice subjected to the thoracic aortic constriction model of cardiac hypertrophy [27]. In a ferric chloride induced thrombosis model, occlusion time was significantly reduced in KLF11 KO mice. Bone marrow transplantation did not correct this phenotype, indicating that vascular KLF11 inhibits arterial thrombosis [28]. Of note, KLF11 deficiency is associated with endothelial cell activation and production of pro-inflammatory molecules [11]. KLF11 directly regulates IL-6 in the brain. Knockdown of KLF11 attenuated hypoxia/regeneration injury in cardiac myocytes [29]. Knockdown of KLF11 reduced apoptosis, caspase3, and cytochrome c and mitochondrial damage. On the other hand, genetic deletion of KLF11 aggravates ischemic brain injury [30]. KLF11 KO mice were associated with progressive fibrosis in a murine model of endometriosis [31]. KLF11 is decreased in endometriosis lesions. Loss of KLF11 mediated repression of Col1a1 expression resulted in increased fibrosis [10]. KLF11 recruited SIN3A/HDAC (histone deacetylase) resulting in Col1a1 promoter deacetylation and repression. TGF-βr1 inhibitor and HAT inhibitor inhibits KLF11 signaling.

KLF11 is a TGF-β inducible immediate early gene (TIEG) and promotes the effects of TGF-β on cell growth by influencing the TGF-β-SMAD signaling pathway and regulating transcription of genes that induce either cell cycle arrest or apoptosis [32].

Of note, we found that Junb is markedly downregulated in KLF11 KO-UUO mice, compared to WT-UUO mice (Table 1). C-Jun and Junb are components of the AP-1 family of transcription factors. Overexpression of Junb inhibits SMAD-specific gene transactivation in keratinocytes and fibroblasts [33]. This finding raises the possibility that the pro-fibrotic effects of KLF11 deficiency may be, at least in part, through inhibition of Junb expression.

Of the enzymes involved in remodeling of extracellular matrix, we found that KLF11 KO-UUO mice showed significant decreases in TIMP3 and Serpina1a expression (Table 1). TIMP3 is a matrix metalloproteinase inhibiter and its deficiency is associated with renal interstitial inflammation and fibrosis [34]. Serpina1a is a serine protease inhibitor which has been shown to be downregulated in mice with diabetes [35]. Increased protease activity may lead to inflammation and tubular epithelial cell death in the UUO model.

KLF11 appears to limit inflammation, in part through binding p65 and inhibiting NF-kB signaling [11]. TGF-β is a key mediator of both fibrosis and inflammation [36, 37]. Both SMAD3/SMAD4 and KLF11 translocate to the nucleus and interact with Sp1 like and other GC-rich sequences of target genes [5, 36, 38, 39]. However, potential interactions between KLF11 and SMAD3 in regulation of matrix production have not been previously defined. As KLF11 is a TGF-β inducible gene, it is likely that tissue fibrosis results from complex interaction between KLF11 and SMAD3/TGF-β signaling. Knockdown of KLF11 attenuates hypoxia/regeneration injury via JAK2/STAT3 signaling in H9c2 [29]. Future studies will be directed towards defining such interactions between KLF11 and TGF-β signaling pathways and how they regulate fibrosis and inflammation.

In summary, we have defined a critical role for KLF11 in regulation of both inflammation and fibrosis in unilateral ureteric obstruction, a well characterized model of renal fibrosis. In particular, KLF11 deficiency is associated with increased renal atrophy, interstitial fibrosis, and interstitial inflammation. Compared to WT-UUO, KLF11 KO-UUO mice show marked upregulation of genes associated with TGF-β signaling, fibrosis, and inflammation. Interventions to increase KLF11 expression may provide a potential therapeutic target to decrease renal inflammation and fibrosis in chronic kidney disease.

Differentially expressed genes of TGF-β/BMP/Fibrotic pathways between the KLF11 KO vs WT.

(PDF)

Click here for additional data file.

Differentially expressed genes of inflammatory response between the KLF11 KO vs WT.

(PDF)

Click here for additional data file.

Renal function parameters in KLF11 KO and WT mice.

(PDF)

Click here for additional data file.

CD3 and CD163 markers quantification in KLF11 KO and WT.

(PDF)

Click here for additional data file.

5 Oct 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Franziska Theilig

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"We would like to acknowledge to Chantal E. McCabe, Ph.D. from the Quantitative Health Sciences Department of Mayo Clinic for all of her assistance and the Department of Laboratory Medicine and Pathology for support of these studies."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"This work was funded by the Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data

4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well

Dear authors,

would you please provide renaul function parameters (Crea, BUN) and Masson Trichrome staining to judge injury?

Best regards,

Franziska Theilig

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study, the authors describe the pathological roles of KLF11 in renal fibrosis. In particular, KLF11 KO mice showed increased kidney injury and fibrosis, accompanied by upregulation of expression of pro-fibrotic and pro-inflammatory genes. There are serious concerns to be addressed.

1. The quality of H&E staining, immunohistochemical staining and heatmaps is of poor quality. For example, I can’t identify the specific marker positive cells in histological images and the gene name on the heatmaps.

2. The authors need to indicate the expression of other KLF family factors in KLF11 KO mice with or without UUO, because deletion of KLF11 may compensatory change the expression of other KLF family factors.

3. Although the authors demonstrated that macrophage infiltration and fibrosis was suppressed in the kidneys of KLF11 global KO mice, it is unclear which KLF11-expressing cells contribute to these phenotypes. Which cells are expressing KLF11 in murine normal kidneys and injured kidneys?

4. It would be interesting to indicate the association of KLF11 with kidney diseases. For example, how does endogenous KLF11 mRNA and protein expression change after UUO?

5. The author describe that the expression of CD3 or CD163-positive cells was examined in methods and results section and the expression of CD68-positive cells was examined in methods section. However, I can’t find these data in this manuscript.

Reviewer #2: 1. In Results Genetic inactivation of KLF11 increases renal injury in UUO model, authors only can get conclusion that genetic inactivation of KLF11 contribute to tubular atrophy, because tubular atrophy can not be equal to renal injury. I suggest authors detect some renal injury biomarkers in this part.

2. The title that KLF11 deficiency enhances chemokine generation and activates the TGF-β/BMP fibrotic pathway in murine unilateral ureteric obstruction is not appropriate, because KLF11 deficiency enhances chemokine generation, and increase inflammation and pro-inflammation cytokines production to result in renal damage from the text, but can not attribute to activate the TGF-β/BMP fibrotic pathway specifically.

3. From the whole study ,authors just observed a phenomenon that KLF11 deficiency results in renal injury, accompanied with chemokine generation enhanced and inflammation and pro-inflammation cytokines production increased, but no specific mechanism.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Dec 2021

PONE-D-21-23487

KLF11 deficiency enhances chemokine generation and activates the TGF-β/BMP fibrotic pathway in murine unilateral ureteric obstruction

PLOS ONE

We would like to thank the reviewers for their thoughtful comments provided in review of our manuscript, “KLF11 deficiency enhances chemokine generation and activates the TGF-β/BMP fibrotic pathway in murine unilateral ureteric obstruction”. We have conducted additional experiments and have made a number of clarifications in response to the constructive comments provided. A summary of the changes is provided below:

Funding information should not be included in the Acknowledgements Section of the Manuscript.

We have removed the statement indicating that the Department of Laboratory Medicine and Pathology provided institutional support for these studies in the acknowledgements section. As suggested, we wish to update the Funding Statement to indicate:

"This work was funded by the Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data

We have provided all data in supporting information files.

Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well

This study does not involve human subjects. We have included the statement that “all animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) prior to conducting any experiments. These animal procedures were conducted in accordance with then National Institutes of Health Guide for the Care and Use of Laboratory Animals”.

Dear authors,

would you please provide renal function parameters (Crea, BUN) and Masson Trichrome staining to judge injury?

In the unilateral ureteral obstruction model, the contralateral kidney provides normal renal function. Therefore, changes in serum creatinine are not anticipated. We have done additional studies to measure BUN and albuminuria and have provided this information in a supplementary table. We have used Sirius Red staining to provide a quantitative assessment of extracellular matrix deposition. Sirius Red staining provides a more accurate assessment of matrix deposition than trichrome staining, as edema and non-collagenous components can stain blue with a trichrome stain. We have compared Sirius Red staining with trichrome staining in our previously published study [1].

Reviewer #1: In this study, the authors describe the pathological roles of KLF11 in renal fibrosis. In particular, KLF11 KO mice showed increased kidney injury and fibrosis, accompanied by upregulation of expression of pro-fibrotic and pro-inflammatory genes. There are serious concerns to be addressed.

1. The quality of H&E staining, immunohistochemical staining and heatmaps is of poor quality. For example, I can’t identify the specific marker positive cells in histological images and the gene name on the heatmaps.

We have revised the figures to provide higher resolution images of the immunohistochemical staining and have enlarged the annotations to the heatmaps.

2. The authors need to indicate the expression of other KLF family factors in KLF11 KO mice with or without UUO, because deletion of KLF11 may compensatory change the expression of other KLF family factors.

We performed additional studies employing RNASeq to assess other KLF family members in KLF11 KO mice with or without UUO. Our data are summarized in a heatmap showing relative expression of the KLF family members in sham and UUO mice (Fig 1 B). We see largest induction of KLF14 and KLF 16 in KLF11 KO UUO compared to WT UUO; other KLF members showed modest changes according to genotype.

3. Although the authors demonstrated that macrophage infiltration and fibrosis was suppressed in the kidneys of KLF11 global KO mice, it is unclear which KLF11-expressing cells contribute to these phenotypes. Which cells are expressing KLF11 in murine normal kidneys and injured kidneys?

We performed additional immunohistochemical studies to identify KLF11 staining cells. We found nuclear staining for KLF11, primarily in proximal and distal tubular epithelial cells, with focal glomerular staining of visceral and parietal epithelial cells. We observed stronger staining in WT mice subjected to UUO, compared to sham, consistent with our RNASeq data showing induction of KLF11 in WT UUO mice compared to sham.

4. It would be interesting to indicate the association of KLF11 with kidney diseases. For example, how does endogenous KLF11 mRNA and protein expression change after UUO?

Although other KLF family members have been implicated in human and experimental kidney disease, to the best of our knowledge, this is the first report associating KLF11 with kidney disease. At the RNA and protein level, as assessed by RNASeq and immunohistochemistry, respectively, we do demonstrate that, in WT mice, KLF11 is induced with UUO.

5. The author describe that the expression of CD3 or CD163-positive cells was examined in methods and results section and the expression of CD68-positive cells was examined in methods section. However, I can’t find these data in this manuscript.

We have provided these data in a supplemental table.

Reviewer #2: 1. In Results Genetic inactivation of KLF11 increases renal injury in UUO model, authors only can get conclusion that genetic inactivation of KLF11 contribute to tubular atrophy, because tubular atrophy can not be equal to renal injury. I suggest authors detect some renal injury biomarkers in this part.

Tubular atrophy is a well-recognized feature of chronic renal injury. For example, ct scores are an integral component of chronic tubular injury scoring according to the Banff Classification of transplant pathology. [2]. We have extensively employed tubular atrophy as an index of chronic tubular injury in our previous publications involving both human and experimental studies [3] [4] [5] [6]. As expected with a unilateral injury model, we did not detect significant differences in serum creatinine among the experimental groups.

2. The title that KLF11 deficiency enhances chemokine generation and activates the TGF-β/BMP fibrotic pathway in murine unilateral ureteric obstruction is not appropriate, because KLF11 deficiency enhances chemokine generation, and increase inflammation and pro-inflammation cytokines production to result in renal damage from the text, but can not attribute to activate the TGF-β/BMP fibrotic pathway specifically.

KLF11 was originally described as a Transforming Growth Factor Beta inducible immediate early gene (TIEG) [7]. Based on this consideration, it was reasonable to focus on the TGF-beta/SMAD pathway; we have shown significant perturbations in this pathway in KLF11 deficient mice. Nevertheless, we have removed “activation of the TGF-beta/BMP fibrotic pathway” from the title.

3. From the whole study, authors just observed a phenomenon that KLF11 deficiency results in renal injury, accompanied with chemokine generation enhanced and inflammation and pro-inflammation cytokines production increased, but no specific mechanism.

To our knowledge, this is the first report linking KLF11 deficiency to renal injury. Characterization of differentially regulated pathways is an important first step in the development of a mechanistic hypothesis whereby KLF11 deficiency results in renal injury. Based on initial characterization of KLF11 as a TIEG, we hypothesized that the renal damage was associated with alterations in TGF-beta signaling. Futures studies will focus on a mechanism whereby KLF11 deficiency promotes kidney injury, with a focus on the TGF-beta pathway.

________________________________________

1. Diaz Encarnacion M, Griffin M, Slezak J, Bergstralh E, Stegall M, Velosa J, et al. Correlation of quantitative digital image analysis with glomerular filtration rate in CAN. Am J Transplantation. 2004;4(2):248-56. PubMed PMID: 14974947.

2. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, et al. The Banff 97 working classification of renal allograft pathology. Kidney International. 1999;55(2):713-23.

3. Helgeson ES, Mannon R, Grande J, Gaston RS, Cecka MJ, Kasiske BL, et al. i-IFTA and chronic active T cell-mediated rejection: A tale of 2 (DeKAF) cohorts. Am J Transplant. 2021;21(5):1866-77. Epub 2020/10/15. doi: 10.1111/ajt.16352. PubMed PMID: 33052625.

4. Grande JP, Helgeson ES, Matas AJ. Correlation of Glomerular Size With Donor-Recipient Factors and With Response to Injury. Transplantation. 2021;105(11):2451-60. Epub 2020/12/05. doi: 10.1097/TP.0000000000003570. PubMed PMID: 33273317; PubMed Central PMCID: PMCPMC8166916.

5. Kashyap S, Osman M, Ferguson CM, Nath MC, Roy B, Lien KR, et al. Ccl2 deficiency protects against chronic renal injury in murine renovascular hypertension. Scientific Reports. 2018;8(1):8598.

6. Kashyap S, Boyilla R, Zaia PJ, Ghossan R, Nath KA, Textor SC, et al. Development of renal atrophy in murine 2 kidney 1 clip hypertension is strain independent. Res Vet Sci. 2016;107:171-7.

7. Lin L, Mahner S, Jeschke U, Hester A. The Distinct Roles of Transcriptional Factor KLF11 in Normal Cell Growth Regulation and Cancer as a Mediator of TGF-beta Signaling Pathway. Int J Mol Sci. 2020;21(8). Epub 2020/04/26. doi: 10.3390/ijms21082928. PubMed PMID: 32331236; PubMed Central PMCID: PMCPMC7215894.

Submitted filename: PONE Response to Reviewers.pdf

Click here for additional data file.

24 Jan 2022

23 Feb 2022

Reviewer #1: In this revision, the authors have satisfactorily addressed my previous comments, and I do not have any additional comments.

We wish to thank this reviewer for the constructive comments that have improved the manuscript.

Reviewer #3:

1. (Figure 1) Expression of KLF11 by the specific cells described in lines 177-178 of the manuscript (tubular epithelial cells, podocytes, and infiltrating mononuclear cells) is difficult to discern in the light micrograph of the WT-UUO kidney section. Is the KLF11 shown in brown? Are glomeruli shown in these micrographs? I would recommend that the authors either provide additional micrographs that more clearly portray the expression and/or use arrows or high-magnification insets to demonstrate the expression by these different cells more clearly. Also, more specific details are required in the figure legend.

The images provided are immunostains for KLF11. The WT-UUO section shows strong nuclear staining for KLF11 (dark brown stain). There is a glomerulus in the micrograph, which we have now indicated.

In the ureteric obstruction model, ligation of the ureter produces pressure induced dilation of tubules, which lead to tubular epithelial cell injury, interstitial fibrosis, tubular atrophy, and interstitial inflammation. Other compartments of the kidney (glomeruli, vasculature, etc.) are secondarily affected (see response to point 3, below).

2. (Table 2) Was the expression of anti-inflammatory or regulatory genes (e.g., FoxP3, IL-10) profiled as well? Did the expression of these genes vary between groups?

We did not find significant differences in anti-inflammatory or regulatory genes between WT and KO UUO groups.

3. This reviewer suggests expanding the Discussion section to include details about how the authors believe KLF11 expression by various cellular subsets (podocytes, tubular epithelial cells, and monocytes) could reduce renal injury following UUO.

We have expanded the discussion and added a reference to support our hypothesis that injury to KLF11 deficient tubular epithelial cells exacerbates pro-inflammatory and pro-fibrotic signaling pathways triggered by ureteric obstruction, a finding supported by our RNA expression data obtained from renal cortex.

In the ureteric obstruction model, the primary/initial target of injury is the tubular epithelial cell (see response to #1, above). In WT-UUO mice, we observed a strong induction of KLF11 staining, primarily within tubular epithelial cells. Based on this observation, we believe that injury to the tubular epithelial cell initiates a cascade of events leading to interstitial inflammation, interstitial fibrosis, and tubular atrophy. We propose that KLF11 deficiency exacerbates tubular injury through upregulation of pro-inflammatory and pro-fibrotic signaling pathways, as outlined in the results section.

Support for the notion that tubular epithelial cells can orchestrate pro-inflammatory and pro-fibrotic signaling pathways was obtained in our previous work using a murine model of renovascular hypertension initiated by placement of a cuff on the right renal artery, restricting blood flow by 75-80% (reference 26, added to address this concern). In this study, we subjected Ccl2-RFP reporter mice to renal artery stenosis surgery. We found a strong induction of Ccl2 (a potent chemoattractant for mononuclear cells) within tubular epithelial cells within 24 hours of renal artery stenosis surgery, a time point at which there was no significant tubular atrophy, interstitial fibrosis, or interstitial inflammation (see reference 26, added to this revision). Based on these considerations, we believe that the tubular epithelial cells—which are the target of UUO and show a striking increase in immunostaining for KLF11 following UUO—are responsible for the recruitment of inflammatory cells and initiate pro-fibrotic signaling pathways.

Minor Issues:

1. (line 3) Suggest changing title from "ureteric" to "ureteral"

Thank you for the suggestion. We have made the recommended change in line 3.

2. (lines 293-295) Suggest changing interpretation of the immunohistochemistry images from "expression" to "infiltration" of macrophages.

We have made the suggested change in line 292.

3. (lines 306-307) Suggest defining CD163 as a marker. Is this meant to detect pro-inflammatory M1 macrophages?

CD163 was used to detect pro-inflammatory M1 macrophages. This has been clarified in line 307-308.

4. Suggest revision for English in the manuscript (especially the revised sections), as there are some errors in grammar and syntax.

We have corrected errors in grammar and syntax.

Submitted filename: Summary of changes to manuscript.docx

Click here for additional data file.

22 Mar 2022

KLF11 deficiency enhances chemokine generation and fibrosis in murine unilateral ureteral obstruction

PONE-D-21-23487R2

Dear Dr. Grande,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Franziska Theilig

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: This reviewer is grateful for the diligence of the authors in addressing each comment directly and completely. I have no remaining concerns.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

31 Mar 2022

PONE-D-21-23487R2

KLF11 deficiency enhances chemokine generation and fibrosis in murine unilateral ureteral obstruction

Dear Dr. Grande:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Franziska Theilig

Academic Editor

PLOS ONE