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.


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 noncollagenous 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.

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.

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.

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.

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.

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.