Clr‑f Expression Regulates Kidney Immune And Metabolic Homeostasis Ⅱ

Kidney tubular and glomerular lesions in Clr‑f‑defcient mice. 

The initial examination of Clr-f−/− kidney sections revealed several alterations in the renal cortex. To evaluate histopathologic lesions, PAS-stained kidney sections from four 12-week-old WT and Clr-f−/− mice were assessed in a blinded fashion (Fig. 2A). A variable presence of proximal and distal tubular lesions was observed in Clr-f−/− kidneys which included, tubular epithelial cell fattening, nuclear displacement, and loss of brush borders (Fig. 2A; top panels). In rare instances, tubular necrosis was found in the form of luminal necrotic debris accumulation and complete epithelial loss in some of the examined areas. The Clr-f−/− kidneys also exhibited glomerular pathologies (Fig. 2A; bottom panels), with areas of focal thickening of glomerular tufs, minimal periglomerular fibroblast proliferation associated with mild interstitial fibrosis, and more severe lesions with mesangiolysis, capillary aneurysms that progressed to pronounced disruption of glomeruli and a constant presence of necrotizing cellular debris in Bowman’s spaces. Glomerular pathology scoring revealed a signifcant number of glomerular lesions in Clr-f-deficient mice in comparison to WT littermates (Fig. 2B). Electron microscopy revealed severe podocyte damage, podocyte foot process effacement, and signifcant thickening of the glomerular basement membrane (GBM) (Fig. 2C,D).

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To assess if kidney immunoglobulin deposits were present, which are characteristically associated with the pathologies observed, we stained renal cortex sections of WT and Clr-f−/− kidneys for the presence of IgA, IgM, and IgG antibodies by IF (Fig. 2E). Clr-f−/− kidneys show clear dominant IF staining of glomerular IgA deposits accompanied by pronounced IgM deposits and a lesser but consistent presence of IgG deposits. Likewise, we detected similar patterns of IgA, IgM, and IgG tubulointerstitial deposits in Clr-f−/− kidneys. Complement protein C3 was also broadly detected in Clr-f−/− kidneys but not in WT kidneys (Fig. 2F).

Clr‑f is required for proper kidney function. 

To evaluate the impact of Clr-f−/− pathologies on kidney function, we analyzed blood and urine composition from 12-week-old male Clr-f−/− and WT littermates. We found a modest, yet statistically signifcant, increase in the urine-protein-to-creatinine (UP/CR) ratio in Clrf −/− mice compared to WT mice, with increased serum creatinine, but similar urine protein levels compared to WT mice, as well as similar fractional excretion of sodium (FENa) (Fig. 2G). Given the tubular expression of Clr-f, we assessed if Clr-f−/− mouse blood pressure in 12-week-old and 24-week-old Clr-f−/− mice was affected. Clr-f−/−mice exhibited a marginal but non-statistically signifcant decrease in blood pressure compared to WT littermates (Fig. 2H). These results show that normal kidney filtration is altered in the absence of Clr-f, but Clr-f shows no signifcant roles in renal electrolyte homeostasis or blood pressure regulation.

Clr‑f−/− kidney transcriptome shows a dysregulation in metabolism. 

To investigate the underlying transcriptional processes responsible for Clr-f−/− kidney pathophenotypes, we performed RNA-sequencing on kidneys of 7-, 13-, and 24-week-old WT and Clr-f−/− mice. Differentially expressed genes (DEGs) in Clr-f−/−mice compared to WT mice were investigated by functional enrichment analysis and transcriptional pathotype analysis (Fig. 3A). We mapped interaction networks of enriched biological processes from gene set enrichment analysis (GSEA) of DEGs identified at each age (Fig. 3B). Our analysis showed an enrichment of cell cycle-related genes upregulated in kidneys of 7-week-old Clr-f−/− mice. At 13 weeks, metabolic processes, transport, cell death, and responses to toxic substances were enriched from DEGs with increased expression in Clr-f−/− kidneys, while non-motile cilium assembly, DNA transcription, and processing were enriched from DEGs with decreased expression. Kidneys of 24-week-old Clr-f−/− mice also exhibited an enrichment of metabolic processes, and transport among DEGs with increased expression, as well as processes that regulate blood circulation, while cell death, immunity, and Transforming growth factor-beta (TGFβ) signaling regulation processes were enriched from DEGs with decreased expression.

To capture age-associated processes that underlie Clr-f−/− kidney defects, we analyzed genes belonging to four dynamic patterns of differential expression: DEGs that increase with age, decrease with age, or transiently increase or decrease at 13 weeks of age. Functional enrichment analysis of biological processes, molecular functions, and defined pathways among these groups revealed that DEGs with increasing expression from 7 to 24 weeks of age in Clr-f−/− kidneys were overrepresented in lipid metabolic pathways, while DEGs related to cell-substrate adhesion decreased in expression from 7 to 24 weeks of age (Fig. 3C). DEGs with a transient increase in expression at 13 weeks of age were mostly associated with mitochondrial respiration, while DEGs with a transient decrease in expression mapped to cell cycle pathways. Within these four categories of dynamic expression were three sub-clusters that represent differential expression patterns in which the magnitude of change in expression between 7 and 13 weeks was either similar, higher, or lower than the degree of change between 13 and 24 weeks of age, respectively (Fig. 3C). Te enriched biological processes and molecular functions from each sub-cluster of genes were largely distinct across the four main dynamic expression patterns identified but refected overlapping or related cellular processes within each group. Overall, the transcriptional profile of Clr-f−/− kidneys reveals a dynamic and evolving dysregulation of cellular metabolism, function, and homeostasis.

Clr‑f−/− mice accumulate fat. 

The identified age-associated increase in DEGs related to lipid metabolism led us to probe expression data of WT and Clr-f−/− mice for known lipid-associated genes. Most of these gene transcripts were elevated in Clr-f−/− mice (Fig. 4A) and aligned with increases in abdominal and perirenal adipose deposits in Clr-f−/− mice at 24 weeks of age (Fig. 4B). Although Clr-f−/− and WT mice exhibited similar body weights (Fig. 4C), the weight of abdominal adipose in Clr-f−/− mice was significantly higher, indicating that adipose tissue accounted for an increased proportion of Clr-f−/− mouse body weight (Fig. 4D). We also detected ectopic lipid accumulation within the kidneys of Clr-f−/− mice, as evidenced by increased ORO staining of Clrf −/− mouse kidneys (Fig. 4E). As perirenal fat and the presence of interstitial fat in the kidney are associated with metabolic risk factors of CKD21, we hypothesized whether Clr-f is protective against etiologies of diabetes or related metabolic disorders associated with CKD.

Clr‑f−/− mouse kidneys have transcriptional profile similarities and diferences to human kidney disease. 

Ectopic lipid accumulation in the kidney is associated with infammation, fibrosis, mitochondrial dysfunction, and cell death22,23. To assess if Clr-f−/− kidneys resemble defned kidney diseases, we compared the transcriptional profle of Clr-f−/− kidneys to published expression profles of kidney tissue and/or blood from human donors with CKD, diabetic nephropathy (DN), lupus nephritis (LN), or IgA nephropathy (IgAN) (Fig. 5A). Clr-f−/− kidney transcriptional profles clustered with blood from IgAN patients and glomerular tissue of DN patients (Fig. 5B). Comparison of Clr-f−/− and IgAN patient expression profles by functional enrichment analysis showed parallel increases in gene expression related to cellular reactive oxide responses, cell death, proteolysis, and amide metabolism, as well as shared decreases in gene expression associated with membrane trafcking processes, cytoskeleton organization, PI3K-AKT signaling, cell cycling, and DNA/RNA metabolism (Fig. 5C). Conversely, Clr-f−/− kidney expression sets difered from IgAN, with increased expression of genes belonging to the AGE-RAGE, ErbB, TNF/MAPK, and Rap1 signaling pathways, and processes of vasopressinregulated water reabsorption, and lysosome activity, and decreased expression of genes belonged to adherens junction, non-motile cilium assembly, cell cycling, DNA metabolism, and TGFβ-signaling. These findings suggest that similar to IgAN, cellular processes of growth control, homeostasis, and repair, are also dysregulated in Clr-f−/− kidneys, while altered metabolism, stress-signaling, and epithelial structure and function are unique to Clr-f−/− kidneys.

Clr‑f‑defcient kidneys exhibit infammation and immune cell infiltration. 

Given the known immunomodulatory roles for Clr proteins, we examined the immune cell contribution to Clr-f−/− kidney pathology. We analyzed the immune cell composition of 12-week-old WT and Clr-f−/− mouse whole kidneys by flow cytometry. Our analysis revealed a signifcant increase in the number of neutrophils, T cells, and B cells, but no difference in renal NK cell, ILC or macrophage numbers (Fig. 6A). Analysis of the periglomerular distribution of immune cells within the kidney cortex of WT and Clr-f−/− kidneys by IF, we observed glomerular accumulations of CD45+ cells (Fig. 6B). Labeling of CD11c, F4/80, NKp46, and CD3, markers indicative of DCs, macrophages, NK/ILCs, and T cells respectively, revealed an intraglomerular accumulation of CD11c+ cells (Fig. 6C), and periglomerular accumulations of F4/80+, NKp46+, and CD3+ cells in Clr-f−/− kidneys (Fig. 6D). Visual quantification of immune cell populations detected at the glomeruli confrmed a signifcant increase in the presence of each of these immune cell populations in kidneys of Clr-f−/− mice compared to WT mice (Fi.g 6E). Immune infiltrate in autoimmune kidney disease is enhanced by the tubular secretion of IL-12 prompting injury by IFNγ secreting cells24–26. We, therefore, examined WT and Clr-f−/− kidneys for the presence of these cytokines and observed staining of both IL-12 and IFNγ in cells of the Clr-f−/− kidney tubulointerstitium (Fig. 6F,G).

Since Clr-f is posited to be a signal of cell health to NKR-P1G-expressing cells, we hypothesized that the renal immune infiltration in Clr-f−/− mice is partly due to an increased presence of NKR-P1G-expressing lymphocytes in the kidney. Analysis of NKR-P1G expression on kidney TCRβ+ T cells and NK1.1+ NK cells in WT and Clrf −/− mice by flow cytometry showed no difference in the mean fluorescence intensity of NKR-P1G staining on renal NK cells, but an increase in NKR-P1G staining of renal T cells in Clr-f−/− mice (Fig. 6H)

Figure 5. Comparison between transcriptional profiles of Clr-f−/− mice and human CKDs. (A) Table of gene expression profile data sets of 15 human renal disorders vs. 13-week-old Clr-f−/− mice. The reference source for the human expression profile data set sources is indicated by the Study column. (B) Similarity matrix showing hierarchical clustering of differential gene expression profiles in panel (A). The red-dashed line within the dendrogram indicates the level of matrix cluster separation. (C) Comparison of 4531 DEGs (Log2 FC; Padj.<0.05) of 13-week-old Clr-f−/− mice (group 0) and IgAN patients. Gene set enrichment from 4 DEG clusters: (I) upregulated in both Clr-f−/− mice and IgAN patients, (II) upregulated in Clr-f−/− mice and downregulated IgAN patients, (III) downregulated in Clr-f−/− mice, and upregulated IgAN patients, and (IV) downregulated in both Clr-f−/− mice and IgAN patients. Heat maps were constructed using Morpheus (https://sofware.broadinstitute. org/morpheus).

Clr‑f deficiency results in T‑ and B‑independent autoimmunity and pathogenesis. To clarify the immune contribution to the kidney pathology of Clr-f−/− mice, we examined Clr-f−/− mice that were also deficient in T- and B-cells, using the Rag1-deficient mouse strain (Rag1−/−Clr-f−/−). As Rag1−/−Clr-f−/− mice lack antibodies, we could evaluate Clr-f-associated pathogenesis in the absence of immunoglobulin deposition. Analysis of the Rag1−/−Clr-f−/− kidneys revealed glomerular lesions indicative of mild proliferative glomerulopathy and areas of glomerular fibrosis (Fig. 7A). Glomerular pathology scoring showed a significantly higher number of glomeruli with lesions in Rag1−/−Clr-f−/− compared to Rag1−/− mice (Fig. 7B). Scoring of Rag1−/−Clr-f−/− kidney sections identified increases in endocapillary proliferation, crescents, glomerulosclerosis or focal segmental sclerosis, and interstitial fibrosis and tubular atrophy, with a signifcant increase in the presence of mesangial cellularity (Fig. 7C).

To assess T and B cell contribution to the lipid accumulation observed in Clr-f−/−mice, we measured total body weight and abdominal adipose weight in Rag1−/−Clr-f−/− relative to Rag1−/− mice. Te Rag1−/−Clr-f−/− body weight was significantly lower than Rag1−/− controls (Fig. 7D), while abdominal lipid weight was significantly increased (Fig. 7E), albeit to a lesser extent than that observed in Clr-f−/− mice compared to WT mice (174 mg of fat gained versus 274 mg on average).

Finally, to investigate the immune contributors to these kidney defects, we examined Rag1−/−Clr-f−/− kidneys for the presence of IL-12 and IFNγ and renal infiltrating immune cells. IF analysis of Rag1−/−Clr-f−/− kidneys revealed an increased presence of IL-12 and IFNγ (Fig. 7F,G). We also observed an extensive periglomerular accumulation of CD45+ cells in Rag1−/−Clr-f−/− kidneys (Fig. 7H), which was found to consist of CD11c+, F4/80+, and Nkp46+ cells. These fndings suggest that T and B cell-independent mechanisms are the primary mediators of renal pathology in mice defcient for Clr-f. 

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