Tubular Epithelia-Specific Deletion of MCP-1 Does Not Afford Protection Against Adriamycin-Induced Kidney Injury

Abstract

The increasing global burden of chronic kidney disease (CKD) magnifies an urgent need to find treatable targets. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a chemokine secreted by kidney tubular epithelia in response to a variety of stimuli. To better understand the effects of tubular MCP-1 in response to kidney injury, we generated tubular epithelia-specific MCP-1 knockout mice (KO; Pax8-Mcp-1^fl/fl). We then exposed these mice and their control littermates to Adriamycin (Adr; 18 mg/kg, IV bolus). Thirty-two days after Adr injection, Mcp-1 transcript and protein levels were suppressed in the KO mice compared to their wild-type (WT) littermates. The KO mice exhibited no effect on survival, change in body weight, albuminuria, kidney function, glomerular or tubular injury, or tubulointerstitial fibrosis compared to WT. Overall, the results suggest that tubule-secreted MCP-1 is not necessary for progression of Adr-induced injury. These findings contribute to our understanding of the role of MCP-1 in kidney injury.

1. Introduction

Monocyte chemoattractant protein-1 (MCP-1/CCL2), a member of the C-C motif chemokine family, plays a pivotal role in proteinuric CKD and renal fibrosis [1,2,3,4,5,6,7,8,9,10,11]. Clinical studies show a correlation between urinary MCP-1 levels and the degree of proteinuria and decline in renal function [6,8,10,12,13,14,15,16,17]. Similarly, in mouse models of diabetic nephropathy, urinary levels correlate with the extent of albuminuria and global knockout of MCP-1 reduces disease severity [9,18,19]. MCP-1 transcripts are upregulated in human glomerulopathies such as focal segmental glomerulosclerosis (FSGS) [20]. Also, in animal models, Adr induces glomerular Mcp-1 expression progressively over the course of 28 days [20]. In addition to FSGS, Adr exposure results in tubulointerstitial fibrosis in humans and rodents [20,21].

Prior research in glomerular diseases has focused on the effects of MCP-1 on podocytes [3,5,9]. Deletion or blockade of the MCP-1 receptor CCR2 alleviated Adr-induced glomerular injury and proteinuria in mice [20,22]. In addition to being produced in podocytes, MCP-1 is also produced by tubular epithelial cells and activates an inflammatory response, with expression mostly located in distal tubule segments [23,24]. In Sprague-Dawley rats, ischemia–reperfusion injury (IRI) increases MCP-1 protein and transcript expression as well as its urinary excretion [25]. Blocking CCR2 in IRI reduces kidney damage [26,27]. Interestingly, unlike in diabetic nephropathy models, global MCP-1 knockout mice are more susceptible to kidney damage and death in an IRI model [23].

Our lab previously demonstrated a lack of protection against Adr-induced glomerular injury in conditional podocyte-specific MCP-1 knockout mice [28]. Since tubules exert significant pathologic effects on glomeruli [29,30], we hypothesized that MCP-1 originating from renal tubules induces podocyte injury. To test this, we generated tubular epithelia-specific conditional MCP-1 knockout (KO) mice and exposed them to Adr-induced injury. We assessed survival, change in body weight, albuminuria, kidney function, glomerular and tubular injury, and tubulointerstitial fibrosis. Our results show that tubular-derived MCP-1 is not necessary for Adr-induced kidney injury.

2. Results

2.1. Generation of Tubular Epithelia-Specific MCP-1 Knockout Mice

To generate the KO mice, we bred Mcp-1 floxed allele mice (B6.Cg-Ccl2^tm1.1Pame/J) with kidney tubule-specific Cre-expressing mice (B6.129P2(Cg)-Pax8^{tm1.1(cre)Mbu}/J). Genotyping confirmed that these mice harbor both the Mcp-1 floxed allele and Pax8-driven Cre recombinase, unlike their control wild-type littermates (WT) (Figure 1A). To further confirm these results and to ensure proper grouping of the mice, we assessed Cre (Cre recombinase) expression. As expected, the knockout mice expressed Cre (1082 ± 81.08 compared to 1.000 ± 0.2020, p < 0.0001). A Pax8^cre mouse (#028196, Jackson Laboratory, Bar Harbor, ME, USA) was used as a positive control (Figure 1B). Since tubular cells release MCP-1 when exposed to Adr [21], we assessed Mcp-1 (monocyte chemoattractant protein-1) transcript expression at 32 days after Adr injection. As expected, it was significantly suppressed in the KO mice compared to WT (0.2914 ± 0.04511 compared to 1.000 ± 0.2421, p = 0.0070) (Figure 1C). Also, we assessed MCP-1 protein levels in tissue lysates, and levels were significantly decreased in KO mice (6.459 ± 0.5095 pg/mg compared to 10.86 ± 1.615 pg/mg, p = 0.0321). This further supports the genotyping and Cre mRNA results of the KO mice. It is not surprising that there is some Mcp-1 expression since other cell types can produce and secrete this chemokine.

2.2. WT and KO Mice Have Similar Survival, Body Weight, Albuminuria, and Renal Function 32 Days After Adr Injection

Unilateral nephrectomy was performed 7 days prior to an IV bolus of Adriamycin (Adr; 18 mg/kg) to induce injury in KO and WT mice. Mice were euthanized on day 32 (Figure 2A). Twelve WT and nine KO mice were exposed to Adr. Ten WT and eight KO mice survived to the study endpoint, and survival proportions were similar between the groups (88.9% compared to 83.3%, p = 0.7411) (Figure 2B). There were no significant differences in change in body weight (−4.625 ± 0.6318 g compared to −5.800 ± 0.7304 g, p = 0.2548), with all mice losing weight after Adr (Figure 2C). Spot urine albumin-to-creatinine levels were elevated at weekly measurement. The last urine collection was on day 28. Even though there was a significant increase in these levels over time (p < 0.01), there were no differences at any measured timepoint between the KO and WT mice (28D: 0.7805 ± 0.3569 mg UAlb/mg UCr compared to 1.944 ± 0.6187 mg UAlb/mg UCr, p = 0.6397) (Figure 2D). Serum creatinine (SCr) (0.3456 ± 0.02307 mg/dL compared to 0.3453 ± 0.03407 mg/dL, p = 0.9943) and blood urea nitrogen (BUN) (29.33 ± 1.433 mg/dL compared to 26.30 ± 1.655 mg/dL, p = 0.1976) were also not significantly different between the groups (Figure 2E,F). Overall, even though Mcp-1 transcript and protein levels were suppressed at 32 days in the KO mice (Figure 1C,D), this had no effect on survival, change in body weight, spot urine albumin levels, or kidney function after Adr.

2.3. Tubular Deletion of MCP-1 Does Not Protect Against Adr-Induced Glomerular Injury

Since Adr induces FSGS, we assessed the extent of Adr-induced glomerular injury by measuring nephrin protein and transcript (Nphs1) expression as well as transcript levels of other podocyte markers. There were no significant differences in nephrin protein (0.8880 ± 0.06035 compared to 1.000 ± 0.07897, p = 0.2789) and transcript expression (0.7112 ± 0.1426 compared to 1.000 ± 0.2018, p = 0.2830) (Figure 3A–C). In support, there were no significant differences in Wt1 (Wilm’s tumor-1) (0.8671 ± 0.08123 compared to 1.000 ± 0.1603, p = 0.5038) and Nphs2 (podocin) (0.9379 ± 0.09793 compared to 1.000 ± 0.1035, p = 0.6746) expression (Figure 3D,E). These results suggest that the tubular knockout did not protect against Adr-induced glomerular injury.

2.4. Tubular Deletion of MCP-1 Does Not Affect Tubular Injury After Adr

To observe if the decrease in Mcp-1 transcript and protein levels (Figure 1C,D) affects tubular injury, we assessed the protein and transcript expression of tubular injury markers, KIM-1 and NGAL. Densitometric analysis revealed no differences between groups for KIM-1 (kidney injury molecule 1) (1.223 ± 0.09261 compared to 1.000 ± 0.06536, p = 0.0730) and NGAL (neutrophil gelatinase-associated lipocalin) (0.7728 ± 0.1987 compared to 1.000 ± 0.2737, p = 0.5144) proteins (Figure 4A–C). Likewise, there were no differences between groups in levels of Havcr1 (gene for KIM-1) (0.1606 ± 0.09474 compared to 1.000 ± 0.4931, p = 0.1541) and Lcn2 (gene for NGAL) (0.2574 ± 0.08497 compared to 1.000 ± 0.4157, p = 0.1365) (Figure 4D,E). These results suggest that tubular epithelia-specific deletion of MCP-1 afforded no protection against Adr-induced tubular injury.

2.5. KO Mice Are Not Protected Against Adr-Induced Tubulointerstitial Fibrosis

To assess Adr-induced tubulointerstitial fibrosis, we measured fibronectin and αSMA (alpha smooth muscle actin) protein and transcript (Fn1 and Acta2) levels. Densitometric analysis revealed no differences between groups for fibronectin (1.140 ± 0.1131 compared to 1.000 ± 0.05787, p = 0.2899) and αSMA (1.142 ± 0.05786 compared to 1.000 ± 0.07229, p = 0.1465) proteins (Figure 5A–C). Similarly, the levels of Fn1 (fibronectin) (0.7112 ± 0.1426 compared to 1.000 ± 0.2018, p = 0.2830) and Acta2 (actin alpha 2, smooth muscle) (0.8671 ± 0.08123 compared to 1.000 ± 0.1603, p = 0.5038) were not significantly different (Figure 5D,E). The percentage of fields with identifiable fibrosis was quantified and was similar between groups (40.00 ± 5.669% compared to 42.50 ± 7.734%, p = 0.7981) (Figure 5F,G). While fibrosis levels were low, this has been previously described in the C57BL/6J strain [31]. These results suggest that knocking out MCP-1 in the tubules did not protect against Adr-induced tubulointerstitial fibrosis.

2.6. Inflammatory Cytokine Expression Is Similar in WT and KO Mice

Since inflammatory cytokines are produced by tubular epithelial cells [32,33], we assessed levels of Tnfa (tumor necrosis factor alpha), Il1b (interleukin 1 beta), and Il6 (interleukin 6). Tnfa (1.044 ± 0.1563 compared to 1.000 ± 0.1912, p = 0.8646), Il1b (1.142 ± 0.1288 compared to 1.000 ± 0.1686, p = 0.5222) and Il6 (0.7327 ± 0.1295 compared to 1.000 ± 0.1337, p = 0.1875) expression was similar between groups (Figure 6A–C). These results suggest that knocking out MCP-1 did not alter the expression of these inflammatory cytokines.

2.7. Mcp-2, Mcp-3, and Mcp-5 Did Not Compensate for Loss of MCP-1

Since tubular MCP-1 KO mice did not exhibit altered disease severity, we tested whether MCP-2, MCP-3 and MCP-5 (related chemokines that can also activate CCR2) were upregulated to compensate for the loss of MCP-1 [33,34]. Therefore, we assessed transcript levels of Mcp-2 (monocyte chemoattractant protein-2), Mcp-3 (monocyte chemoattractant protein-3), and Mcp-5 (monocyte chemoattractant protein-5). No differences between the groups were detected for Mcp-2 (0.7330 ± 0.1723 compared to 1.000 ± 0.2000, p = 0.3410), Mcp-3 (1.752 ± 0.5828 compared to 1.000 ± 0.2149, p = 0.2063), and Mcp-5 (0.7688 ± 0.2054 compared to 1.000 ± 0.2355, p = 0.4825) (Figure 7A–C).

3. Discussion

Previous studies have demonstrated a critical role for the MCP-1/CCR2 axis in podocyte injury [1,2,3,4,5,6,7,8,9,10,11]. However, such studies do not identify the source of MCP-1 activating CCR2. Our previous work determined that podocyte-derived MCP-1 is not necessary for Adr-induced kidney injury [28]. This led us to hypothesize that other cell-type sources of MCP-1 are likely involved. It is known that MCP-1 promotes disease in CKD and is produced by a variety of kidney cell types, including tubular epithelia [5,6,23,35,36,37], in proteinuric diseases [6,35]. Furthermore, it is known that tubular epithelia can have profound effects on glomerular biology through the elaboration of MMP-7, NMN, and other secreted proteins [30,38,39,40]. Therefore, using a variety of methods, we sought to evaluate the role of tubular epithelia-derived MCP-1 in response to Adr-induced injury by knocking out tubular MCP-1.

Adriamycin is known to induce proteinuric CKD and tubulointerstitial fibrosis in humans and mice by increasing reactive oxygen species and mitochondrial dysfunction, leading to an inflammatory response and the release of MCP-1 and other inflammatory mediators that we assessed in this study [21,31,32,41,42]. Since our knockout mice had an Adr-resistant C57BL/6J background, we sensitized its effects by performing a unilateral nephrectomy prior to injection as well as using a higher dose (18 mg/kg) known to cause injury in this strain [21,31,41]. Important to our investigation, tubular cells release MCP-1 when exposed to Adr [21]. These findings provided a compelling rationale for its usage in our study. We have also shown that there is high upregulation of Mcp-1 expression after Adr, which was abrogated in our MCP-1 knockout mice (Figure 1C). Likewise, MCP-1 tissue expression followed a similar pattern (Figure 1D). Mcp-1 mRNA and protein levels were not completely suppressed, likely due to the expression of MCP-1 by other cell types, including podocytes, inflammatory cells, and extra-renal sources. Although Adr accumulates in the kidney in rodents, it also causes hepatoxicity and cardiotoxicity [21] by injuring hepatocytes and cardiomyocytes, resulting in an inflammatory response and release of MCP-1 [43,44]. Any MCP-1 that reaches the circulatory system could be filtered by the glomerulus and excreted into the urine, which could compensate for the knockout levels. Unfortunately, we were unable to characterize our mice with existing antibodies targeting MCP-1 or its red fluorescent protein tag in immunofluorescent tissue staining. However, we are confident that our genotyping, qPCR, and ELISA results demonstrate MCP-1 knockdown.

In vitro and in vivo studies have demonstrated that MCP-1 affects nephrin expression and induces proteinuria possibly through actions at its receptor [5,7,9,20]. In our study, the knockout mice had similar nephrin protein and transcript expression compared to WT littermates (Figure 3). It is possible that non-tubular sources of MCP-1 such as podocytes, inflammatory cells, and extra-renal sources are responsible for this effect. We have previously shown that podocyte-specific MCP-1 knockout has no effect, but it is possible that podocyte and tubular sources of MCP-1 serve redundant functions, with either source being enough to cause podocyte dysfunction. Future studies can test whether combined podocyte and tubular knockout leads to a different result. Indeed, since global MCP-1 knockout mice are protected from disease [9,18,19], it is possible that at least a double podocyte/tubular knockout would be necessary to identify the same effect. It is also possible that other cell types, including those originating outside the kidney, could be the key source of MCP-1 in the Adr model. These possibilities require further study.

Advanced stages of CKD express both glomerular and tubular injury. The proximal tubule plays a pivotal role in reabsorbing filtered albumin through receptor-mediated endocytosis by megalin or cubilin [45,46,47]. Therefore, it is possible that injury or dysfunction of the proximal tubule impairs reabsorption and could contribute to the albuminuria observed in our experiment.

MCP-1 is also involved in the development of renal fibrosis [6,23,48]. For example, in streptozotocin-induced diabetic mice, knocking out MCP-1 decreases fibronectin protein expression [49]. However, our knockout mice showed similar fibronectin and αSMA protein and transcript expression between groups, indicating a lack of protection against tubulointerstitial fibrosis (Figure 5). Since the proteinuria in our Adr model was unaffected in our KO mice, it may be expected that tubulointerstitial fibrosis was unaffected. The causes of renal fibrosis in glomerular disease can be multifactorial. It can be the result of proteinuric injury to the tubules, tubular atrophy, inflammation, and many other factors [2,3,4,6]. In addition to MCP-1, tubular epithelial cells generate and release inflammatory cytokines such as TNF-α, IL-1, and IL-6 [32,42]. Transcript expression of these cytokines was similar between groups, suggesting that the inflammatory state in the KO mice was not attenuated by knocking out MCP-1 (Figure 6). Our work shows that tubular-derived MCP-1 is not critical for any secondary tubular injury resulting directly from proteinuria or glomerular injury.

The rationale for knocking out MCP-1 instead of its receptor, CCR2, from the tubules was to identify the role of tubular epithelia-produced MCP-1 in response to injury. Besides MCP-1, other chemokines such as MCP-2, MCP-3 and MCP-5 also bind to CCR2 to elicit effects [33,34]. Therefore, knocking out or blocking CCR2 does not allow specific identification of the effects of MCP-1 alone and additionally fails to capture any receptor-independent activities of MCP-1. However, it is possible that these related chemokines replace the function of MCP-1. We found that transcript levels of the other chemokines were similar between groups, suggesting a lack of a compensatory upregulation in their expression (Figure 7). We cannot rule out compensation by these chemokines in the absence of a simultaneous knockout of all these MCPs. However, our study demonstrates that any potential MCP-1 effects that do not rely on CCR2 are also not necessary for Adr disease progression [24]. However, in an IRI model, global MCP-1 knockout mice were more susceptible to death and kidney damage [23]. This indicates a more important role for MCP-1 in direct tubular injuries. Indeed, the high expression of MCP-1 in a wide variety of kidney diseases suggests important roles in pathophysiology. Although expressed nearly universally after injury, it is now clear that MCP-1 has disease-specific functions.

In our model of Adr-induced glomerular injury and proteinuria, tubular-specific knockout of MCP-1 did not affect the course of the disease. Animal survival, body weight, glomerular/tubular injury markers, and fibrosis were similar between WT and KO mice. This indicates a dispensable role for MCP-1 in Adr disease progression.

4. Materials and Methods

4.1. Animals

The Institutional Animal Care and Use Committee protocol was approved by the University of Pittsburgh. Pax8-Mcp-1^fl/fl (KO) mice and control wild-type littermates (WT) were treated with the ethical and scientific standards recommended by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The investigators followed ARRIVE guidelines [50]. Eight- to twelve-week-old male mice were housed in the animal care facility at the University of Pittsburgh on a constant 12:12 h light–dark cycle with water and Prolab^® IsoPro^® RMH 3000 5P75 diet (PMI Nutrition International, LLC, Arden Hills, MN, USA) provided ad libitum.

The KO mice were created by crossing female B6.Cg-Ccl2^tm1.1Pame/J mice (#016849, Jackson Laboratory, Bar Harbor, ME, USA) containing the loxP sites flanking exons 2 and 3 of the C-C-motif ligand 2 (Ccl2/Mcp1) [51] with male B6.129P2(Cg)-Pax8^{tm1.1(cre)Mbu}/J mice expressing Cre recombinase in renal tubules (#028196, Jackson Laboratory, Bar Harbor, ME, USA) [52]. All mice possessed a C57BL/6J background.

4.2. Antibodies

Primary antibodies utilized for immunoblotting (IB) were alpha smooth muscle actin (αSMA) (IB: 1:1000; #14395-1-AP, Proteintech, Rosemont, IL, USA), fibronectin (IB: 1:1000; #F3648, MilliporeSigma, Burlington, MA, USA), kidney injury marker-1 (KIM-1) (IB: 1:1000; 30948-1-AP, Proteintech, Rosemont, IL, USA), neutrophil gelatinase-associated lipocalin (NGAL) (IB: 1:1000; AF1857, R&D Systems, Minneapolis, MN, USA), and nephrin (IB: 1:1000; #20R-NP002, Fitzgerald Industries International, Acton, MA, USA).

4.3. Adriamycin Model

Sample size was based on the results of a preliminary study and breeding capacity. After genotyping, wild-type and knockout mice were randomized and housed according to matching body weight. Group allocation was known to C.D.B. at the different stages of the experiment. An intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) was used to anesthetize the mice before they were subjected to abdominal incision and unilateral nephrectomy of the left kidney 7 days prior to a single intravenous, retro-orbital injection of Adriamycin (Adr) (18 mg/kg, doxorubicin-HCl; #D1515, MilliporeSigma) [41,53]. The numbers of mice that received Adr exposure were 12 WT and 9 KO mice. The mice were monitored for pain, suffering, and distress by the investigators and animal care facility technicians. The mice were euthanized 32 days after Adr injection with a lethal dose of ketamine (300 mg/kg) and xylazine (30 mg/kg), followed by cervical dislocation. The kidneys and sera were harvested during daylight hours.

4.4. Biochemical Measurements

Spot urine was collected, and urine albumin levels were assayed with a mouse albumin ELISA kit (Bethyl Laboratories, Worthington, TX, USA). Serum and urine creatinine levels were assayed with the Creatinine (Enzymatic) Reagent set (#C7548-120, Pointe Scientific, Canton, MI, USA). Blood urea nitrogen (BUN) level was assayed with the QuantiChrom™ Urea Assay kit (#DIUR-100, BioAssay Systems, Hayward, CA, USA). Protein homogenates were assayed with the mouse CCL2/JE/MCP-1 Quantikine ELISA kit (#MJEOOB, R&D Systems, Minneapolis, MN, USA).

4.5. Histology

Kidney tissue was fixed in formalin, paraffin-embedded and then sectioned at 3 µM before being stained with Masson’s Trichrome Stain (MTS). Tissue processing and MTS were performed by the University of Pittsburgh Medical Center Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core. MTS sections were scored by an investigator (J.L.G.) masked to group assignment. The percentage of fibrosis-positive fields (e.g., the number of fibrosis-positive fields/10 total fields × 100) for each mouse was calculated.

4.6. Immunoblotting

Homogenates were generated by douncing the pole end of a kidney section in pre-chilled radioimmunoprecipitation buffer (RIPA: 50 mM Tris, pH 8.0; 150 mM NaCl; 0.1% SDS; 0.5% sodium deoxycholate; 1% Triton X-100) with added Halt™ Protease & Phosphatase Single-Use Inhibitor Cocktail (ThermoScientific, Rockford, IL, USA) and then centrifuged at 16,000× g for 15 min at 4 °C. Protein concentrations were determined with the Pierce™ BCA Protein Assay Kit (ThermoScientific). Homogenates were boiled in Laemmli sample buffer containing dithiothreitol for 10 min. Equivalent protein concentrations were loaded into Criterion TGX Stain-free gels (Bio-Rad Laboratories, Hercules, CA, USA). A pre-transfer image of each gel was captured for measurement of total protein. Proteins were transferred to a PVDF membrane (Trans-Blot Turbo Transfer Pack, #1704157, Bio-Rad Laboratories) using the Trans-Blot Turbo System (Bio-Rad Laboratories). The membrane was blocked in 5% nonfat milk for 1 h before incubation overnight in primary antibody at 4 °C. After repeated washing in TRIS-buffered saline with 0.1% Tween-20 (TBS-T), the membrane was incubated in the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. Chemiluminescent detection was performed with Pierce™ SuperSignal^® West Pico Chemiluminescent Substrate (ThermoScientific). Electronic images were captured with Image Lab Touch Software 3.0.1.14 (Bio-Rad Laboratories) on the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories), and band intensity was quantified with ImageJ 1.53e (NIH, Bethesda, MD, USA).

4.7. Quantitative Real-Time PCR (qPCR)

Total RNA was extracted from the pole end of a kidney section with TRIzol^® Reagent (Ambion^®, Carlsbad, CA, USA) before being reverse transcribed with the RevertAid Reverse Transcriptase kit (ThermoFisher Scientific, Pittsburgh, PA, USA). Reactions contained cDNA, iTAQ™ Universal SYBR^® Green Supermix (Bio-Rad Laboratories), nuclease-free water, and one of the following primer pairs: Acta2 (actin alpha 2, smooth muscle) NM_007392.3 (mouse) (forward: 5′-GAGGCACCACTGAACCCTAA-3′; reverse: 5′-CATCTCCAGAGTCCAGCACA-3′), Cre (Cre recombinase; enterobacteria phage P1) NC_005856.1 (forward: 5′-AGCCGAAATTGCCAGGATCA-3′; reverse: 5′-AACCAGCGTTTTCGTTCTGC-3′), Fn1 (fibronectin) NM_010233.2 (mouse) (forward: 5′-CGAGGTGACAGAGACCACAA-3′; reverse: 5′-CTGGAGTCAAGCCAGACACA-3′), Havcr1 (hepatitis A virus cellular receptor 1; kidney injury molecule 1) NM_134248.2 (mouse) (forward: 5′-TTCAGGAAGCTGAGCAAACAT-3′; reverse: 5′-CCCCAACATGTCGTTGTGATT-3′) [54]. Il1b (interleukin 1 beta) NM_008361.4 (mouse) (forward: 5′-TGCCACCTTTTGACAGTGATG-3′; 5′-ATGTGCTGCTGCGAGATTTG-3′), Il6 (interleukin 6) NM_031168.2 (forward: 5′-CTTGGGACTGATGCTGGTG-3′; reverse: 5′-TCCACGATTTCCCAGAGAAC-3′), Lcn2 (lipocalin 2; neutrophil gelatinase-associated lipocalin) NM_008491.1 (mouse) (forward: 5′-CCATCTATGAGCTACAAGAGAACAAT-3′; reverse: 5′-TCTGATCCAGTAGCGACAGC-3′), Mcp-1 (C-C motif chemokine ligand 1; monocyte chemoattractant protein-1) NM_011333.3 (mouse) (forward: 5′-CACTCACCTGCTGCTACTCA-3′; reverse: 5′-GCTTGGTGACAAAAACTACAGC-3′), Mcp-2 (C-C motif chemokine ligand 8; monocyte chemoattractant protein-2) NM_021443.3 (mouse) (forward: 5′-TCAGCCCAGAGAAGCTGACT-3′; reverse: 5′-GGGGGATCTTCAGCTTTAGTACA-3′) [23], Mcp-3 (C-C motif chemokine ligand 7; monocyte chemoattractant protein-3) NM_013654.3 (mouse) (forward: 5′-AGGATCTCTGCCACGCTTC-3′; reverse: 5′-TTGACATAGCAGCATGTGGAT-3′) [23], Mcp-5 (C-C motif chemokine ligand 12; monocyte chemoattractant protein-5) NM_011331.3 (mouse) (forward: 5′-CCACCATCAGTCCTCAGGTATT-3′; reverse: 5′-CGGACGTGAATCTTCTGCTT-3′) [23], Nphs1 (nephrin) NM_019459.2 (mouse) (forward: 5′-CCCAGGTACACAGAGCACAA-3′; reverse: 5′-CTCACGCTCACAACCTTCAG-3′), Nphs2 (podocin) NM_130456.4 (mouse) (forward: 5′-CACTTTGGCCTGTCTTTGTG-3′; reverse: 5′-GCCCAAGATGTAAAGGTTGC-3′), Ppia (peptidylprolyl isomerase A; cyclophilin A) NM_008907.2 (mouse) (forward: 5′-AGGTGAAAGAAGGCATGAAC-3′; reverse: 5′-ACAGTCGGAAATGGTGATCT-3′) [55], Tnfa (tumor necrosis factor alpha) NM_013693.3 (mouse) (forward: 5′-TCGTAGCAAACCACCAAGTG-3′; reverse: 5′-CTTTGAAGAGAACCTGGGAG-3′), or Wt1 (Wilm’s tumor-1) NM_144783.4 (mouse) (forward: 5′-CATCCCAGGCAGGAAAGTGT-3′; reverse: 5′-TGCAGTCAATCAGGTGTGCT-3′). Quantitative PCR (qPCR) was performed using the CFX Connect™ Real-Time System (Bio-Rad Laboratories). Results were normalized to Ppia, and fold changes were determined with the 2^−ΔΔCt method [56]. Melt curves were assessed to ensure specific product amplification.

4.8. Statistical Analysis

Data were subjected to the ROUT outlier test. To compare survival distributions, a log-rank test was performed. For two independent group comparisons, an unpaired Student’s t-test (two-tailed) was performed. To assess between-group differences in longitudinal results, mixed-effects analysis (two-way ANOVA) was performed followed by Bonferroni’s multiple comparisons test. Each point on the graphs represents a mouse. Results are reported as the mean ± S.E.M. and were analyzed with GraphPad Prism 10.1.1 software (GraphPad Software Inc., La Jolla, CA, USA). The threshold for significance was p < 0.05.

5. Conclusions

Our study used a clinically relevant experimental model to induce kidney injury and tubulointerstitial fibrosis in conditional knockout mice generated to investigate the role of tubular epithelia-derived MCP-1. No significant differences were found between control and knockout mice. These findings suggest that tubular epithelia-derived MCP-1 is not necessary for disease development in an Adr model. Although these results did not support our initial hypothesis, these findings nonetheless provide a valuable contribution to our understanding of MCP-1 in CKD pathogenesis.