Reducing GEF-H1 Expression Inhibits Renal Cyst Formation, Inflammation, and Fibrosis via RhoA Signaling in Nephronophthisis

Nephronophthisis (NPHP) is the most prevalent monogenic disease leading to end-stage renal failure in childhood. RhoA activation is involved in NPHP pathogenesis. This study explored the role of the RhoA activator guanine nucleotide exchange factor (GEF)-H1 in NPHP pathogenesis. We analyzed the expression and distribution of GEF-H1 in NPHP1 knockout (NPHP1KO) mice using Western blotting and immunofluorescence, followed by GEF-H1 knockdown. Immunofluorescence and renal histology were used to examine the cysts, inflammation, and fibrosis. A RhoA GTPase activation assay and Western blotting were used to detect the expression of downstream GTP-RhoA and p-MLC2, respectively. In NPHP1 knockdown (NPHP1KD) human kidney proximal tubular cells (HK2 cells), we detected the expressions of E-cadherin and α-smooth muscle actin (α-SMA). In vivo, increased expression and redistribution of GEF-H1, and higher levels of GTP-RhoA and p-MLC2 in renal tissue of NPHP1KO mice were observed, together with renal cysts, fibrosis, and inflammation. These changes were alleviated by GEF-H1 knockdown. In vitro, the expression of GEF-H1 and activation of RhoA were also increased, with increased expression of α-SMA and decreased E-cadherin. GEF-H1 knockdown reversed these changes in NPHP1KD HK2 cells. Thus, the GEF-H1/RhoA/MLC2 axis is activated in NPHP1 defects and may play a pivotal role in NPHP pathogenesis.


Introduction
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that is the main genetic cause of end-stage renal disease in childhood [1][2][3][4]. Pathogenic genes encode proteins associated with the structure and function of the primary cilia. To date, over 25 pathogenic genes have been identified, the most prevalent of which is NPHP1 [5][6][7][8][9]. Renal histopathological changes in NPHP are characterized by corticomedullary tubular dilation, cyst formation, diffuse interstitial fibrosis, and inflammation, as well as thickening and layering of the tubular basement membrane [10][11][12][13][14]. Importantly, the pathogenesis of NPHP is not yet fully understood [15]. RhoA activation and a perturbed cytoskeleton have been observed in a variety of ciliopathies, including NPHP, suggesting that RhoA may be implicated in the pathogenesis of NPHP [16,17]. However, the mechanism underlying RhoA activation in ciliopathies remains unclear.
RhoA belongs to the Rho GTPase family and functions as a binary switch regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs activate RhoA by promoting the release of GDP and binding of GTP, whereas GAPs hydrolyze GTP to GDP and switch off RhoA to an inactive conformation [18]. RhoA activation initiates a large number of biological processes essential for cytoskeletal organization, cilium assembly, cell motility, proliferation, polarization, morphology, and contraction [19,20], as well as lumen formation, fibrosis, and inflammation [21][22][23].

The GEF-H1/RhoA Pathway Was Activated in the Renal Tissue of NPHP1 KO Mice and NPHP1 KD HK2 Cells
To investigate alterations in the GEF-H1/RhoA pathway in diseases caused by NPHP1 gene defects, NPHP1 KO mice and NPHP1 KD HK2 cells were chosen for this study. Western blot analysis revealed that the expression of GEF-H1 increased in the kidneys of 24-weekold NPHP1 KO mice ( Figure 1A-C). By immunofluorescence analysis, the intracellular distribution of GEF-H1 in tubular cells was altered, and GEF-H1 was more concentrated on the luminal side of the tubules in NPHP1 KO mice compared with the wild-type (WT) mice. The expression and intracellular distribution of microtubules remained unchanged, while Pearson's correlation coefficients revealed that the degree of microtubule and GEF-H1 colocalization significantly decreased compared with WT mice ( Figure 1D,E). Furthermore, downstream GTP-RhoA activity was elevated, indicating activation of the GEF-H1/RhoA pathway in NPHP1 KO mice ( Figure 1F,G).
In vitro, NPHP1 KD HK2 cells were established following the successful transfection of lenti-shRNA. As shown by the Western blotting results, nephrocystin-1 (the protein encoded by the NPHP1 gene) expression was significantly reduced (p < 0.0001), whereas the GEF-H1 levels were increased in NPHP1 KD HK2 cells (p < 0.01) ( Figure 1H-J). Meanwhile, the expression of GTP-RhoA, the active form of RhoA, was increased ( Figure 1K-L).

Knockdown of GEF-H1 Alleviated Renal Histological Injuries in NPHP1 KO Mice
To explore the effect of the GEF-H1/RhoA pathway on the pathogenesis of NPHP, adeno-associated virus-9 (AAV9) carrying shGEF-H1 or negative control vector (eGFP) was constructed and injected into the bilateral kidneys of mice at approximately 5 weeks, and the mice were sacrificed at 24 weeks ( Figure 2B,C). Green eGFP was observed in the kidney tissues of mice transfected with AAV-shGEF-H1 and AAV-eGFP using immunofluorescence imaging ( Figure 2A). Decreased GEF-H1 expression was detected in kidney specimens from the AAV9-shGEF-H1 transfected mice using Western blotting ( Figure 2D-F). Cyst formation and renal tubular dilatation in the distal convoluted tubules (THP + ) and collecting ducts (DBA + ) were observed in NPHP1 KO mice ( Figure 2G). Renal cyst formation was alleviated ( Figure 2G-I), and renal fibrosis and inflammation improved ( Figure 3A,B) in GEF-H1 knockdown NPHP1 KO mice compared with control NPHP1 KO mice. The improvement in interstitial fibrosis and inflammation was further verified by comparing the amount of collagen deposition, fibroblasts, and macrophage infiltration in the kidney specimens of mice using Masson staining and immunostaining for α-smooth muscle actin and F4/80, respectively ( Figure   We observed a slight elevation in blood urea nitrogen and creatinine levels in NPHP1 KO mice compared with wild-type (WT) mice, which decreased with the AAV-shGEF-H1 intervention; however, these differences were not significant ( Figure 3H-I). Differences in the bilateral kidney weight to body weight ratio (2KW/BW) of mice between the different groups were not significant ( Figure 3J) (p < 0.05).

GEF-H1 Knockdown Alleviated Epithelial-Mesenchymal Transition (EMT) in NPHP1 KD HK2 Cells
We constructed the NPHP1 KD HK2 cell model by transfecting HK2 cells with lentivirus and visualizing eGFP under a fluorescence microscope ( Figure 4A). Increased α-smooth muscle actin (α-SMA) expression and decreased E-cadherin expression in NPHP1 KD HK2 cells were detected using Western blotting compared with WT HK2 cells ( Figure 4B-E), which indicated that NPHP1 KD HK2 cells were in the process of EMT.
To determine the effect of GEF-H1 knockdown on NPHP1 KD HK2 cells, we constructed a GEF-H1 knockdown siRNA (siGEF-H1) vector and a negative control vector (siNC). Compared with siNC-transfected cells, the expression of GEF-H1 protein was decreased in both NPHP1 KD and WT HK2 cells after siGEF-H1-transfected (p < 0.01). At 72 h after transfection with siGEF-H1, NPHP1 KD HK2 cells showed increased E-cadherin expression and decreased α-SMA expression ( Figure 4F-J). These results indicated that GEF-H1 knockdown could reverse EMT in NPHP1 KD HK2 cells.

The GEF-H1/RhoA Pathway Was Associated with MLC2 Phosphorylation in NPHP1 KO Mice
MCL2 is a downstream target of the GEF-H1/RhoA pathway and is a regulator of cell cytoskeletal reorganization, cell morphological changes, and inflammation [36]. To determine if MLC2 was activated in NPHP1 defects, we measured the phosphorylation of MLC2 (p-MCL2) in the kidneys of NPHP1 KO mice. As expected, p-MCL2 was upregulated in the kidneys of NPHP1 KO mice compared with WT mice. This upregulation was decreased after GEF-H1 knockdown in NPHP1 KO mice ( Figure 5A-D). These trends are consistent with those observed for cysts, inflammation, and fibrosis. These results indicate that the GEF-H1/RhoA/MLC2 pathway may play an important role in the pathogenesis of NPHP.     in the kidneys of NPHP1 KO mice compared with WT mice. This upregulation was de creased after GEF-H1 knockdown in NPHP1 KO mice ( Figure 5A-D). These trends are con sistent with those observed for cysts, inflammation, and fibrosis. These results indicate that the GEF-H1/RhoA/MLC2 pathway may play an important role in the pathogenesis o NPHP.

Discussion
Our results show that the expression of GEF-H1 increased and that its intracellular distribution was altered in NPHP1 KO mice and NPHP1 KD HK2 cells. The expression and intracellular distribution of the microtubules remained unchanged. Pearson's correlation coefficients revealed that the degree of microtubule and GEF-H1 colocalization signifi cantly decreased. Downstream, RhoA was activated while the knockdown of GEF-H1 re versed these effects. These results indicated that not only did the expression of GEF-H1 increase but also that GEF-H1 detached from microtubules due to the defects of NPHP1 The GEF-H1/RhoA pathway was activated.
RhoA activation is tightly regulated by GEFs and GAPs. Aberrantly increased RhoA activity has been observed in many ciliopathies. However, the mechanisms underlying RhoA activation in ciliopathies remain largely unknown [37]. In autosomal dominant pol ycystic kidney disease (ADPKD), Streets et al. found that PKD1 defects decreased the ex pression of centrosomal ARHGAP35, which is attributed to persistent RhoA activation

Discussion
Our results show that the expression of GEF-H1 increased and that its intracellular distribution was altered in NPHP1 KO mice and NPHP1 KD HK2 cells. The expression and intracellular distribution of the microtubules remained unchanged. Pearson's correlation coefficients revealed that the degree of microtubule and GEF-H1 colocalization significantly decreased. Downstream, RhoA was activated while the knockdown of GEF-H1 reversed these effects. These results indicated that not only did the expression of GEF-H1 increase but also that GEF-H1 detached from microtubules due to the defects of NPHP1. The GEF-H1/RhoA pathway was activated.
RhoA activation is tightly regulated by GEFs and GAPs. Aberrantly increased RhoA activity has been observed in many ciliopathies. However, the mechanisms underlying RhoA activation in ciliopathies remain largely unknown [37]. In autosomal dominant polycystic kidney disease (ADPKD), Streets et al. found that PKD1 defects decreased the expression of centrosomal ARHGAP35, which is attributed to persistent RhoA activation and its downstream signals and associated cellular biological changes, such as actin cytoskeleton disorganization, ciliogenesis disturbance, and cell morphology changes [16].
NPHP1 is a major pathogenic gene involved in NPHP. Its encoded protein, nephrocytin-1, normally resides in the transition zone of the cilium and cell-cell junctions. The mechanism by which NPHP1 deficiency leads to GEF-H1 detachment from microtubules and is activated remains unclear and merits further study. GEF-H1 normally localizes to the microtubules through its N-and C-termini or through its PH domain in quiescent cells. GEF-H1 can also bind to microtubules by associating with other proteins, such as dynein motor light-chain Tctex-1 [29] and FAM123A, which interact with microtubule end-binding 1 and 3 proteins (EB1 and EB3) [38]. The binding of GEF-H1 to microtubules sequesters it and inhibits GEF activity on RhoA. GEF-H1 activation is usually achieved by microtubule depolymerization. However, depolymerization-independent activation of GEF-H1 has also been reported [38]. In this study, no apparent change in microtubules was observed, indicating that the activation of GEF-H1 in NPHP1 defects is a depolymerizationindependent mechanism.
The association between nephrocystin-1 and microtubules has been observed in previous studies. Otto et al. found that nephrocystin-1 interacts with β-tubulin both in vivo and in vitro. This interaction localizes at the C-terminal region between amino acid residues 237 and 670 of nephrocystin-1 [39]. Mollet et al. showed that nephrocystin-1 and nephrocystin-4 associate with α-tubulin in polarized and ciliated tubular MDCK cells [31]. These studies indicate that both α-tubulin and β-tubulin interact with nephrocystin-1. Defects in nephrocytin-1 may affect microtubules; however, more studies are needed to clarify this.
GEF-H1 can also bind to apical junctions (AJs) by integration with cingulin or paracingulin in polarized epithelial cells [31], which sequester it to interact with RhoA. Cell-cell junctions are also involved in the localization of nephrocystin-1. Therefore, nephrocystin-1 defects may also affect GEF-H1 in AJs. However, the association of nephrocytin-1 with GEF-H1 and its role in the pathogenesis of NPHP1 defects deserves further study.
Our results revealed that the knockdown of GEF-H1 not only restored the activation of RhoA and phosphorylation of MCL2 but also reversed cyst formation, interstitial fibrosis, and inflammation in NPHP1 KO mice, and inhibited EMT changes in NPHP1 KD HK2 cells by downregulating the expression of α-SMA and upregulating the expression of E-cadherin. EMT may be involved in the pathogenesis of renal fibrosis in cilia-related cystic disease [40][41][42], as observed in kidneys from other NPHP patients and in mouse models [41]. Aberrant activation of RhoA, disorganized actin filaments, and disturbed ciliogenesis have long been observed in various ciliopathies [37]. Blocking of RhoA/ROCK signaling by the ROCK inhibitor Y-27632 restores cilia length and the disorganized actin filaments in PKD1 KD cells and reduces cyst formation in PKD1 KO mice [16]. Recently, Garcia H. et al. validated prostaglandin signaling as a target of NPHP1-defective NPHP. PGE1 treatment can decrease the upregulated RhoA activity and attenuate the renal manifestations in NPHP1 KO mice, including tubular dilatation, early upregulation of fibrosis markers and extracellular matrix components, and abnormal cilia [17].
Cyst formation, interstitial fibrosis, and inflammation are the characteristic pathological changes in NPHP. Cyst formation is a process influenced by many factors, such as cytoskeletal dynamics, intracellular and extracellular mechanical forces, and polarized membrane transport [43][44][45]. Crosstalk between different cytoskeletons is necessary to ensure a constant subcellular lumen diameter and to prevent cyst formation [45]. Cytoskeletal disturbances are common features of cyst formation. ROCK is a key actin-remodeling regulator responsible for the formation of stress fibers, and RhoA is an activator of ROCK [37]. Dysregulation of the RhoA/ROCK pathway leads to a highly disorganized cytoskeleton in cyst cells in ADPKD, as well as in other ciliopathies [16,37]. ROCK inhibitors have successfully improved cyst formation in ADPKD, suggesting that the cytoskeletal-associated RhoA/ROCK pathway may be a major factor in cyst initiation [16].
Fibrosis and inflammation are common pathophysiological processes that contribute to disease progression in chronic kidney diseases (CKDs). TGF-β plays a central role in renal fibrosis in most CKDs. In our previous study, we observed the activation of the TGF-β/Smad signaling pathway and its role in inducing EMT in NPHP1-defective MDCK cells [46]. Increased expression of TGF-β1 and EMT at the site of renal interstitial fibrosis were observed in NPHP7-mutant patients and mouse models [41]. However, GEF-H1 has been observed to be a downstream effector of TGF-β, and blockage of GEF-H1 can reverse TGF-β-induced cytoskeleton reorganization and EMT changes in vitro [47,48]. Inactivation of GEF-H1 in patients with mechanical trauma and uveitis can ameliorate disease progression by inhibiting fibrosis and inflammation [48].
In addition to actin remodeling, the GEF-H1/RhoA/MLC2 pathway is also implicated in mediating inflammation, intercellular permeability, and cell adhesion, which are associated with reperfusion injury, epithelial or endothelial barrier failure, and tumor metastasis [49][50][51].
In the present study, the knockdown of GEF-H1 abrogated RhoA and MLC2 activation in NPHP1 KO mice, with alleviated renal pathological changes, including cyst formation, interstitial fibrosis, and inflammation, as indicated by macrophage infiltration. In vitro, EMT changes in NPHP1 KD HK2 cells were reversed by GEF-H1 knockdown. These results indicate that the GEF-H1/RhoA/MLC2 axis plays a key role in the pathogenesis of NPHP1defective NPHP. However, the detailed mechanism requires further investigation.

Mice and Antibodies
NPHP1 KO C57BL/6J mice were generated by our group by deleting exons 2 to 20 of NPHP1 using the CRISPR/Cas9 technique [52]. All animal experiments were performed according to the ARRIVE1 guidelines, and protocols were approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University. The

Urea and Creatinine Measurements
Blood was collected from the retroorbital vein. The serum was then centrifuged and separated at room temperature. An automatic biochemical analyzer was used to detect urea and creatinine levels (Biobase BK280, Shandong, China).

Cell Culture
The HK2 cell line was purchased from the Public Medical Laboratory Center of Nanfang Hospital, Southern Medical University. The cells were routinely cultured and maintained at 37 • C in a humidified 5% CO2 incubator in DMEM/F12 (Gibco, Billings, MT, USA) containing 10% fetal bovine serum (Gibco, Billings, MT, USA) and 1% antibiotics (Gibco, Billings, MT, USA).

Lentiviral Transfection
For NPHP1 knockdown, we used high-titer lentiviral shRNA vectors prepared by GenePharma Co. (Shanghai, China). Transfection was performed according to the protocol. Briefly, cells cultured in 24-well plates (1 × 10 4 cells/well) were transduced with lentivirus for 24 h. Cells were then selected and expanded with puromycin (4 µg/mL) for 6 to 10 days after 48 h of transduction. The efficiency of infection was determined using Western blotting. We used a medium containing 2 µg/mL puromycin to establish stable knockdown (shNPHP1) or negative control (shNC) HK2 cell lines.

Protein Extraction and Western Blotting
Proteins were extracted from the tissue and cell samples and used for Western blot analysis. RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail, and a phosphatase inhibitor cocktail was used to lyse the cells and tissues (CWBIO, Beijing, China). The amount and quality of proteins were determined using a bicinchoninic acid assay (BCA) (Beyotime, Shanghai, China) and a Synergy H1 Microplate Reader (BioTek, Shoreline, WA, USA). The diluted proteins were incubated at 100 • C for 5 min. Proteins were separated using 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to polyvinylidene difluoride membranes for Western blotting. The membranes were blocked with Tris-buffered saline supplemented with 0.1% (v/v) Tween 20 (TBST) and freshly-prepared 100 g/L non-fat milk for 2 h at 25 • C, followed by incubation at 4 • C overnight in the presence of various primary antibodies. The following day, the membranes were washed 3-5 times and incubated with fluorescein-conjugated secondary antibodies at room temperature for 1 h. An Odyssey scanner (Li-Cor Biosciences, Lincoln, NE, USA) was used to scan the immunoblots. Band intensities were quantified using the Image Studio software (Ver5.0, LI-COR, Lincoln, NE, USA), and GAPDH was used as the reference.

RhoA GTPase Activation Assay
GTP loading of RhoA was measured using a RhoA Activation Kit (NewEast Biosciences, Upper Merion Township, PA, USA) according to the manufacturer's instructions. Briefly, a small fraction of the protein lysate was isolated and used to measure the total RhoA levels. The remaining protein lysate was incubated with glutathione S-transferase (GST) bound to Rhotekin RBD beads for 1.5 h. The beads were centrifuged, washed, and resuspended in a loading buffer. After boiling for 5 min, all samples were subjected to SDS-PAGE. Western blot analysis revealed the presence of RhoA.

Immunofluorescence
Samples on glass slides were fixed for 20 min with ice-cold 4% paraformaldehyde, washed twice with phosphate-buffered saline (PBS), and permeabilized for 5 min with 0.1% Triton X-100 in PBS. Following a PBS wash, the cells were blocked with 10% goat serum for 1 h before incubation with primary antibodies overnight at 4 • C. Following 3 PBS washes, cells were incubated with secondary antibodies at room temperature for 1 h. Slides were mounted with an anti-fade reagent (Invitrogen SlowFade Gold) and photographed using a super-resolution microscope (N-SIM, Nikon, Tokyo, Japan) and an inverted microscope (OLYMPUS DP80, Tokyo, Japan).

Histopathological Analysis
The kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson's trichrome staining were performed, and the results were observed under a light microscope. The ImageJ software (Ver1.8.0, NIH, Bethesda, MD, USA) was used to analyze the HE and Masson trichrome-stained areas.

Statistical Analysis
Experiments were independently repeated at least three times. Data were normally distributed, and a two-tailed t-test was used to compare the two groups. One-way analysis of variance (ANOVA) followed by the Tukey test was used for comparison among multiple groups. The data are presented as mean ± SEM. p < 0.05 was considered statistically significant. GraphPad Prism 9.3.0 (Dotmatics, San Diego, CA, USA) was used for the statistical analysis.

Conclusions
Our results indicate that NPHP1 defects induce GEF-H1 expression and alter its intracellular distribution. Altered GEF-H1 is a key regulator in the pathogenesis of NPHP1defective NPHP, and GEF-H1-associated RhoA activation and MLC2 phosphorylation may play pivotal roles in the disease process. Our study provides novel insights into the pathogenesis of NPHP and a foundation for developing targeted treatments.