The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfβ), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.

Tubulointerstitial injuries are known to predict the deterioration of renal function in chronic kidney disease (CKD). We recently reported the protective role of Vasohibin-1(VASH-1), a negative feedback regulator of angiogenesis, in diabetic nephropathy, but its impact on tubulointerstitial injuries remains to be elucidated. In the present study, we evaluated the role of endogenous VASH-1 in regulating the tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO), and assessed its role on fibrogenesis and the activation of Smad3 signaling in renal fibroblasts. UUO was induced in female Vasohibin-1 heterozygous knockout mice (VASH-1(+/-)) or wild-type (WT) (VASH-1(+/+)) littermates. Mice were sacrificed on Day 7 after left ureter ligation, and the kidney tissue was obtained. Interstitial fibrosis, the accumulation of type I and type III collagen and monocytes/macrophages infiltration in the obstructed kidneys (OBK) were significantly exacerbated in VASH-1(+/-) mice compared with WT mice (Day 7). The increases in the renal levels of TGF-β1, pSmad3, NF-κB pp65, CCL2 mRNA, and the number of interstitial fibroblast-specific protein-1 (FSP-1)(+) fibroblasts in the OBK were significantly aggravated in VASH-1(+/-) mice. In addition, treatment with VASH-1 siRNA enhanced the TGF-β1-induced phosphorylation of Smad3, the transcriptional activation of the Smad3 pathway and the production of type I/type III collagen in fibroblasts, in vitro. Taken together, our findings demonstrate a protective role for endogenous VASH-1 on tubulointerstitial alterations via its regulation of inflammation and fibrosis and also show the direct anti-fibrotic effects of VASH-1 on renal fibroblasts through its modulation of TGF-β1 signaling.

Metformin is today the first choice treatment for type-2 diabetes, but has also protective effects in several renal disease models. Previously, we have demonstrated that the protective effects in response to unilateral ureteral obstruction (UUO) are independent of organic cation transporters (OCTs), the transporters responsible for the metformin uptake into the renal cells. The mechanisms behind the renoprotective effects are incompletely understood, but our previous results indicate that the renoprotective effects at least partly could be dependent on actions of metformin outside the renal cells. In this study, we investigate whether the renoprotective effects of metformin can be mediated via systemic immunomodulatory actions. We demonstrated that metformin can affect the immune system in the kidney as well as in the peripheral blood and spleen following UUO. UUO kidneys showed infiltration of immune cells including monocytes, B cells, and T cells, but metformin limited infiltration of all cell types. UUO animals had increased spleen sizes, but this increase was attenuated by metformin. Metformin treatment surprisingly resulted in a higher proportion of monocytes with infiltratory capacity 7 days after UUO. Other studies have suggested that metformin regulates monocyte maturation through signal transducer and activator of transcription 3 (STAT3) activation, as also indicated by our results. In conclusion, our results demonstrate that metformin limits the infiltration of immune cells into the kidney, as well as modulates immune cell composition at a systemic level.

Renal fibrosis is tightly associated with chronic kidney disease, irrespective of the underlying pathogenesis. We previously demonstrated mild antifibrotic effects of targeting the P2X7 receptor in a pyelonephritis model. Reduced P2X7 R-activation elevated the neutrophil-to-macrophage ratio, resulting in less matrix accumulation without affecting the initial tissue healing. Here, we test if this P2X7 R-dependent modification of matrix accumulation also applies to a noninfectious fibrosis model of unilateral ureteral obstruction (7dUUO) and whether the response is gender-dependent. We found that P2X7 -/- mice show reduced fibrosis compared to wild type after 7dUUO: the effect was most pronounced in females, with a 55% decrease in collagen deposition after 7dUUO (p < 0.0068). P2X7 R deficiency did not affect early fibrosis markers (TGF-β, α-SMA) or the renal infiltration of neutrophils. However, a UUO-induced increase in macrophages was observed in wildtypes only (p < 0.001), leaving the P2X7 -/- mice with ≈50% fewer CD68+ cells in the renal cortex (p = 0.018). In males, 7dUUO triggered an increase in diffusely interstitial scattering of the profibrotic, macrophage-attracting metalloproteinase MMP8 and showed significantly lower MMP8 tissue expression in both male and female P2X7 -/- mice (p < 0.0008). Thus, the P2X7 R is advocated as a late-stage fibrosis moderator by reducing neutrophil-dependent interstitial MMP8 release, resulting in less macrophage infiltration and reduced matrix accumulation.

Renal obstruction is frequently found in adults and children. Mechanical stimuli, including pressure and stretch in the obstructed kidney, contribute to damage; animal models of obstruction are characterized by increased cellular proliferation. We were interested in the direct effects of pressure and stretch on renal tubular cell proliferation. Human HKC-8 or rat NRK-52E proximal tubule cells were subjected to either pressure [0, 60 or 90 mmHg] or static stretch [0 or 20%] for 24 or 48 h. Cell proliferation was measured by cell counting, cell cycle analyzed by flow cytometry, and PCNA and Skp2 expression were determined by qPCR or western blot. Blood gases were determined in an iSTAT system. Proliferation was also assessed in vivo after 24 h of ureteral obstruction. There was a significant increase in HKC-8 cell number after 48 h of exposure to either 60 or 90 mmHg pressure. Western blot and qPCR confirmed increased expression of PCNA and Skp2 in pressurized cells. Cell cycle measurements demonstrated an increase in HKC-8 in S phase. Mechanical stretching increased PCNA protein expression in HKC-8 cells after 48 h while no effect was observed on Skp2 and cell cycle measurements. Increased PCNA expression was found at 24 h after ureteral obstruction. We demonstrate direct transduction of pressure into a proliferative response in HKC-8 and NRK-52E cells, measured by cell number, PCNA and Skp2 expression and increase in cells in S phase, whereas stretch had a less robust effect on proliferation.

Human GWAS and Mendelian genetic studies have linked polymorphic variants and mutations in the human uromodulin gene (UMOD) with chronic kidney disease. The primary function of this kidney-specific and secreted protein remains elusive. This study investigated whether UMOD deficiency modified responses to unilateral ureteral obstruction (UUO)-induced kidney injury. Kidneys harvested from groups of wild-type (UMOD+/+) and knockout (UMOD-/-) male mice (n = 7-10 each) were studied on days 7, 14, and 21. Compared to sham kidneys, UMOD protein levels increased 9-13x after UUO and were associated with increased urinary UMOD levels. Kidney KIM-1 protein levels were higher in the UMOD-/- groups at all time-points (4-14x). The UMOD-/- groups also had higher KIM-1 kidney-to-urine relative ratios (5-35x). In vitro studies using KIM-1 expressing 769-P cells showed lower KIM-1 levels in the presence of UMOD protein. Levels of proapoptotic genes and the epithelial cell apoptotic protein marker M30 were significantly lower in the UMOD-/- groups. Both M30 and KIM-1 colocalized with intraluminal UMOD protein deposits. Interstitial inflammation was less intense in the UMOD-/- groups. Renal fibrosis severity (kidney collagen mRNA and protein) was similar in both genotypic groups on days 7, 14, and 21. Our findings suggest a role for UMOD-dependent inhibition of KIM-1 expression and its apoptotic cell scavenging responses during chronic obstruction-associated tubular injury.

Plasma membrane calcium pump isoform 1 (PMCA1) is encoded by ATPase plasma membrane Ca2+transporting 1 (ATP2B1), the most likely candidate gene responsible for hypertension. Although PMCA1 is highly expressed in the kidney, little is known about regulation of its renal expression in various pathological conditions in vivo. Our study was designed to elucidate regulation of renal PMCA1 expression in mice. We employed three mouse models for kidney disease. These were the unilateral ureteral obstruction (UUO), the remnant kidney using 5/6 nephrectomy, and chronic angiotensin II administration models. Mice were assessed for systolic blood pressure and renal injury in accordance with the damage induced in the specific model. Kidney PMCA1 mRNA levels were measured in all mice. The UUO model showed renal fibrosis but no changes in blood pressure or renal PMCA1 mRNA expression. Similarly, the 5/6 nephrectomy model exhibited declined renal function without changes in blood pressure or renal PMCA1 mRNA expression. In contrast, chronic angiotensin II administration increased albuminuria and blood pressure as well as significantly increasing renal PMCA1 mRNA and protein expression. These results suggest that renal PMCA1 has a role as one of the molecules involved in angiotensin II-induced hypertension and kidney injury.

Short-chain fatty acids (SCFAs) are the end products of the fermentation of dietary fibers by the intestinal microbiota and reported to exert positive effects on host physiology. Acetate is the most abundant SCFA in humans and is shown to improve acute kidney injury in a mouse model of ischemia-reperfusion injury. However, how SCFAs protect the kidney and whether SCFAs have a renoprotective effect in chronic kidney disease (CKD) models remain to be elucidated. We investigated whether acetate and other SCFAs could attenuate the kidney damage. In in vitro experiments, cell viability of acetate-treated human kidney 2 (HK-2) cells was significantly higher than that of vehicle-treated in an oxidative stress model, and acetate reduced cellular reactive oxygen species (ROS) production. In mitochondrial analysis, the MitoSOX-positive cell proportion decreased, and transcription of dynamin-1-like protein gene, a fission gene, was decreased by acetate treatment. In in vivo experiments in mice, acetate treatment significantly ameliorated fibrosis induced by unilateral ureteral obstruction, and the oxidative stress marker phosphorylated histone H2AX (γH2AX) was also reduced. Further, acetate treatment ameliorated dysmorphic mitochondria in the proximal tubules, and ROS and mitochondrial analyses suggested that acetate improved mitochondrial damage. Our findings indicate a renoprotective effect of acetate in CKD.

Kidney fibrosis is a histological hallmark of chronic kidney disease (CKD) and is believed to be involved in the progression of CKD. Therefore, inhibition of kidney fibrosis is a potential strategy for slowing CKD progression. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated by interleukin-6 and is reported to be involved in fibrosis. Previously, S3I-201, an inhibitor of STAT3 phosphorylation, was shown to inhibit renal fibrosis in a mouse model, but its mechanism was not clarified completely. In this study, we investigated whether STX-0119, a new inhibitor of STAT3 dimerization, suppressed kidney fibrotic gene expression using a mouse model of kidney fibrosis and examined the underlying mechanisms. Kidney fibrosis was induced by unilateral ureteral obstruction (UUO), which was accompanied by upregulation of STAT3 target genes. STX-0119 administration suppressed the expression of fibrotic genes in UUO kidneys without affecting STAT3 phosphorylation. STX-0119 decreased Cxcr4 mRNA in cultured rat kidney fibroblasts and Ccr1 mRNA in blood cells from UUO mice, both of which are reported to be involved in the progression of kidney fibrosis. These results suggest that STX-0119 inhibits fibrotic gene expression in kidney by suppressing Cxcr4 and Ccr1 expression. This is the first report to indicate a part of the mechanism of the antifibrotic effects of a STAT3 inhibitor and suggests that STX-0119 may be a lead compound for the treatment of kidney fibrosis.

IL-22, a member of the IL-10 cytokine family, accelerates tubule regeneration upon acute kidney injury, hence we speculated on a protective role also in chronic kidney disease. We quantified intrarenal IL-22 expression after unilateral ureteral (UUO) in wild-type mice and performed UUO in IL-22 knock-out animals. Obstruction phenotypic differences between IL22+/+ and IL22-/- mice were assessed by histology, immunohistochemistry, immunofluorescence as well as western blotting and reverse-transcriptase quantitative PCR ex vivo. Additionally, we performed in vitro experiments using both murine and human tubular cells to characterize IL-22 effects in epithelial healing. We found increasing IL-22 positivity in infiltrating immune cells over time upon UUO in wild-type mice. UUO in IL22-/- mice caused more tubular cell injury as defined by TUNEL positive cells and loss of tetragonolobus lectin staining. Instead, tubular dilation, loss of CD31+ perivascular capillaries, and interstitial fibrosis were independent of the Il22 genotype as assessed by standard histology, immunostaining, and mRNA expression profiling. In vitro experiments showed that recombinant human IL-22 significantly enhanced human tubular epithelial cell proliferation and wound closure upon mechanical injury, and electric cell-substrate impedance sensing studies revealed that recombinant IL-22 sustained tubular epithelial barrier function upon injury. In contrast, IL-22 had no such direct effects on human fibroblasts. Together, in progressive kidney remodeling upon UUO, infiltrating immune cells secrete IL-22, which augments tubular epithelial integrity and epithelial barrier function, but does not affect vascular rarefaction or fibrogenesis. We conclude that IL-22 could represent a molecular target to specifically modulate tubular atrophy.

The major sphingolipid metabolite, sphingosine-1-phosphate (S1P), has important biological functions. S1P serves as a ligand for a family of five G-protein-coupled receptors with distinct signaling pathways regulating important biological pathways. S1P induces renal fibrosis through an inflammatory pathway. However, its direct fibrosis-inducing effect on the kidney has not been shown. The role of S1P as a direct mediator of renal fibrosis was investigated in normal rat kidney interstitial fibroblast (NRK-49F) cells (in vitro) and kidneys of a unilateral ureteral obstruction (UUO) mouse model (in vivo). To clarify the role of S1P in renal fibrosis, we adopted nude UUO mice with immune response deficits. NRK-49F cells were stimulated with various concentrations of exogenous S1P and FTY720 (a S1P receptor agonist) or N,N-dimethylsphingosine (DMS; a sphingosine kinase inhibitor). C57BL6 and nude UUO mice were pretreated with FTY720, DMS, or saline. Expression levels of alpha-smooth muscle actin (a-SMA), E-cadherin, collagen type 1 (COL1), collagen type 4 (COL4), tissue inhibitor of matrix metalloproteinase-1 (TIMP1), and plasminogen activator inhibitor-1 (PAI1) were examined. S1P stimulated fibrosis in NRK-49F cells and UUO mice. Increased a-SMA, COL1, COL4, TIMP1, and PAI1 and decreased E-cadherin expression levels were observed in both the S1P-stimulated cells and UUO mice. Nude UUO mouse kidneys expressed fibrotic markers. Fibrotic changes were successfully induced in both UUO and nude UUO mice, evident through prominent fibronectin and COL1 staining. These S1P-induced fibrotic changes were suppressed by FTY720 and DMS both in vitro and in vivo. Thus, S1P essentially and directly mediates renal fibrosis.