Hormonal regulation of postnatal development of renal tubular transport processes

Renal tubular transport of p-aminohippurate (PAH) is immature at birth. Repeated saturation of transport sites by treatment with various organic anions is without any influence on the postnatal development of kidney transport capacity. Hormonal regulation of postnatal maturation of PAH transport must therefore be taken into consideration. It was tried to stimulate immature PAH transport by treating rats of different ages with thyroid hormones, corticosteroids or testosterone, respectively. In rats with immature kidney function, renal PAH excretion can be stimulated by daily treatment with thyroid hormones. Experiments on renal cortical slices have shown that PAH excretion is preferentially stimulated by an increase of transport capacity. Whereas thyroid hormones stimulate the renal excretion of PAH both in young and in adult rats, dexamethasone treatment is more effective in rats with immature kidney function. Dexamethasone treatment is without any influence on PAH accumulation in renal cortical slices. Kidney weight and the protein content of kidney tissue was increased after dexamethasone treatment. Repeated testosterone administration did not stimulate the PAH transport in rats of different ages. The data have demonstrated the influence of thyroid hormones or of dexamethasone on renal tubular transport processes in rats with immature kidney function. Treatment with such hormones could be useful in the management of renal insufficiency in full-term and pre-term neonates with immature kidney function.     

Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Effect of S-(2-chloroethyl)-DL-cysteine on the transport of p-aminohippurate ion in renal plasma membrane vesicles

The effects of S-(2-chloroethyl)-DL-cysteine (CEC) (a potent nephrotoxin) on the transport of p-aminohippurate ion (PAH) in renal plasma membrane vesicles isolated from rat renal cortex were studied in vitro. The uptake of PAH was significantly reduced in a dose-dependent manner in both the brush border membrane (BBM) and basolateral membrane (BLM) vesicles. These results demonstrate that CEC is capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. Probenecid, a typical inhibitor of the organic anion transport system, showed the highest inhibition of PAH uptake in both the membranes vesicles. These data indirectly suggest that transport by renal tubular cells may result in the accumulation of CEC in renal cellular organelles eventually in toxic concentrations. Thus, CEC showed both dose- and time-dependent inhibition of the activities of gamma-glutamyl transferase (a BBM marker enzyme) and Na+, K(+)-ATPase (a BLM marker enzyme), while no such inhibition was noticed with probenecid. Pretreatment with probenecid prevented the inhibition of the gamma-glutamyl transferase activity due to CEC in BBM, but failed to do so for the Na+,K(+)-ATPase activity in BLM vesicles. Thus, the data suggest that the inhibition of the activities of these membrane-specific enzymes by CEC could lead to the initial development of its nephrotoxicity.     

Stimulation of renal phosphate secretion in the stenohaline freshwater teleost: Carassius auratus gibelio Bloch

1. Renal excretion of phosphate in the Prussian carp was modulated by tubular reabsorption and tubular secretion. 2. Under the conditions of an i.v. phosphate-load during which the plasma concentration of phosphate was doubled, 65% of the phosphate load was excreted by the kidney, mainly by tubular secretion. 3. The renal clearance of PAH markedly exceeded the clearance of polyfructosan which could be used for the measurement of the GFR. 4. A transport maximum for tubular PAH secretion was reached at plasma concentrations of about 250 mumol/l.     

Stimulation of renal tubular transport by ethacrynic acid in rats of different ages.

The transport system for organic acids in the kidney is not fully developed in the neonatal period. The effect of repeated administrations of ethacrynic acid on the renal excretion of p-aminohippurate (PAH) was studied in rats of different ages. Pretreatment with ethacrynic acid was followed by an increase in the renal excretion of PAH in 33-, 55-, 105- and 240-day-old rats but not in newborn rats. In 55-day-old rats the increase in renal excretion of PAH after pretreatment with ethacrynic acid was not associated with any consistent change of the glomerular filtration rate. It is concluded from these results that the stimulation of transport processes in the kidney by ethacrynic acid and some other drugs is linked with their affinity to tissue proteins.     

Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione

The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.     

Renal tubular transport of delta-aminolevulinic acid in rat

delta-Aminolevulinic acid (ALA) interferes with cell membrane and metabolic functions in a variety of tissues. To determine if ALA interacts with renal tubular transport functions, we examined concentrative transport of this heme precursor in rat kidneys. ALA was accumulated against a concentration gradient in rat renal cortical slices. Section freeze-dry autoradiography demonstrated selective accumulation in cells of proximal tubules. Concentrative uptake of ALA was inhibited by KCN, probenecid and p-aminohippurate (PAH). ALA inhibited slice uptake of PAH but failed to block slice accumulation of galactose, cycloleucine, lysine, glycine, proline, or alpha-aminoisobutyric acid and did not alter O2 utilization. Massive intraperitoneal injection of ALA did not increase 24 hr fractional excretion of amino acids in vivo. Concentrative transport of ALA in proximal tubules does not lead to generalized renal tubular transport defects but ALA appears to share the organic acid secretory system in rat kidney.     

Size selected mRNA induces expression of P-aminohippurate transport in Xenopus oocytes

Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7-1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 microM and 66.6 nmoles.oocyte-1.min-1, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na+ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.     

Renal transport of organic acids and bases in genetically obese mice.

Accumulation of p-aminohippurate (PAH) and N-methylnicotinamide (NMN) by renal cortical slices was used to estimate transport capacity for organic anions and cations, respectively. In a previous study, renal organic anion transport appeared to be selectively depressed in animals rendered obese by high fat feeding. However, the effects of obesity could not be discretely separated from the effects of the 30% fat diet used to produce the obesity. Genetically obese hyperglycemic mice provided a model to determine the effect of obesity on renal transport systems without the complication due to diet. Accumulation of both PAH and NMN was depressed in renal cortical slices from genetically obese mice. Addition of plasma from thin or obese animals increased PAH accumulation by slices from thin animals. Accumulation by slices from obese animals was unaffected by addition of plasma. Oxygen consumption with acetate in the medium was less in kidneys from obese mice than kidneys from thin mice. Thus, in addition to inhibition of transport capacity, renal cortex of genetically obese mice has a biochemical defect that prevents response to stimulators. It is concluded that several renal functions are depressed in the genetically obese hyperglycemic mouse. Whether the depressed function results from the obesity or is concomitant with the gene for obesity is as yet undertermined.     

Effects of penicillin pretreatment on renal tubular para-aminohippurate transport in the immature rat.

The effect of pretreatment with penicillin on para-aminohippurate (PAH) transport by the kidney of the immature rat was evaluated in vivo. After 3 days of penicillin administration, renal clearances of inulin (CIN), PAH (CPAH), and the renal tubular transport maximum (Tm) for PAH were measured in rats as young as 17 days of age. The CPAH in 19- to 21-day-old rats pretreated with procaine penicillin was 54% greater than that of their littermate controls. Similarly, CPAH of rats that received sodium penicillin was 31% greater than control. CIN was not increased after penicillin pretreatment. Pretreatment of rats older than 24 days did not change CIN or CPAH. The Tm for PAH of 17-day-old rats pretreated with sodium penicillin was 51% greater than that of control rats. It was concluded that pretreatment with penicillin enhances the renal secretion of organic anions by the immature rat.     

Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant

The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive ⁸⁶Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.     

Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.     

Inhibitory effect of KW-3902, an adenosine A(1) receptor antagonist, on p-aminohippurate transport in OK cells.

KW-3902 (8-(noradamantan-3-yl)-1,3-dipropylxanthine) is a novel potent and selective adenosine A(1) receptor antagonist. We examined the effect of KW-3902 on p-aminohippurate (PAH) transport in opossum kidney (OK) epithelial cells. Pretreatment for 3 h with KW-3902 inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side. The uptake of PAH across the basolateral membrane of OK cells was inhibited by KW-3902 pretreatment in a time- and concentration-dependent manner. A kinetic analysis revealed that the inhibitory effect of KW-3902 on the basolateral PAH uptake was due to an increase in the Michaelis constant (K(m)) as well as a decrease in the maximum uptake rate (V(max)), showing that the inhibition was a mixed type. Pretreatment with adenosine deaminase or 8-cyclopentyl-1,3-dipropylxanthine, another selective adenosine A(1) receptor antagonist, also decreased the basolateral PAH uptake. KW-3902 pretreatment had no effect on the concentration of intracellular alpha-ketoglutarate which exchanges for PAH across the basolateral membrane of OK cells. These results suggest that KW-3902 has an inhibitory effect on PAH transport in OK epithelial cells.     

Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions when cultured in a hormonally defined serum-free medium

Summary A primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cell were observed to retain in vitro a Na⁺-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na⁺-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitivep-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK acivity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by Grant 9 RO1 DK40286-07 from the National Institutes of Health, Bethesda, MD, and NIH Research Career Development Award 1 K04 CA 0088-01 to M.T.     

Protection of glomerular filtration rate by the thromboxane receptor antagonist L655,240 during low dose cyclosporine administration

Cyclosporin A (CsA) alters the production of prostaglandins (PG) by the kidney. CsA causes an increase in renal vascular resistance, a decrease in renal blood flow, a decrease in glomerular filtration rate (GFR), and increases the renal production of the vasoconstrictor thromboxane. Recently, low dose CsA has been utilized in the treatment of refractory autoimmune diseases. To determine if low dose CsA administration could produce renal hemodynamic alterations and to determine if the thromboxane receptor antagonist L655,240 could prevent these alterations, we administered groups of rats either CsA, 5 mg/kg, subcutaneously and the L655,240 vehicle NaHCO₃ (CsA-NaHCO₃), or CsA and L655,240 (CsA-L655,240), or CsA vehicle and L655,240. The rats were administered the drugs for 7 days and then subjected to inulin and PAH clearances or kidneys were harvested for prostaglandin production studies. CsA significantly depressed GFR and renal plasma flow when compared to the L655,240 treated groups. There was no difference in inulin or PAH clearance between the CsA-L655,240 and CsA vehicle L655,240 groups. Glomerular prostaglandin production including thromboxane was depressed by CsA administration. No histologic alterations were noted in the glomeruli or the medullary portions of the kidney. We conclude that administration of low dose CsA, 5 mg/kg, for 7 days results in a decrease in renal blood flow and GFR without histologic alterations. Administration of the thromboxane receptor antagonist L655,240 prevents the renal hemodynamic alterations induced by CsA in this rat model.     

Saikosaponin B2 attenuates kidney fibrosis via inhibiting the Hedgehog Pathway

BackgroundRenal interstitial fibrosis is a common pathway through which chronic kidney disease progresses to end-stage renal disease. There are currently no effective drugs available to treat kidney fibrosis, so traditional medicine is likely to be a candidate. The therapeutic potential of saikosaponin B2 (SSB2), a biologically active ingredient of Radix Bupleuri, on renal fibrosis has not been reported.MethodsA unilateral ureteral obstruction (UUO) model was conducted to induce renal interstitial fibrosis in mice. SSB2’s effect was valuated by histological staining and exploring the changes in expression of relative proteins and mRNAs. A conditional medium containing sonic hedgehog variant protein stimulating normal rat kidney interstitial fibroblast cells (NRK-49F) was used in an in vitro model to determine the possible mechanism. The molecular target of SSB2 was verified using several mutation plasmids.ResultsSSB2 administration reduced kidney injury and alleviated interstitial fibrosis by decreasing excessive accumulation of extracellular matrix components in UUO mice. It could also reduce the expression of α-SMA, fibronectin and Gli1, a crucial molecule of the hedgehog (Hh) signaling pathway both in vivo and in vitro. In NIH-3T3 cells simulated by conditional medium containing sonic hedgehog variant protein, SSB2 showed the ability to decrease the expression of Gli1 and Ptch1 mRNA. Using a dual-luciferase reporter assay, SSB2 suppressed the Gli-luciferase reporter activity in NIH-3T3 cells, and the IC₅₀ was 0.49 μM, but had no effect on the TNF-α/NF-κB and Wnt/β-catenin signaling pathways, indicating the inhibition selectivity on the Hh signaling pathway. Furthermore, SSB2 failed to inhibit the Hh pathway activity evoked by ectopic expression of Gli2ΔN and Smo D473H, suggesting that SSB2 might potentially act on smoothened receptors.ConclusionSSB2 could attenuate renal fibrosis and decrease fibroblast activation by inhibiting the Hh signaling pathway.