Smad2 Protects against TGF-β/Smad3-Mediated Renal Fibrosis

Smad2 and Smad3 interact and mediate TGF-β signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-β1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-β/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-β1. Conversely, overexpression of Smad2 attenuated TGF-β1–induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-β–mediated fibrosis by counteracting TGF-β/Smad3 signaling.TGF-β/Smad signaling has been shown to play a critical role in renal fibrosis.^1–4 It is now clear that TGF-β1 signals through the heteromeric complex of TGF-β type I receptor and TGF-β type II receptor to activate two key downstream mediators, Smad2 and Smad3, to exert its biological activities such as cell growth, differentiation, extracellular matrix (ECM) production, and apoptosis.⁵ Although it is known that Smad2 and Smad3 physically interact and are structurally similar with >90% homology in their amino acid sequences,⁶ the distinct functions of these two genes in embryonic development has been noted. Genetic deletion of Smad2 in mice results in embryonic lethality at an early stage of development, whereas mice null for Smad3 survive with impaired immunity.^7,8 Although the functional role of Smad3 in cell growth, differentiation, apoptosis, tissue repair and fibrosis, and immune responses has been well studied,^8–10 the pathophysiologic role of Smad2 in these processes remains largely unclear. This may be attributed to the unavailability of Smad2 knockout (KO) mice for such studies as a result of the early embryonic lethality.In the context of tissue repair and fibrosis, Smad3 has been studied extensively, but little attention has been paid to the functional role of Smad2. It is now well accepted that Smad3 is a key mediator of TGF-β signaling in ECM production and tissue fibrosis. This may be associated with the finding that Smad3-binding elements are found in most collagen promoters^11–13; therefore, TGF-β1–induced collagen matrix expression is Smad3 dependent. Emerging evidence has shown that Smad3 plays an important role in tissue repair and fibrosis including wound healing,¹⁴ epithelial-to-mesenchymal transition (EMT),¹⁵ and tissue scar formation under various disease conditions in skin,¹³ lung,¹⁶ heart,¹⁷ kidney,¹⁵ and liver¹⁸; however, the functional importance of Smad2 and the interaction between Smad2 and 3 in the profibrotic response to TGF-β1 remain largely unclear. Thus, this study investigated the functional role of Smad2 in ECM production and renal fibrosis in vivo in a mouse model of unilateral ureteral obstruction (UUO) and in vitro in tubular epithelial cells (TECs) with knockdown or overexpression of Smad2 and in mouse embryonic fibroblasts (MEFs) lacking Smad2. The mechanisms of Smad2 in regulating fibrosis in response to TGF-β1 were explored.     

miR-192 Mediates TGF-β/Smad3-Driven Renal Fibrosis

TGF-β/Smad3 promotes renal fibrosis, but the mechanisms that regulate profibrotic genes remain unclear. We hypothesized that miR-192, a microRNA expressed in the kidney may mediate renal fibrosis in a Smad3-dependent manner. Microarray and real-time PCR demonstrated a tight association between upregulation of miR-192 in the fibrotic kidney and activation of TGF-β/Smad signaling. Deletion of Smad7 promoted miR-192 expression and enhanced Smad signaling and fibrosis in obstructive kidney disease. In contrast, overexpression of Smad7 to block TGF-β/Smad signaling inhibited miR-192 expression and renal fibrosis in the rat 5/6 nephrectomy model; in vitro, overexpression of Smad7 in tubular epithelial cells abolished TGF-β1–induced miR-192 expression. Furthermore, Smad3 but not Smad2 mediated TGF-β1–induced miR-192 expression by binding to the miR-192 promoter. Last, overexpression of a miR-192 mimic promoted and addition of a miR-192 inhibitor blocked TGF-β1–induced collagen matrix expression. Taken together, miR-192 may be a critical downstream mediator of TGF-β/Smad3 signaling in the development of renal fibrosis.TGF-β is a profibrogenic cytokine that mediates renal fibrosis positively by activating its downstream mediators called Smad2 and Smad3 but negatively by its inhibitory factor Smad7^1–4; however, the mechanisms as to how the Smads regulate the fibrogenic genes during renal fibrosis remain unclear.MicroRNAs (miRNAs) are small, noncoding RNAs with approximately 22 nucleotides. The mature miRNAs can complementarily bind to the mRNA 3′ untranslated region to regulate the gene expression by translational repression or induction of mRNA degradation.⁵ Increasing evidence shows that TGF-β may act by regulating miRNAs to exhibit its biological effects such as epithelial-to-mesenchymal transition (EMT),⁵ suggesting that TGF-β regulates the expression of these miRNAs to promote EMT.Several miRNAs, including miR-192, -194, -204, -215, and -216, are highly expressed in the kidney, as compared with other organs.^6,7 In the context of renal fibrosis, expression levels of miR-192 increased significantly in glomeruli isolated from diabetic mice.⁸ In vitro, miR-192 is induced by TGF-β1 and mediates TGF-β–induced collagen expression in mesangial cells (MCs) by downregulating ZEB2 expression.⁸ In contrast, a recent study also found that TGF-β1 suppresses miR-192 expression in human tubular epithelial cells (TEC) and loss of miR-192 promotes fibrogenesis in diabetic nephropathy.⁹ The discrepancy in these two studies with opposite findings and understanding of miR-192 in diabetic nephropathy necessitates further investigation of the potential role of miR-192 and the mechanisms that regulate miR-192 expression during renal fibrosis under various disease conditions. Thus, this study tested the hypothesis that TGF-β1 may act by stimulating Smad3 to regulate miR-192 expression during renal fibrosis. This was tested in rodent models of obstructive and remnant kidney diseases induced in mice that lacked Smad3 or Smad7, had conditional knockout (KO) for Smad2, or overexpressed renal Smad7. In addition, TGF-β–induced miR-192 expression via the Smad3-dependent mechanism was determined in TECs overexpressing Smad7 or knocking down for Smad2 or Smad3 and in Smad2 or Smad3 KO mouse embryonic fibroblasts (MEFs).     

Advanced Glycation End-Products Induce Tubular CTGF via TGF-β–Independent Smad3 Signaling

Advanced glycation end-products (AGEs) can induce expression of connective tissue growth factor (CTGF), which seems to promote the development of diabetic nephropathy, but the exact signaling mechanisms that mediate this induction are unknown. Here, AGEs induced CTGF expression in tubular epithelial cells (TECs) that either lacked the TGF-β1 gene or expressed dominant TGF-β receptor II, demonstrating independence of TGF-β. Furthermore, conditional knockout of the gene encoding TGF-β receptor II from the kidney did not prevent AGE-induced renal expression of CTGF and collagen I. More specific, AGEs induced CTGF expression via the receptor for AGEs-extracellular signal–regulated kinase (RAGE-ERK)/p38 mitogen-activated protein kinase–Smad cross-talk pathway because inhibition of this pathway by several methods (anti-RAGE antibody, specific inhibitors, or dominant negative adenovirus to ERK1/2 and p38) blocked this induction. Overexpressing Smad7 abolished AGE-induced Smad3 phosphorylation and CTGF expression, demonstrating the necessity for activation of Smad signaling in this process. More important, knockdown of either Smad3 or Smad2 demonstrated that Smad3 but not Smad2 is essential for CTGF induction in response to AGEs. In conclusion, AGEs induce tubular CTGF expression via the TGF-β–independent RAGE-ERK/p38-Smad3 cross-talk pathway. These data suggest that overexpression of Smad7 or targeting Smad3 may have therapeutic potential for diabetic nephropathy.Connective tissue growth factor (CTGF; CCN2), a member of CCN family of growth factors, plays an important role in connective tissue homeostasis and fibroblast proliferation, migration, adhesion, and extracellular matrix expression.¹ Clinically, renal expression of CTGF is increased in patients with diabetic nephropathy (DN), and its expression correlates closely with the degree of albuminuria.^2,3 In addition, studies in human renal biopsy show that CTGF expression significantly augments glomerular and tubulointerstitial injury with α-smooth muscle actin cell accumulation.⁴ Several pieces of evidence from recent rodent studies further support the notion that CTGF is important in the pathogenesis of DN. For example, the thickening of glomerular basement membrane is attenuated in CTGF^+/− mice.⁵ In type 1 diabetic mouse model, cell-specific overexpression of CTGF in podocytes of CTGF transgenic mice is able to intensify proteinuria and mesangial expansion.⁶ The co-localization of increased renal CTGF expression and AGE accumulation in diabetic rats indicates a causal link between AGE deposition and CTGF expression.⁷ This is supported by the ability of the AGE inhibitor to suppress CTGF expression and reduce renal fibrosis.⁷ Although the mechanisms that regulate renal CTGF function are not clearly understood, CTGF should play an essential role in DN.Engagement of AGEs to the receptor (RAGE) has been shown to play a critical role in diabetic complications, including DN.⁸ Indeed, AGE-induced tubular epithelial-to-mesenchymal transition (EMT) and renal fibrosis are RAGE dependent.^8,9 Under diabetic conditions, although treatments with high glucose and angiotensin II are also able to upregulate CTGF expression in glomerular mesangial cells (MCs) and TECs,^2,10–12 it is clear that AGEs mediate CTGF expression by stimulating TGF-β expression.^13,14 It is generally believed that TGF-β/Smad signaling should be responsible for inducing CTGF expression because CTGF is a downstream mediator of TGF-β signaling^{11,12,15–17}; however, the exact mode of signaling mechanisms by which AGEs induce CTGF expression remains largely unclear.Our previous study of MCs, TECs, and vascular smooth muscle cells (VSMCs) showed that AGEs are able to induce Smad2/3 phosphorylation markedly in TGF-β receptor I (TβRI) and TβRII mutant cell lines via the extracellular signal–regulated kinase (ERK)/p38 mitogen-activate protein kinase (MAPK)-dependent mechanism.¹⁸ This demonstrates a critical role for the TGF-β–independent Smad pathway in AGE-mediated fibrotic response. This is further supported by the finding that blockade of TGF-β1 with specific small hairpin RNA (shRNA) and a neutralizing antibody is unable to inhibit significantly AGE-induced CTGF mRNA expression.¹⁹ All of these studies suggest a TGF-β–independent mechanism in regulating CTGF expression in response to AGEs. Because AGEs are capable of activating the TGF-β/Smad signaling pathway via the ERK/p38 MAPK-dependent mechanism and because CTGF is a target gene of TGF-β/Smad signaling,^7,15,19–22 we thus hypothesized that AGEs might induce CTGF expression via the TGF-β–independent Smad3 signaling pathway. This was tested in mouse TECs lacking TGF-β1 gene²³ and rat TEC lines overexpressing the dominant negative TβRII or Smad7 or having a knockdown of Smad2 or Smad3. Finally, the functional importance of the TGF-β–independent signaling pathway in AGE-mediated CTGF expression and renal fibrosis was tested in mice that had conditional knockout (KO) for TβRII from the kidney.     

Autophagy Regulates TGF-β Expression and Suppresses Kidney Fibrosis Induced by Unilateral Ureteral Obstruction

Autophagy is an evolutionarily conserved process that cells use to degrade and recycle cellular proteins and remove damaged organelles. During the past decade, there has been a growing interest in defining the basic cellular mechanism of autophagy and its roles in health and disease. However, the functional role of autophagy in kidney fibrosis remains poorly understood. Here, using GFP-LC3 transgenic mice, we show that autophagy is induced in renal tubular epithelial cells (RTECs) of obstructed kidneys after unilateral ureteral obstruction (UUO). Deletion of LC3B (LC3^−/− mice) resulted in increased collagen deposition and increased mature profibrotic factor TGF-β levels in obstructed kidneys. Beclin 1 heterozygous (beclin 1^+/−) mice also displayed increased collagen deposition in the obstructed kidneys after UUO. We also show that TGF-β1 induces autophagy in primary mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. LC3 deficiency resulted in increased levels of mature TGF-β in primary RTECs. Under conditions of TGF-β1 stimulation and autoinduction, inhibition of autolysosomal protein degradation by bafilomycin A1 increased mature TGF-β protein levels without alterations in TGF-β1 mRNA. These data suggest a novel intracellular mechanism by which mature TGF-β1 protein levels may be regulated in RTECs through autophagic degradation, which suppresses kidney fibrosis induced by UUO. The dual functions of TGF-β1, as an inducer of TGF-β1 autoinduction and an inducer of autophagy and TGF-β degradation, underscore the multifunctionality of TGF-β1.In the kidney, fibrosis is responsible for chronic progressive kidney failure, and the prevalence of CKD is increasing worldwide.^1,2 Extracellular matrix (ECM) protein production and progressive accumulation are hallmarks of renal tubulointerstitial fibrosis in progressive kidney disease. Collagens are the main components of the ECM in the kidney, and type I collagen (Col-I) is the major type associated with disease states.^3,4 The cellular mechanisms that facilitate tubulointerstitial fibrosis after injury remain incompletely defined. Recent lineage tracing or genetic fate mapping studies have strongly challenged the theory that renal tubular epithelial cells (RTECs) traverse the tubular basement membrane to become myofibroblasts in a process of epithelial-to-mesenchymal transition (EMT), but rather, that interstitial pericytes/perivascular fibroblasts are the myofibroblast progenitor cells.^5–7 It also has been proposed that profibrotic factors, such as TGF-β1, are upregulated in the tubular interstitial area on injury, leading to kidney fibrosis.⁸ TGF-β1 induces production of ECM proteins, including fibronectin and collagens, and inhibits degradation of ECM proteins mainly by matrix metalloproteinases.^9–11 Given the recent evidence that casts doubts about the role of EMT in vivo, how RTECs contribute to the development of renal tubulointerstitial fibrosis is not entirely clear.TGF-β is synthesized as a single polypeptide precursor that includes a preregion signal peptide, which is removed by proteolytic cleavage, and pro–TGF-β, containing a proregion called the latency-associated peptide and a mature TGF-β, and it converts to homodimeric pro–TGF-β through disulfide bonds.¹² After cleavage by proprotein convertases, such as furin, latency-associated peptide remains noncovalently associated with the dimeric form of mature TGF-β as the small latent complex (SLC).¹³ SLC formation occurs in the Golgi apparatus, and mature TGF-β is secreted as part of SLC and associated with latent TGF-β–binding protein to form TGF-β large latent complex, which interacts with ECM. On stimulus, the dimeric form of mature TGF-β is dissociated from large latent complex and becomes the bioactive mature TGF-β ligand, which can then bind TGF-β receptors to trigger downstream Smad-dependent or -independent signaling pathways.^12,13 Thus, the availability of mature TGF-β is the limiting factor of TGF-β activity and not TGF-β synthesis per se, because the body generates more pro–TGF-β than necessary. Whereas TGF-β/TGF-β receptor downstream signaling pathways have been extensively investigated, the regulation of TGF-β maturation and bioavailability has not been well studied but may serve as an important target for fibrotic diseases that alter TGF-β signaling.Macroautophagy, hereafter referred to as autophagy, is a fundamental cellular homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles. The process of autophagy involves the formation of double membrane–bound vesicles called autophagosomes that envelop and sequester cytoplasmic components, including macromolecular aggregates and cellular organelles, for bulk degradation by a lysosomal degradative pathway.¹⁴ Autophagy can be induced in response to either intracellular or extracellular factors, such as amino acid or growth factor deprivation, hypoxia, low cellular energy state, endoplasmic reticulum stress or oxidative stress, organelle damage, and pathogen infection.^15–22 To date, over 30 genes involved in autophagy have been identified in yeast, and they have been termed autophagy-related genes (Atgs). The mammalian ortholog of Atg8 is comprised of a family of proteins known as microtubule-associated protein 1 light chain 3 (LC3) that functions as a structural component in the formation of autophagosomes.²³ LC3B (herein referred to as LC3) is the best characterized form and the most widely used as an autophagic marker. The conversion of the cytosolic form of LC3 (LC3-I) to lipidated form (LC3-II) indicates autophagosome formation. In contrast to LC3, Beclin 1, encoded by the beclin 1 gene, is the mammalian ortholog of yeast Atg6 that is required for the initiation of autophagy through its interaction with Vps34. Homozygous deletion of beclin 1 (beclin 1^−/−) exhibits early embryonic lethality, whereas heterozygous deletion (beclin 1^+/−) results in increased incidence of spontaneous tumorigenesis, abnormal proliferation of mammary epithelial cells and germinal center B lymphocytes, and increased susceptibility to neurodegeneration.^24–27We previously reported that autophagy promotes intracellular degradation of Col-I induced by TGF-β1 in glomerular mesangial cells.²⁸ In the present study, we explored the functional role of autophagy in an in vivo model of progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) in autophagy-deficient LC3 null (LC3^−/−) and heterozygous (beclin 1^+/−) mice and green fluorescent protein (GFP)-LC3 transgenic mice. We also performed functional studies in primary cultured mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. We hypothesized that induction of autophagy in RTECs promotes TGF-β degradation and thereby reduces TGF-β secretion and suppresses development of kidney fibrosis.     

All-Trans Retinoic Acid Attenuates the Renal Interstitial Fibrosis Lesion in Rats but Not By Transforming Growth Factor-β1/Smad3 Signaling Pathway

All-trans retinoic acid (ATRA) is an important therapeutic agent for prevention of the renal diseases. Transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway is a key signaling pathway which takes part in the progression of renal interstitial fibrosis (RIF). This investigation was performed to study the effect of ATRA in RIF rats and its effect on the TGF-β1/Smad3 signaling pathway. Sixty Wistar male rats were divided into three groups at random: sham operation group (SHO), model group subjected to unilateral ureteral obstruction (GU), model group treated with ATRA (GA), n = 20, respectively. RIF index, protein expression of TGF-β1, collagen-IV (Col-IV) and fibronectin (FN) in renal interstitium, and mRNA and protein expressions of Smad3 in renal tissue were detected at 14-day and 28-day after surgery. The RIF index was markedly elevated in group GU than in SHO group (p < 0.01), and the RIF index of GA group was alleviated when compared with that in GU group (p < 0.01). Compared with in group SHO, the mRNA/protein expression of Smad3 in renal tissue was significantly increased in group GU (p < 0.01). However, the mRNA and protein expressions of Smad3 in renal tissue in GA group were not markedly alleviated by ATRA treatment when compared with those in GU (each p > 0.05). Protein expressions of TGF-β1, Col-IV, and FN in GU group were markedly increased than those in SHO group (each p < 0.01), and their expressions in GA group were markedly down-regulated by ATRA treatment than those of GU group (all p < 0.01). The protein expression of Smad3 was positively correlated with RIF index, protein expression of TGF-β1, Col-IV or FN (each p < 0.01). In conclusion, ATRA treatment can alleviate the RIF progression in UUO rats. However, ATRA cannot affect the signaling pathway of TGF-β1/Smad3 in the progression of RIF.     

Losartan Alleviates Renal Fibrosis by Down-Regulating HIF-1α and Up-Regulating MMP-9/TIMP-1 in Rats with 5/6 Nephrectomy

Background: This study investigated the effects of losartan intervention on the expressions of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in renal fibrosis in rats with 5/6 nephrectomy. Methods: Sprague Dawley rats were randomly divided into three groups. Rats in the losartan group were gavaged with losartan (33.3 mg/kg/day) from 1 week after 5/6 nephrectomy, and those in the sham group and the model group only received an equal volume of saline solution by gavage. Rats were sacrificed at the ends of the 4, 8 and 12 weeks, respectively. Urinary N-acetyl-glucosaminidase (NAG), 24-h urinary protein, serum cystatin C, blood urea nitrogen (BUN), and serum creatinine (Scr) levels were assessed. Kidney tissues were observed under light and electron microscope. The expressions of HIF-1α, transforming growth factor-β1 (TGF-β1), MMP-9, and TIMP-1 were determined by immunohistochemistry and Western blotting. Results: Twenty-four hour urinary protein, urinary NAG, serum cystatin C, BUN, and Scr levels in the model group were significantly higher than those in the sham group (p < 0.05), but losartan treatment improved these changes. The apparent glomerular sclerosis and tubulointerstitial fibrosis were also found in the model group, which were ameliorated by losartan. The expressions of HIF-1α, TGF-β1, MMP-9, and TIMP-1 were elevated and MMp-9/TIMP-1 ratio was lowered in the model group (p < 0.05), but losartan increased the expression of MMP-9 and MMp-9/TIMP-1 ratio (p < 0.05) and lessened the expressions of HIF-1α, TGF-β1, and TIMP-1 (p < 0.05). Conclusion: Losartan may ameliorate renal fibrosis partly by down-regulating HIF-1α and up-regulating MMP-9/TIMP-1 in rats with 5/6 nephrectomy.     

Low-intensity pulsed ultrasound protects subchondral bone in rabbit temporomandibular joint osteoarthritis by suppressing TGF-β1/Smad3 pathway

Transforming growth factor β1(TGF-β1)/Smad3 pathway promotes the pathological progression of subchondral bone in osteoarthritis. The aim of this study is to determine the effect of low-intensity pulsed ultrasound (LIPUS) on the pathological progression and TGF-β1/Smad3 pathway of subchondral bone in temporomandibular joint osteoarthritis (TMJOA). Rabbit TMJOA model was established by type II collagenase induction. The left joint in this model was continuously stimulated with LIPUS for 3 and 6 weeks (1 MHz; 30 mW/cm²) for 20 min/day. The morphological and histological features of subchondral bone were respectively examined by microcomputed tomography and Safranin-O staining. The number of osteoclasts was quantitatively assessed by tartrate-resistant acid phosphatase staining. Immunohistochemistry and Western blot analysis were conducted to evaluate the protein expression of Cathepsin K and TGF-β1/Smad3 pathway. The results indicated that LIPUS could improve the trabecular microstructure and histological characteristics of subchondral bone in rabbit TMJOA. It also suppressed abnormal subchondral bone resorption and activation of TGF-β1/Smad3 pathway, characterized by the number of osteoclasts, protein expression levels of Cathepsin K, TGF-β1, type II TGFβ receptor, and phosphorylated Smad3 (pSmad3) were decreased. In conclusion, LIPUS promoted the quality of subchondral bone by suppressing osteoclast activity and TGF-β1/Smad3 pathway in rabbit TMJOA.     

Angiotensin II Dose-Dependently Stimulates Human Renal Proximal Tubule Transport by the Nitric Oxide/Guanosine 3′,5′-Cyclic Monophosphate Pathway

Stimulation of renal proximal tubule (PT) transport by angiotensin II (Ang II) is critical for regulation of BP. Notably, in rats, mice, and rabbits, the regulation of PT sodium transport by Ang II is biphasic: transport is stimulated by picomolar to nanomolar concentrations of Ang II but inhibited by nanomolar to micromolar concentrations of Ang II. However, little is known about the effects of Ang II on human PT transport. By functional analysis with isolated PTs obtained from nephrectomy surgery, we found that Ang II induces a dose-dependent profound stimulation of human PT transport by type 1 Ang II receptor (AT₁)-dependent phosphorylation of extracellular signal-regulated kinase (ERK). In PTs of wild-type mice, the nitric oxide (NO) /cGMP/cGMP-dependent kinase II (cGKII) pathway mediated the inhibitory effect of Ang II. In PTs of cGKII-deficient mice, the inhibitory effect of Ang II was lost, but activation of the NO/cGMP pathway failed to phosphorylate ERK. Conversely, in human PTs, the NO/cGMP pathway mediated the stimulatory effect of Ang II by phosphorylating ERK independently of cGKII. These contrasting responses to the NO/cGMP pathway may largely explain the different modes of PT transport regulation by Ang II, and the unopposed marked stimulation of PT transport by high intrarenal concentrations of Ang II may be an important factor in the pathogenesis of human hypertension. Additionally, the previously unrecognized stimulatory effect of the NO/cGMP pathway on PT transport may represent a human-specific therapeutic target in hypertension.Among the several regulatory systems for BP, the renin-angiotensin system (RAS) plays a key role. Angiotensin II (Ang II) can regulate BP by type 1 (AT₁) Ang II receptors in either the kidney or the extrarenal tissues. Using a kidney cross-transplantation strategy, Crowley et al.¹ revealed that AT_1A in the kidney is critical for the pathogenesis of Ang II-mediated hypertension and its cardiovascular complications. Crowley et al.¹ also showed that genetic abrogation AT_1A in renal proximal tubules (PTs) alone is sufficient to reduce BP and provide substantial protection against Ang II–induced hypertension in mice, suggesting that the stimulation of PT sodium transport by Ang II has a major impact on BP regulation.²Paradoxically, however, the regulation of PT sodium transport by Ang II is biphasic: transport is stimulated by low (picomolar to nanomolar) concentrations of Ang II, whereas it is inhibited by high (nanomolar to micromolar) concentrations of Ang II.^3,4 Notably, the concentrations of Ang II within the kidney are known to be much higher than the concentrations in plasma, although the measurement of urinary Ang II may be less valuable.^5,6 Therefore, the inhibitory effect of high concentrations of Ang II could also have some physiologic relevance to the regulation of in vivo PT transport.⁵ Ang II regulates the major PT sodium transporters the apical Na⁺/H⁺ exchanger isoform 3 (NHE3), the basolateral Na⁺-HCO₃⁻ cotransporter (NBCe1), and the basolateral Na⁺/K⁺ ATPase in the biphasic manner.^7–12 Although controversial results had been reported of the receptor subtype(s) mediating the biphasic effects of Ang II,^13,14 the studies using isolated PTs obtained from AT_1A knockout (KO) mice have established that AT_1A mediates both the stimulatory and inhibitory effects of Ang II.^11,15 The stimulation by Ang II has been attributed to the activation of protein kinase C and/or the decrease in the intracellular cAMP concentration, resulting in the activation of extracellular signal-regulated kinase (ERK) pathway.^16–18 However, the inhibition by Ang II has been attributed to the activation of the phospholipase A₂/arachidonic acid/5,6-epoxyeicosatrienoic acid (EET) pathway and/or the nitric oxide (NO)/guanosine 3′,5′-cyclic monophosphate (cGMP) pathway.^7,12,17,19 Although the biphasic regulation of PT transport by Ang II has been reported in several species, such as rats, mice, and rabbits,^3,4,15 whether Ang II has similar biphasic effects on human PT transport remains unknown. Moreover, the signaling mechanisms mediating the Ang II actions on human kidney have not been clarified at all.To clarify these issues, we examined the effects of Ang II on isolated human PTs obtained from nephrectomy surgery. Unexpectedly, our findings revealed that Ang II, unlike in the other species, dose-dependently stimulates human PT transport. The different modes of transport regulation by Ang II can be largely explained by the contrasting roles of the NO/cGMP pathway, which as a downstream mediator of Ang II signaling in PTs, is stimulatory in human subjects but inhibitory in the other species. The unopposed marked stimulation of PT transport by the high local concentrations of Ang II may be an important factor in the pathogenesis of human hypertension. Furthermore, the stimulatory effect of the NO/cGMP pathway on human PT transport may represent a previously unrecognized therapeutic target of hypertension.     

Guanosine 3′,5′-monophosphate in bone: Microscopic visualization by an immuno-histochemical technique

Summary Guanosine 3,5-monophosphate (cyclic GMP, cGMP) was localized in bone cells by the use of an immunoglobulin-enzyme bridge method. We observed that in cat alveolar bone most osteoblasts did not stain for cGMP, while adjacent periodontal cells displayed cytoplasmic as well as nuclear staining. Numerous osteocytes contained diffuse reaction products over most or all of the cellular area. The method used in this study may be helpful in identifying specific hard tissue cell types whose function(s) involve cGMP.