Inhibition of Autoregulated TGFβ Signaling Simultaneously Enhances Proliferation and Differentiation of Kidney Epithelium and Promotes Repair Following Renal Ischemia

We studied autocrine transforming growth factor (TGF)β signaling in kidney epithelium. Cultured proximal tubule cells showed regulated signaling that was high during log-phase growth, low during contact-inhibited differentiation, and rapidly increased during regeneration of wounded epithelium. Autoregulation of signaling correlated with TGFβ receptor and Smad7 levels, but not with active TGFβ, which was barely measurable in the growth medium. Confluent differentiated cells with low receptor and high Smad7 levels exhibited blunted responses to saturating concentrations of exogenously provided active TGFβ, suggesting that TGFβ signaling homeostasis was achieved by cell density-dependent modulation of signaling intermediates. Antagonism of Alk5 kinase, the TGFβ type I receptor, dramatically accelerated the induction of differentiation in sparse, proliferating cultures and permitted better retention of differentiated features in regenerating cells of wounded, confluent cultures. Alk5 antagonism accelerated the differentiation of cells in proximal tubule primary cultures while simultaneously increasing their proliferation. Consequently, Alk5-inhibited primary cultures formed confluent, differentiated monolayers faster than untreated cultures. Furthermore, treatment with an Alk5 antagonist promoted kidney repair reflected by increased tubule differentiation and decreased tubulo-interstitial pathology during the recovery phase following ischemic injury in vivo. Our results show that autocrine TGFβ signaling in proliferating proximal tubule cells exceeds the levels that are necessary for physiological regeneration. To that end, TGFβ signaling is redundant and maladaptive during tubule repair by epithelial regeneration.Regeneration of an adult epithelium such as those lining the kidney tubules involves not only proliferation but also de-differentiation, followed by growth arrest and re-differentiation.^1,2,3 The signaling cues that coordinate these processes are largely unknown. Endocrine and paracrine factors affect epithelial repair following injury in vivo. Nevertheless, epithelial homeostasis is also regulated by density-dependent contact-inhibition and attendant differentiation. The mechanisms that mediate these processes are poorly understood, but likely involve transforming growth factor (TGF)β, in addition to signals generated by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. These considerations prompted us to investigate how endogenously generated signals might control epithelial regeneration in terms of cell proliferation and differentiation.Increased TGFβ signaling can lead to apoptosis, growth inhibition, or epithelial-mesenchymal transitions (EMT) of epithelial cells, including kidney epithelial cells.^4,5,6,7,8,9 Prolonged exposure to high concentrations of active TGFβ is frequently used to model these alterations. While these effects of sustained high intensity TGFβ signaling are well studied, much less is known about physiologically regulated TGFβ signals and how they become increased by epithelial injury and subsequent regeneration. Autocrine TGFβ signals are antiproliferative for epithelial cells and cultures from TGFβ₁ null kidney tubules display enhanced proliferative rates.¹⁰ Nevertheless, TGFβ signaling was found to be increased rather than decreased during the proliferation of surviving kidney epithelium following cell loss by ischemia, and this was accompanied by increased expression of TGFβ and its receptors in regenerating cells.¹¹ Similarly, proliferating keratinocytes in skin wounds show enhanced TGFβ signaling¹². It has been puzzling why antiproliferative TGFβ signaling becomes enhanced in rapidly growing cells under pathological conditions.In this study, we have investigated the functional relevance of cell-autonomous, ie, endogenously generated, TGFβ signals for regenerating kidney epithelial cells in culture and in vivo. Fully differentiated proximal tubule (PT) cells retain the ability to undergo mitotic division,^13,14,15 and, following cell loss by injury, survivors dedifferentiate, proliferate, and then redifferentiate to reconstitute the lost cell mass.^1,2,3,13,16 We found that cell-autonomous TGFβ signals are tightly autoregulated during repeated cycles of proliferation and contact-inhibition in PT cultures. Signaling was high during log phase growth and became progressively suppressed as cultures became contact-inhibited and differentiated. It was decreased in growing subconfluent cultures by neutralizing TGFβ antibodies, indicating a requirement for extracellular ligand. However, in the absence of TGFβ antibodies, increases and decreases of signaling were determined solely by cell density, and occurred independently of the concentrations of barely measurable active TGFβ in growth medium. Instead, the signaling fluctuations were associated with increased and decreased expression of TGFβ receptor and reciprocal alterations of inhibitory Smad7. Moreover, saturating concentrations of exogenous TGFβ were found to elicit blunted signaling responses from contact-inhibited differentiated cells relative to growing undifferentiated cells. These observations suggested that: (1) extracellular TGFβ ligand played a permissive role but did not, by itself, determine the intensity of signaling fluctuations during the epithelial growth cycle; and (2) signaling homeostasis during growth and quiescence was related to the modulation of TGFβ receptors and Smad7. Functionally, we found that inhibition of cell-autonomous TGFβ signals resulted in remarkably accelerated differentiation and concurrent stimulation of proliferation in growing PT cultures. Importantly, we extended our observations to demonstrate that treatment with small molecule Alk5 inhibitors not only promoted differentiation in regenerating PT epithelium during wound healing in vitro, but also improved the repair of kidney damage with greater restoration of epithelial differentiation and tubule integrity following ischemia in vivo. These unprecedented findings have direct relevance to the development of treatments that might promote repair and recovery following loss of epithelium by acute kidney injury (AKI).     

Hitchhiking and selective sweeps of Plasmodium falciparum sulfadoxine and pyrimethamine resistance alleles in a population from central Africa

Sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum is encoded by a number of mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes. Here, we have characterized point mutations in dhfr and dhps and microsatellite loci around dhfr on chromosome 4 and dhps on chromosome 8 as well as neutral markers on chromosomes 2 and 3 in 332 samples from Yaoundé, Cameroon. The triple mutant dhfr haplotype that originated in Southeast Asia is the most predominant in this sample set, but we also find additional independent haplotypes at low frequency and an incipient process of genetic differentiation among alleles of Southeast Asian origin. As reported for other African populations, we find evidence of a selective sweep for resistant dhfr mutants in this Cameroonian population due to drug selection. Although we find evidence for a selective sweep in dhps mutants associated with SP resistance, the dynamics of dhps mutants appear different than those observed for dhfr mutants. Overall, our results yield support for the use of microsatellite markers to track resistant parasites; however, the detection of resistant dhfr alleles in low frequency, the evidence of divergence among dhfr alleles that share a common evolutionary origin, and the distinct dynamics of resistant dhps alleles emphasize the importance of comprehensive, population-based investigations to evaluate the effects of drug selection on parasite populations.     

Reconstruction of traumatically avulsed parotid duct using buccal mucosa flap: report of a new technique

Injury to parotid duct is frequently overlooked especially in multiple injuries settings. Consequently they are rarely reported in the literature. Treatment of these injuries is highly controversial. Short series and anecdotal case studies claim success with various approaches such as nonsurgical management, simple ligation of the proximal duct with or without antisialagogues, microsurgical primary repair, creation of sialodocho-oral fistula, and vein-graft replacement. A novel technique of sialodochoplasty using buccal-mucosa pedicle flap is described in this report. Short-term result of this method in a 4-year-old boy with traumatic loss of right parotid duct was encouraging. Hypothetical advantages of this new operation are reviewed in the background of alternative procedures. It is concluded that buccal-mucosa flap technique appears to be a promising alternative in the management of parotid duct injuries.     

En plaque intradural extramedullary spinal tuberculoma and concurrent intracranial tuberculomas: paradoxical response to antituberculous therapy. Case report

Spinal intradural extramedullary tuberculoma is a rare entity. Rarer still are extensive en plaque intradural extramedullary tuberculomas occurring concurrently with multiple intracranial tuberculomas as a paradoxical response to chemotherapy for tuberculosis (TB). The authors describe the case of a 21-year-old man who was treated for tuberculous meningitis. Three months after the episode of meningitis, while undergoing chemotherapy for TB, he developed features of thoracic myelopathy. Investigations revealed an extensive en plaque intradural extramedullary lesion spanning seven segments in the lower thoracic spine. Magnetic resonance imaging of the brain revealed multiple asymptomatic intracranial tuberculomas. Even after further treatment with antituberculous chemotherapy was initiated, the lesion failed to respond. The authors performed a laminectomy and excised the en plaque intradural extramedullary lesion. The patient's condition responded well to this treatment. Although the appearance of intracranial tuberculoma as a paradoxical response to chemotherapy has been previously reported, no authors have reported on the development of an extensive en plaque intradural extramedullary tuberculoma in conjunction with asymptomatic multiple intracranial tuberculomas as a paradoxical response. In cases in which patients present with compressive myelopathy following therapy for tuberculous meningitis, it is important to consider in the differential diagnosis that intradural extramedullary tuberculoma may be a paradoxical response to chemotherapy. The authors' experience and their review of the literature indicate that surgery has a definitive role to play in the management of spinal intradural extramedullary tuberculoma.     

Preclinical toxicity and pharmacokinetics of the Bruton's tyrosine kinase-targeting anti-leukemic drug candidate, alpha-cyano-beta-hydroxy-beta-methyl-N- (2,5-dibromophenyl) propenamide (LFM-A13)

The leflunomide (CAS 75706-12-6) metabolite (LFM) analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13, DDE-28, CAS 244240-24-2) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK). LFM-A13 exhibited favorable pharmacokinetics in CD-1 mice, BALB/c mice, rats, and dogs. The intraperitoneal bioavailability was estimated to be -100%, while the oral bioavailability was -30%. LFM-A13 enters, but does not bind to multiple tissues followed by a rapid elimination from most of the tissues. Limited distribution of LFM-A13 to extravascular tissues and its corresponding low volume of distribution could be attributed to its plasma protein binding. LFM-A13 was not toxic to mice, rats, or dogs at daily dose levels as high as 100 mg/kg. LFM-A13 formulated as a suspension and hard gelatin capsules for oral administration showed a rapid absorption and favorable pharmacokinetic features. These preclinical research studies provide the basis for future pre-IND studies and clinicaldevelopment of LFM-A13 as an intravenously or orally administered new anti-leukemia agent targeting BTK.