Response of Steroid-Refractory Acute GVHD to α1-Antitrypsin.

α1-Antitrypsin (AAT) is a serine protease inhibitor with anti-inflammatory, antiapoptotic, and immunomodulatory properties. It has therapeutic efficacy in animal models of autoimmune diseases, inflammatory disorders, and transplantation. In a phase I/II open-label single-center study, we administered AAT (Glassia; Baxalta/Kamada, New Ziona, Israel) as salvage therapy to 12 patients with steroid-refractory acute graft-versus-host disease (GVHD). AAT was given i.v. at 2 dose levels over a 15-day course. All patients had grades III or IV GVHD with stage 4 gut involvement. After treatment, plasma AAT levels increased in both cohorts and remained within 2 to 4 mg/mL for the duration of treatment. No clinically relevant toxicities attributable to AAT were observed. GVHD manifestations improved in 8 of 12 patients, and 4 responses were complete. Six patients (50%) were alive at last follow-up (>104 to >820 days). These findings show that AAT is well tolerated and has efficacy in the treatment of steroid-refractory severe acute GVHD. Further studies are warranted.

Current drug development challenges in chronic kidney disease (CKD)--identification of individualized determinants of renal progression and premature cardiovascular disease (CVD).

Chronic kidney disease (CKD) and end-stage renal disease (ESRD) are currently considered as major health burdens. Notably, CKD can be regarded as an interesting clinical model of accelerated cardiovascular disease (CVD) and ageing, which offers exciting new perspectives and challenges for pharmaceutical drug development. However, during the last decades, therapeutic advances to slow down the progression of CKD and reduce CVD risk have largely failed due to several possible reasons including (i) the lack of profound understanding of the pathophysiology of chronic renal damage and its associated CVD; (ii) an inadequate characterization of molecular mechanisms of currently approved therapies such as renin-angiotensin-aldosterone-system (RAAS) blockade; (iii) the unclear biochemical property needs required for novel therapeutic approaches; (iv) the missing quantity and quality of clinical trials in the nephrology field; and, most importantly, (v) the absence of prognostic renal biomarkers that reflect the severity of the structural organ damage and predict ESRD as well as CVD mortality. There is clearly an insufficient understanding of why a significant proportion of CKD patients progress to ESRD and/or die from CVD whereas others rather remain stable. In this article, we urge renal researchers to develop novel experimental and clinical tools for rational and translational drug discovery. Identification of individualized determinants of CKD progression and/or premature CVD will enable personalized medicine and lead to novel innovative nephro- and/or cardioprotective pharmacological treatment in these high-risk patients.

Expression of components of the renin-angiotensin system in autosomal recessive polycystic kidney disease.

Hypertension is a common complication in children with autosomal recessive polycystic kidney disease (ARPKD) who have survived the neonatal period. No information is available regarding the mechanism of hypertension in this condition. The renin-angiotensin system (RAS) is thought to play a role in hypertension associated with the more common autosomal dominant polycystic kidney disease (ADPKD). Occasional reports have documented increased activity of the intrarenal RAS in ADPKD, with ectopic renin expression within cysts and dilated tubules. Because of similarities between ARPKD and ADPKD, we hypothesized that increased intrarenal RAS activity might also be found in ARPKD. We performed immunohistochemical studies on kidney tissues from two infants with ARPKD and two control kidneys. The cystic dilated tubules showed staining with the peanut lectin arachis hypogaea, a marker of distal tubules and collecting ducts, but not with lotus tetragonolobus, a marker of proximal tubules. Strong renin staining was seen in many cysts and tubules of ARPKD kidneys, but only in the afferent arterioles of the normal control kidneys. Angiotensinogen staining was also observed in some cysts and in proximal tubules. Staining for angiotensin-converting enzyme, angiotensin II type 1 receptor, and angiotensin II peptide was present in many cystic dilated tubules. These immunohistochemical studies document for the first time ectopic expression of components of the RAS in cystic-dilated tubules of ARPKD and suggest that overactivity of RAS could result in increased intrarenal angiotensin II production, which may contribute to the development of hypertension in ARPKD.

Safety of new phosphate binders for chronic renal failure.

Phosphate (Pi) retention is a common problem in patients with chronic kidney disease, particularly in those who have reached end-stage renal disease (ESRD). In addition to causing secondary hyperparathyroidism and renal osteodystrophy, recent evidence suggests that, in ESRD patients, high serum phosphorus concentration and increased calcium and phosphorous (Ca x P) product are associated with vascular and cardiac calcifications and increased mortality. Dietary phosphorus restriction and Pi removal by dialysis are not sufficient to restore Pi homeostasis. Reduction of intestinal Pi absorption with the use of Pi binders is currently the primary treatment for Pi retention in patients with ESRD. The use of large doses of calcium-containing Pi binders along with calcitriol administration may contribute to over-suppression of parathyroid hormone secretion and adynamic bone disease as well as to a high incidence of vascular calcifications. When used in patients with impaired renal function, aluminium salts were found to accumulate in bone and other tissues, resulting in osteomalacia and encephalopathy.Sevelamer, an aluminium- and calcium-free Pi binder can reduce serum phosphorus concentration and is associated with a significantly lower incidence of hypercalcaemia, while maintaining the ability to suppress parathyroid hormone production. An additional benefit of sevelamer is its ability to lower low density lipoprotein-cholesterol and total cholesterol levels. Sevelamer attenuates the progression of vascular calcifications in haemodialysis patients, which may lead to lower mortality. The use of sevelamer in non-dialysed patients might aggravate metabolic acidosis, common in these patients. Several other calcium-free Pi binders are in development. Lanthanum carbonate has shown significant promise in clinical trials in ESRD patients. Magnesium salts do not offer a significant advantage over currently available Pi binders. Their use is restricted to patients receiving dialysis since excess magnesium must be removed by dialysis. Iron-based compounds have shown variable efficacy in short-term clinical trials in small numbers of haemodialysis patients. Mixed metal hydroxyl carbonate compounds have shown efficacy in animals but have not been studied in humans. Major safety issues include absorption of the metal component with possible tissue accumulation and toxicity.

Medication noncompliance in patients with chronic disease: issues in dialysis and renal transplantation.

For many chronic conditions, poor patient compliance with prescribed medications and other aspects of medical treatment can adversely affect the treatment outcome. Compliance with long-term treatment for chronic asymptomatic conditions such as hypertension is on the order of 50%. Although drugs with a longer therapeutic half-life may ease the burden of repeated daily dosing, the efficacy of any self-administered medication depends to a large extent on patient compliance. This article addresses the compliance issues in patients undergoing renal replacement therapy and in those with a successful renal transplant. A focused discussion of compliance in dialysis and renal transplant patients is followed by a general review of the literature on patient compliance. Many factors associated with poor compliance in this patient population are identified via a review of the recent literature. The difficulties in monitoring medication compliance and the methods used are discussed. Among factors associated with poor compliance, the following have been identified in several studies: frequent dosing, patient's perception of treatment benefits, poor patient-physician communication, lack of motivation, poor socioeconomic background, lack of family and social support, and younger age. Many strategies have been suggested to improve medication compliance, most without scientific validation. Strategies to improve compliance in dialysis and transplant patients are similar to those described for other chronic conditions and include simplifying the treatment regimen, establishing a partnership with the patient, and increasing awareness through education and feedback.

Effect of hemodialysis and hemofiltration on plasma C.E.R.A. concentrations.

C.E.R.A., a continuous erythropoietin receptor activator, is a long-acting erythropoiesis-stimulating agent approved for the treatment of anemia in patients with chronic kidney disease. Although the large molecular weight and the carbohydrate chain make it unlikely that C.E.R.A. could be removed during hemodialysis or hemofiltration, no such data have been published. In vitro studies were performed to assess the removal of C.E.R.A. during hemodialysis and hemofiltration, using both low-flux and high-flux membranes and parameters very similar to those used in clinical practice. Clinical pharmacokinetic studies of plasma C.E.R.A. concentrations in patients undergoing hemodialysis were also performed following subcutaneous injection of C.E.R.A. In the in vitro studies, plasma C.E.R.A. concentrations were not significantly different from baseline values in the primed blood reservoir over a 4-hour period during hemodialysis (P = 0.12). C.E.R.A. concentrations in the plasma obtained from the venous end of the hemofilter increased proportionally with the plasma total protein concentrations, reflecting the consequence of hemoconcentration and suggesting that C.E.R.A and plasma total proteins were retained by hemofiltration membranes to a similar degree. These in vitro studies showed that C.E.R.A. was not removed by simulated hemodialysis or hemofiltration either via transmembrane transport or adsorption to the membrane. The results were corroborated by the clinical pharmacokinetic data, which showed no detectable changes in plasma C.E.R.A. concentrations during hemodialysis using either low-flux or high-flux dialyzers. These results suggest that C.E.R.A. can be administered to patients at any time during hemodialysis or hemofiltration without appreciable loss in the extracorporeal circuit.

Detection and management of nephrotoxicity during drug development.

INTRODUCTION: The kidneys are vulnerable to injury due to their high filtration capacity and high metabolic activity, and most drugs, especially hydrophilic drugs and their metabolites, are eliminated largely by kidneys in urine, thus increasing the risk of drug-induced nephrotoxicity (DIN). DIN accounts for 18 - 27% of community- and hospital-acquired episodes of acute kidney injury (AKI) and is a serious concern during development of novel therapeutic drugs. AREAS COVERED: This review provides an overview of definitions, classification, risk factors, complications, and outcomes of DIN. In addition, it gives a practical source of information when dealing with nephrotoxicity issues during drug development and provides guidance on renal safety risk evaluation for clinical studies. Lastly, current research focus in the search for novel biomarkers of DIN is also provided. EXPERT OPINION: To enable early detection of DIN and to ensure patients' safety through appropriate risk minimization and mitigation strategies, future research should focus on identification and validation of novel predictive biomarkers of kidney injury and development of DIN-specific classification and staging system. This could help in reducing the current high rate of attrition during drug development, and reduce marketing and post-marketing withdrawals of nephrotoxic drugs.

Loss of polycystin-1 in human cyst-lining epithelia leads to ciliary dysfunction.

A "two-hit" hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1(null/null) mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1(+/null) cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts.

Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts.

Autosomal dominant polycystic kidney disease (ADPKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by "second hit" somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with ADPKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37 degrees C in the WT cell lines and at 33 degrees C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39 degrees C. The majority (29) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD.

The intrarenal renin-angiotensin system in autosomal dominant polycystic kidney disease.

Hypertension is a common complication of autosomal dominant polycystic kidney disease (ADPKD), often present before the onset of renal failure. A role for the renin-angiotensin system (RAS) has been proposed, but studies of systemic RAS have failed to show a correlation between plasma renin activity and blood pressure in ADPKD. Ectopic renin expression by cyst epithelium was first reported in 1992 (Torres VE, Donovan KA, Sicli G, Holley KE, Thibodeau ST, Carretero OA, Inagami T, McAteer JA, and Johnson CM. Kidney Int 42: 364-373, 1992). It is not known, however, whether other RAS components are also expressed by cysts in ADPKD. We show that, in addition to renin, angiotensinogen (AGT) is produced by some cysts and dilated tubules. Angiotensin-converting enzyme, ANG II type 1 receptor, and ANG II peptide are also present within cysts and in many tubules; and some cyst fluids contain high ANG II concentrations. Additionally, cyst-derived cells in culture continue to express the components of the RAS at both the protein and mRNA levels. We further show that renin is expressed primarily in cysts of distal tubule origin and in cyst-derived cells with distal tubule characteristics, whereas AGT is expressed primarily in cysts of proximal tubule origin and in cyst-derived cells with proximal tubule characteristics. Renin production by cyst-derived cells appears to be regulated by extracellular Na+ concentration. Based on these observations, we propose a model of an autocrine/paracrine RAS in polycystic kidney disease, whereby overactivity of the intrarenal system results in sustained increases in intratubular ANG II concentrations.

Aberrant polycystin-1 expression results in modification of activator protein-1 activity, whereas Wnt signaling remains unaffected.

Polycystin-1, the polycystic kidney disease 1 gene product, has been implicated in several signaling complexes that are known to regulate essential cellular functions. We investigated the role of polycystin-1 in Wnt signaling and activator protein-1 (AP-1) activation. To this aim, a membrane-targeted construct encoding the conserved C-terminal region of mouse polycystin-1 reported to mediate signal transduction activity was expressed in human embryonic and renal epithelial cells. To ensure specificity and minimal cotransfection effects, we focused our study on the endogenous proteins that actually transduce the signals, beta-catenin and T-cell factor/lymphoid-enhancing factor for Wnt signaling and (phosphorylated) c-Jun, ATF2, and c-Fos for AP-1. Our data indicate that the C-terminal region of polycystin-1 activates AP-1 by inducing phosphorylation and expression of at least c-Jun and ATF2, whereas c-Fos was not affected. Under our experimental conditions, polycystin-1 did not modulate Wnt signaling. AP-1 activity was aberrant in human autosomal dominant polycystic kidney disease (ADPKD) renal cystic epithelial cells and in renal epithelial cells expressing transgenic full-length polycystin-1, resulting in decreased Jun-ATF and increased Jun-Fos activity, whereas Wnt signaling remained unaffected. Since our data indicate that aberrant polycystin-1 expression results in altered AP-1 activity, polycystin-1 may be required for adequate AP-1 activity.