Impact of sirolimus, tacrolimus and mycophenolate mofetil on osteoclastogenesis--implications for post-transplantation bone disease.

BACKGROUND: Post-transplantation bone disease is associated with a high degree of morbidity including pain and fractures. Glucocorticoid-induced osteoporosis on top of pre-existing renal osteodystrophy is considered the major pathogenic factor, while the role of non-glucocorticoid immunosuppressants is less well defined. METHODS: In this study, we investigated the influence of sirolimus (SRL) versus calcineurin inhibitor (CI)-based immunosuppressive regimens on biomarkers of bone resorption in renal transplant patients. In addition, the impact of SRL, tacrolimus and mycophenolate mofetil (MMF) on osteoclast activation and function was assessed in cell culture systems. RESULTS: Using this approach, we demonstrated reduced serum levels of bone resorption markers in patients treated with SRL after kidney transplantation compared to a CI-based regimen. In line with this observation, we detected profoundly reduced osteoclast differentiation and subsequently diminished hydroxyapatite resorption in the presence of SRL compared to MMF and tacrolimus in vitro. Moreover, SRL significantly reduced osteoclast precursor proliferation in vitro compared to tacrolimus and led to augmented apoptosis in osteoclast precursors. CONCLUSIONS: Taken together, SRL was shown to inhibit osteoclast formation in vivo and in vitro. SRL thus may have the potential to balance osteoclast promoting effects of glucocorticoids and CI, thereby counteracting the development of accelerated osteoporosis in renal transplant recipients.

Real-world data confirm the effectiveness of caplacizumab in acquired thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare but life-threatening condition. In 2018, the nanobody caplacizumab was approved for the treatment of adults experiencing an acute episode of aTTP, in conjunction with plasma exchange (PEX) and immunosuppression for a minimum of 30 days after stopping daily PEX. We performed a retrospective, observational analysis on the use of caplacizumab in 60 patients from 29 medical centers in Germany during acute disease management. Caplacizumab led to a rapid normalization of the platelet count (median, 3 days; mean 3.78 days). One patient died after late treatment initiation due to aTTP-associated complications. In 2 patients with initial disease presentation and in 4 additional patients with laboratory signs of an exacerbation or relapse after the initial therapy, PEX-free treatment regimens could be established with overall favorable outcome. Caplacizumab is efficacious in the treatment of aTTP independent of timing and ancillary treatment modalities. Based on this real-world experience and published literature, we propose to administer caplacizumab immediately to all patients with an acute episode of aTTP. Treatment decisions regarding the use of PEX should be based on the severity of the clinical presentation and known risk factors. PEX might be dispensable in some patients.

ADAMTS13 and VWF activities guide individualized caplacizumab treatment in patients with aTTP.

Introduction of the nanobody caplacizumab was shown to be effective in the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in the acute setting. The official recommendations include plasma exchange (PEX), immunosuppression, and the use of caplacizumab for a minimum of 30 days after stopping daily PEX. This study was a retrospective, observational analysis of the use of caplacizumab in 60 patients from 29 medical centers in Germany. Immunosuppressive treatment led to a rapid normalization of ADAMTS13 activities (calculated median, 21 days). In 35 of 60 patients, ADAMTS13 activities started to normalize before day 30 after PEX; in 11 of 60 patients, the treatment was extended beyond day 30; and in 5 patients, it was extended even beyond day 58 due to persistent autoimmune activity. In 34 of 60 instances, caplacizumab was stopped before day 30 with a favorable outcome whenever ADAMTS13 activities were >10%. In contrast, 11 of 34 patients with ADAMTS13 activities <10% at the time of stopping caplacizumab treatment developed a nonfavorable outcome (disease exacerbation or relapse). In some cases, prolongation of the treatment interval to every other day was feasible and resulted in a sustained reduction of von Willebrand factor activity. ADAMTS13 activity measurements are central for a rapid diagnosis in the acute setting but also to tailor disease management. An ADAMTS13 activity-guided approach seems safe for identifying the individual time point when to stop caplacizumab to prevent overtreatment and undertreatment; this approach will result in significant cost savings without jeopardizing the well-being of patients. In addition, von Willebrand factor activity may serve as a biomarker for drug monitoring.

Conditional gene targeting in the kidney.

Complete mapping of the genome in a number of organisms provides a challenge for experimental nephrologists to identify potential functions of a vast number of new genes in the kidney. Since knockout technologies have evolved in the early eighties the mouse has become a valuable model organism. Researchers can now artificially eliminate the expression of specific genes in a mammalian organism and examine the phenotype. New developments have emerged that allow investigators to knock out a gene specifically in the kidney. Several kidney-specific promoters provide valuable tools and bacterial artificial chromosome (BAC) based techniques like recombineering will enhance both number and accuracy of new mouse lines with spatially controlled gene expression. In addition to spatial control, tetracycline- or tamoxifen-inducible systems, provide the possibility of influencing the temporal expression pattern of a gene enabling researchers to dissect its functions in adult organisms. Knocking out a gene will continue to be the gold standard for defining the role of a specific gene whereas tissue-specific gene knockdown using RNA interference represents an alternative approach for generating lower-priced and fast loss of function models. In addition to reverse genetic approaches, forward genetic techniques like random mutagenesis in mice continue to evolve and will enhance our understanding of disease mechanisms in the kidney.

Angiopoietin-1 is essential in mouse vasculature during development and in response to injury.

Angiopoietin-1/Tek signaling is a critical regulator of blood vessel development, with conventional knockout of angiopoietin-1 or Tek in mice being embryonically lethal due to vascular defects. In addition, angiopoietin-1 is thought to be required for the stability of mature vessels. Using a Cre-Lox conditional gene targeting approach, we have studied the role of angiopoietin-1 in embryonic and adult vasculature. We report here that angiopoietin-1 is critical for regulating both the number and diameter of developing vessels but is not required for pericyte recruitment. Cardiac-specific knockout of angiopoietin-1 reproduced the phenotype of the conventional knockout, demonstrating that the early vascular abnormalities arise from flow-dependent defects. Strikingly, deletion in the entire embryo after day E13.5 produced no immediate vascular phenotype. However, when combined with injury or microvascular stress, angiopoietin-1 deficiency resulted in profound organ damage, accelerated angiogenesis, and fibrosis. These findings redefine our understanding of the biological roles of angiopoietin-1: it is dispensable in quiescent vessels but has a powerful ability to modulate the vascular response after injury.

Deciphering the renal code: advances in conditional gene targeting.

Several powerful new techniques can examine gene function in mammals. Recombinase systems and kidney-specific promoters enable gene knockout and overexpression. Genetic systems induced on administration or removal of antibiotics or hormones permit control of gene expression. Gene silencing using short interfering RNA expression systems should accelerate loss-of-function studies. Thorough characterization of animals that have undergone conditional gene targeting has already provided insights into renal development and diseases. Here we discuss the advantages and pitfalls of currently available gene-targeting systems.

Selective inhibition of inducible nitric oxide synthase enhances intraglomerular coagulation in chronic anti-Thy 1 nephritis.

BACKGROUND: A particular Lewis rat substrain (LEW/Maa) develops chronic glomerulonephritis in the anti-Thy 1 model (aThy 1-GN) characterized by increased microaneurysm formation, chronic glomerular sclerosis and persistent albuminuria. This phenotype is accompanied by increased and prolonged glomerular induction of inducible nitric oxide synthase (iNOS) when compared to the LEW/Moe substrain, in which aThy 1-GN resolves quickly. We investigated the effect of selective iNOS inhibition by l-N6-(1-iminoethyl)-lysine (L-NIL) administration on aThy 1-GN in LEW/Maa rats. METHODS: Nephritic rats were studied over a period of 7 days. L-NIL-treated animals received 20 mg/day L-NIL in the drinking water starting two days prior to disease induction. iNOS activity was determined in cultured glomeruli and in urine samples, respectively. Severity of aThy 1-GN was determined by scoring glomerular matrix expansion and microaneurysm formation, and by albuminuria measurements (ELISA). Immunohistochemical evaluation was performed including staining for macrophages (ED-1), platelets (PL-1) and fibrin deposition. RESULTS: L-NIL treated rats (+NIL) showed a significant decrease in peak nitrate production by ex vivo cultured glomeruli, and in urinary nitrate excretion versus untreated nephritic rats (-NIL). Mean arterial pressure remained unchanged in both +NIL and -NIL rats. +NIL rats developed significantly increased albuminuria (+44%) associated with a significant increase in glomerular platelet (+45%) and fibrin deposition (+48%). CONCLUSIONS: Selective inhibition of iNOS aggravated albuminuria in chronic aThy 1-GN in LEW/Maa rats. Induction of iNOS during the inflammatory phase of this model may be a partially protective mechanism by interfering with intraglomerular coagulation processes.

Inducible nitric oxide synthase-derived nitric oxide promotes glomerular angiogenesis via upregulation of vascular endothelial growth factor receptors.

The vascular endothelial growth factor (VEGF) system is of major importance for glomerular endothelial repair in glomerulonephritis (GN) and is significantly affected by nitric oxide (NO) release. For investigating whether glomerular upregulation of inducible NO synthase (iNOS) in GN might affect VEGF-mediated repair, a selective iNOS inhibitor, L-N6-(1-iminoethyl)-lysin (L-NIL), was administered to rats with anti-Thy 1.1 GN from day -2 until day 5 after GN induction. Compared with untreated nephritic rats, L-NIL-treated nephritic rats showed similar mean arterial BP, significantly decreased de novo peak nitrate production, and increased albuminuria on day 6. This was preceded by a significant decrease of glomerular endothelial cell proliferation and endothelial area on day 2 in L-NIL-treated nephritic rats. Upregulation of glomerular VEGF mRNA and protein expression, in particular of the VEGF(164) splicing variant, occurred similarly in L-NIL-treated and untreated nephritic rats on days 2 and 7. However, the upregulation of glomerular VEGF receptor 1 and 2 mRNA expression on day 2 was reduced by 77 and 67%, respectively, in L-NIL-treated nephritic rats as compared with untreated nephritic rats. In parallel, glomerular VEGF(165) binding was reduced by 34% in L-NIL-treated nephritic rats on day 2. Glomerular upregulation of the VEGF(164) co-receptor neuropilin-1 mRNA in nephritic rats was reduced by L-NIL treatment only on day 7. Healthy untreated or L-NIL-treated controls showed no significant differences in any parameter analyzed. In conclusion, impaired repair of glomerular endothelium and downregulation of glomerular VEGF receptor expression was observed after selective iNOS inhibition in experimental GN. These data identify iNOS-derived NO production as the first in vivo regulator of the glomerular VEGF system and as an important mechanism promoting glomerular healing.

Nitric oxide synthase isoform expression in acute versus chronic anti-Thy 1 nephritis.

BACKGROUND: Two inbred Lewis rat substrains (LEW/Moe, LEW/Maa) were identified responding differently to induction of anti-Thy 1 glomerulonephritis (aThy 1-GN). LEW/Moe rats show an acute mesangioproliferative glomerulonephritis with rapid healing of glomerular lesions within four weeks, while LEW/Maa rats develop severe glomerular injury followed by chronic glomerular sclerosis and persistent albuminuria. We investigated whether the glomerular expression pattern of nitric oxide synthase (NOS) isoforms could explain these substrain-related differences. METHODS: Rats (N = 5 to 7 per group) were investigated in a time course experiment. Severity of aThy 1-GN was determined by albuminuria measurements, glomerular matrix score and microaneurysm formation. Glomerular gene expression of NOS isoforms was determined by semiquantitative RT-PCR. Inducible NOS (iNOS) activity was determined in cultured glomeruli and peritoneal macrophages. Neuronal NOS (nNOS) protein expression was detected by Western blotting and enzyme histochemistry. Plasma renin activity (PRA) was measured by RIA. RESULTS: Induction of iNOS expression and activity was found significantly increased and sustained in LEW/Maa vs. LEW/Moe rats associated with an increased number of infiltrating macrophages and with an increased capacity of iNOS-expression and iNOS-activation by isolated macrophages in LEW/Maa rats. Glomerular nNOS mRNA and nNOS protein expression were constitutively increased in LEW/Maa rats. Renal nNOS localization was restricted to the macula densa region in both substrains and associated with increased PRA in LEW/Maa rats. No difference in glomerular endothelial NOS-mRNA expression between the substrains was observed. CONCLUSIONS: Increased glomerular iNOS and nNOS expression were associated with chronic anti-Thy 1 glomerulonephritis in LEW/Maa rats and may contribute to glomerular damage by separate mechanisms.