Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/ß-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection.

BACKGROUND: We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/ß-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2. AIM: To determine the effects of osmolarity/tonicity changes, Wnt/ß-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells. METHODS: Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. ß-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS). RESULTS: Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/ß-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, ß-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability. CONCLUSIONS: Lcn2-R upregulation and Lcn2 downregulation via Wnt/ß-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.

Renal Contrast-Enhanced Sonography Findings in a Model of Acute Cellular Allograft Rejection.

Noninvasive methods to diagnose and differentiate acute cellular rejection from acute tubular necrosis or acute calcineurin inhibitor toxicity are still missing. Because T lymphocytes play a decisive role in early states of rejection, we investigated the suitability and feasibility of antibody-mediated contrast-enhanced ultrasound by using microbubbles targeted to CD3(+) , CD4(+) , or CD8(+) T cells in different models of renal disease. In an established rat renal transplantation model, CD3-mediated ultrasound allows the detection of acute rejection as early as on postoperative day 2. Ultrasound signal intensities increased with the severity of inflammation. Further, an early response to therapy could be monitored by using contrast-enhanced sonography. Notably, acute tubular necrosis occurring after ischemia-reperfusion injury as well as acute calcineurin inhibitor toxicity could easily be differentiated. Finally, the quantified ultrasound signal correlated significantly with the number of infiltrating T cells obtained by histology and with CD3 mRNA levels, as well as with chemokine CXCL9, CXCL11, and CCL19 mRNA but not with KIM-1 mRNA expression, thereby representing the severity of graft inflammation but not the degree of kidney injury. In summary, we demonstrate that antibody-mediated contrast-enhanced ultrasound targeting T lymphocytes could be a promising tool for an easy and reproducible assessment of acute rejection after renal transplantation.

Calmodulin-associated post-translational regulation of rat organic cation transporter 2 in the kidney is gender dependent.

In this work, regulation of organic cation transporter type 2 from rat (rOCT2) stably transfected in HEK293 cells was investigated by microfluorimetry with 4-(4-(dimethylamino)styryl)-N-methylpyridinium as substrate. The transport mediated by rOCT2 was specifically stimulated by PKA, phosphatidylinositol-3-kinase, p56(lck) tyrosine kinase, mitogen-extracellular-signal-regulated-kinase-1/2, calmodulin (CaM), and CaM-kinase-II. The regulatory pattern of rOCT2 differs markedly quantitatively and qualitatively from that of other OCT isoforms. Only CaM-dependent upregulation is conserved throughout the OCT family. For this reason, CaM regulation of rOCT2 was also investigated in isolated S3-segments (known to express only rOCT2) of male and female rat proximal tubules. Inhibition of CaM by calmidazolium significantly decreased rOCT2 activity (-49.0 +/- 13.6%, n = 4) in male but not female (9.0 +/- 13.0%, n = 4) rats. Real-time PCR and Western blot investigations of CaM expression in rat kidneys showed that male animals have significantly higher CaM expression. This is the first study describing post-translational gender-dependent rOCT2 regulation.

MATE1 regulates cellular uptake and sensitivity to imatinib in CML patients.

Although imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), 25-30% patients do not respond or relapse after initial response. Imatinib uptake into targeted cells is crucial for its molecular response and clinical effectiveness. The organic cation transporter 1 (OCT1) has been proposed to be responsible for this process, but its relevance has been discussed controversially in recent times. Here we found that the multidrug and toxin extrusion protein 1 (MATE1) transports imatinib with a manifold higher affinity. MATE1 mainly mediates the cellular uptake of imatinib into targeted cells and thereby controls the intracellular effectiveness of imatinib. Importantly, MATE1 but not OCT1 expression is reduced in total bone marrow cells of imatinib-non-responding CML patients compared with imatinib-responding patients, indicating that MATE1 but not OCT1 determines the therapeutic success of imatinib. We thus propose that imatinib non-responders could be identified early before starting therapy by measuring MATE1 expression levels.

Ht31: the first protein kinase A anchoring protein to integrate protein kinase A and Rho signaling.

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin-mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor proto-lymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24-q25). It encodes Ht31 and the smaller splice variants Brx and proto-Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti-Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin-mediated water reabsorbtion.