Hypercalcemia after transplant nephrectomy in a hemodialysis patient: a case report

<p>Abstract</p> <p>Introduction</p> <p>Hypercalcemia is a complication often seen in chronic hemodialysis patients. A rare cause of this condition is sarcoidosis. Its highly variable clinical presentation is challenging. Especially in patients suffering chronic kidney graft failure the nonspecific constitutional symptoms of sarcoidosis like fever, weight loss, arthralgia and fatigue may be easily misleading.</p> <p>Case presentation</p> <p>A 51 year old male developed hypercalcemia, arthralgia and B-symptoms after explantation of his kidney graft because of suspected acute rejection. The removed kidney showed vasculopathy and tubulointerstitial nephritis, which had not been overt in the biopsy taken half a year earlier. Despite explantation and withdrawal of the immunosuppression the patient's general condition deteriorated progressively. A rapid rise in serum calcium finally provoked us to check for sarcoidosis. CT scans of the lungs, broncho-alveolar-lavage and further lab tests confirmed the diagnosis.</p> <p>Conclusion</p> <p>This case demonstrates that withdrawal of immunosuppressive drugs sometimes unmasks sarcoidosis. It should be considered as differential diagnosis even in hemodialysis patients, in whom other reasons for hypercalcemia are much more common.</p

Klonierung und Sequenzierung des Ssp (S. saprophyticus surface-associated protein\textit {S. saprophyticus surface-associated protein}) und Identifizierung des Proteins als eine oberflächenassoziierte Lipase

Das $\textit {Staphylococcus saprophyticus surface-associated protein}$ (Ssp) ist ein fibrilläres, leicht ablösbares Oberflächenprotein, das von klinischen $\textit {S. saprophyticus}$-Isolaten produziert wird. Im Rahmen dieser Arbeit wurde es kloniert und als eine Lipase identifiziert. Es wurde eine Phagen-Genbibliothek von $\textit {S. saprophyticus}$ mittels Plaque-Hybridisierung und mittels eines Antikörper-Screening durchsucht. Diese Klonierungsansätze blieben erfolglos. Das Protein wurde anschließend einem proteolytischen Partialverdau unterzogen und das dabei erhaltene Fragment N terminal sequenziert. Mit zwei degenerierten Primern konnte eine PCR durchgeführt und die dabei erhaltene Teilsequenz mittels weiterer Versuche vervollständigt werden. Die aus einem Sequenzvergleich theoretisch hergeleitete Funktion des Proteins wurde experimentell belegt. Dabei wurde das $\it ssp$-Gen in einen Lipase-negativen Staphylokokken- sowie $\textit {E. coli}$-Stamm transformiert und diesen dadurch eine lipolytische Aktivität verliehen

The Surface-Associated Protein of Staphylococcus saprophyticus Is a Lipase

Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus

Effect of recipient-donor sex and weight mismatch on graft survival after deceased donor renal transplantation.

This study evaluated the combined effect of recipient-to-donor weight and sex mismatch after deceased-donor renal transplantation in a German transplant cohort and the evolution of recipient-to-donor weight difference over a 13-year observation period. The association of absolute weight and sex difference with graft failure was explored in an outpatient cohort of deceased-donor transplant recipients who underwent kidney transplantation between 2000 and 2012. Graft failure was defined as repeated need for dialysis or death with a functioning graft. Recipient and donor sex pairings were classified as sex concordant (MDMR/FDFR) or discordant (MDFR/FDMR). These classes were further stratified into four groups according to recipient-to-donor weight mismatch ≥10 kg (recipient > donor) or <10 kg (recipient < donor). Multivariable Cox proportional hazards models were applied to evaluate the time to graft loss adjusting for donor, immunologic, surgical, organizational, and recipient predictors. Sex-concordant transplant pairings <10 kg weight difference served as the reference group. Among 826 transplant recipients, 154 developed graft failure (18.6%). Median graft survival time was 3.9 years; first quartile (0.2-1.2), second quartile (1.2-2.9), third quartile (2.9-5.8), and fourth quartile (5.8-12.4). After multivariable adjustment, the highest relative hazard for graft failure was observed for sex-discordant transplant pairings with a ≥10 kg weight difference between recipient and donor (compared to the reference group MDMR/FDFR with weight difference <10 kg, MDMR/FDFR with weight difference ≥10 kg, hazard ratio 1.86, 95% confidence interval 1.07-3.32-p = 0.029; MDFR/FDMR with weight difference <10 kg, hazard ratio 1.14, 95% confidence interval 0.78-1.68-p = 0.507, and MDFR/FDMR with weight difference ≥10 kg, hazard ratio 2.00, 95% confidence interval 1.15-3.48-p = 0.014). A recipient-to-donor weight mismatch of ≥10 kg was associated with an increased risk of graft loss or recipient death with a functioning graft. Concurrent sex discordance seemed to enhance this effect as indicated by an increase in the hazard ratio. We detected no significant tendency for increasing recipient-to-donor weight differences from 2000 to 2012

A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease.

Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis

AT1 receptors in the collecting duct directly modulate the concentration of urine

10.1681/ASN.2010101095Journal of the American Society of Nephrology22122237-2246JASN

Nephrin surface abundance in adriamycin nephropathy at day 7.

<p><b>(A)</b> Coomassie gel shows albuminuria in ADR mice at day 7 (ADR 7d), which was not detected in healthy mice (control). <b>(B)</b> Quantitative analysis of the albumin to creatinine ratio in healthy mice (control) versus ADR mice on days 1 and 7 (ADR1 and ADR 7). Statistical analysis: <i>t</i>-test with Welch’s correction was performed on day 1 and 7. **** p < 0.0001 (non-significant differences (ns); control: n = 9; ADR: n = 12). <b>(C, D)</b> Immunofluorescence staining of glomeruli from healthy mice (control) or ADR mice (ADR 7 d). Staining was performed with an anti-nephrin antibody (red), and nuclei were stained with DAPI (blue). White arrows indicate colocalization of nephrin with EEA1 positive vesicles. <b>(E)</b> Western blot analysis of surface nephrin (streptavidin) and total nephrin immunoprecipitates and lysates (WB nephrin). In comparison to healthy mice (control), less surface nephrin (WB: streptavidin) was detected in ADR mice at day 7 (ADR 7d). Total nephrin immunoprecipitates and lysates (WB: nephrin) showed less expression of total nephrin at day 7. β-actin (WB: actin) was used as loading control. <b>(F, G)</b> Densitometric analysis of western blots: <b>(F)</b> Amount of cell surface nephrin graphed as biotinylated nephrin to total nephrin ratio; <b>(G)</b> Amount of total nephrin graphed as total nephrin to β-actin ratio. <b>(H)</b> Immunohistochemistry: staining of p57 indicates podocytes in healthy mice (control) and NTN mice (NTN d1). <b>(I)</b> Number of p57 positive cells per glomerular tuft <b>(J)</b> Glomerular tuft area (μm²) <b>(K)</b> Ratio of p57 positive cells in relation to tuft area (control n = 2, ADR n = 3, 40 gloms per mouse quantified). Western blot data show means ± SD. Podocyte counts show means ± SEM. Statistical analysis: unpaired <i>t</i>-test with Welch’s correction. Significance level was set to 5%, *** p < 0.0001, **** p<0.00001, (non-significant differences (ns)).</p

Albuminuria and nephrin surface abundance in early nephrotoxic nephritis.

<p><b>(A)</b> Coomassie gel shows albuminuria in mice injected with NTN serum (NTN 1d). No albuminuria was detected in mice injected with normal saline (control). <b>(B)</b> Quantitative evaluation of albumin-creatinine excretion in healthy mice (control) and NTN mice (NTN 1d). Statistical analysis: Mann-Whitney U test, ** p < 0.01 (control: n = 4; NTN: n = 6). <b>(C, D)</b> Immunofluorescence staining of murine kidney sections of healthy mice (control) and mice injected with NTN serum (NTN 1d). Staining was performed with an anti-nephrin antibody (red), an anti-EEA1-antibody (green) and nuclear DNA with Draq5 (blue). White arrows indicate colocalization of nephrin with EEA1 positive vesicles. <b>(E)</b> Representative western blot of total and biotinylated nephrin detected with streptavidin (streptavidin). In comparison to untreated mice (control) less surface nephrin (WB: streptavidin) was detected in NTN mice (NTN 1 d). Total nephrin immunoprecipitates and lysates (WB: nephrin) showed equal expression of total nephrin in both groups. β-actin (WB: actin) was used as loading control. <b>(F, G)</b> Densitometric analysis of western blots: <b>(F)</b> Amount of nephrin at the cell surface graphed as biotinylated nephrin to total nephrin ratio; <b>(G)</b> Amount of total nephrin graphed as total nephrin to β-actin ratio. <b>(H)</b> Immunohistochemistry: staining of p57 indicates podocytes in healthy mice (control) and NTN mice (NTN d1). <b>(I)</b> Number of p57 positive cells per glomerular tuft <b>(J)</b> Glomerular tuft area (μm²) <b>(K)</b> Percentage of p57 positive cells in relation to tuft area. (40 gloms per mouse quantified). Western Blot data show means ± SD. Podocyte counts show means ± SEM. Statistical analysis: unpaired <i>t</i>-test with Welch’s correction. Significance level was set to 5%, *p < 0.01, ** p<0.001, (non-significant differences (ns)).</p