Assessment of In Vivo Kidney Cell Death: Acute Kidney Injury.

The kidney has been studied as an organ to investigate cell death in vivo for a number of reasons. The unique vasculature that does not contain collateral vessels favors the kidney over other organs for the investigation of ischemia-reperfusion injury. Unilateral uretic obstruction has become the most prominently studied model for fibrosis with impact far beyond postrenal kidney injury. In addition, the tubular elimination mechanisms render the kidney susceptible to toxicity models, such as cisplatin-induced acute kidney injury. During trauma of skeletal muscles, myoglobulin deposition causes tubular cell death in the model of rhabdomyolysis-induced acute kidney injury. Here, we introduce these clinically relevant in vivo models of acute kidney injury (AKI) and critically review the protocols we use to effectively induce them.

Assessment of In Vivo Kidney Cell Death: Glomerular Injury.

The glomerulus functions as the filtration unit of the kidney. The mesangial, endothelial, and podocyte cells of the glomerulus exhibit the three clinically most important cell types, which are involved in diverse pathologic processes. Cell death has hardly been investigated in these cells but may be of critical importance to the pathogenesis of nephrotic syndrome, nephritic syndrome, focal segmental glomerulosclerosis (FSGS), mesangial proliferation, and thrombonic microangiopathy (which involves dysfunction and death of glomerular endothelial cells). The complexity of the glomerulus is frequently affected in autoimmune disorders, which may elicit cell death in mesangial cells and glomerular endothelia. Artificial antisera are used to induce anti-mesangial cell serum-induced mesangiolysis and selective endothelial cell injury, respectively. Genetic variations result in loss of function of podocytes and nephrotic syndrome, which may encompass similar cell death mechanisms as the ones that are observed in the model of secondary focal segmental glomerulosclerosis (FSGS). The following protocols describe our current arsenal to target glomerular cells in vivo.

Progenitor Renin Lineage Cells are not involved in the regeneration of glomerular endothelial cells during experimental renal thrombotic microangiopathy.

Endothelial cells (EC) frequently undergo primary or secondary injury during kidney disease such as thrombotic microangiopathy or glomerulonephritis. Renin Lineage Cells (RLCs) serve as a progenitor cell niche after glomerular damage in the adult kidney. However, it is not clear whether RLCs also contribute to endothelial replenishment in the glomerulus following endothelial injury. Therefore, we investigated the role of RLCs as a potential progenitor niche for glomerular endothelial regeneration. We used an inducible tet-on triple-transgenic reporter strain mRen-rtTAm2/LC1/LacZ to pulse-label the renin-producing RLCs in adult mice. Unilateral kidney EC damage (EC model) was induced by renal artery perfusion with concanavalin/anti-concanavalin. In this model glomerular EC injury and depletion developed within 1 day while regeneration occurred after 7 days. LacZ-labelled RLCs were restricted to the juxtaglomerular compartment of the afferent arterioles at baseline conditions. In contrast, during the regenerative phase of the EC model (day 7) a subset of LacZ-tagged RLCs migrated to the glomerular tuft. Intraglomerular RLCs did not express renin anymore and did not stain for glomerular endothelial or podocyte cell markers, but for the mesangial cell markers α8-integrin and PDGFRß. Accordingly, we found pronounced mesangial cell damage parallel to the endothelial injury induced by the EC model. These results demonstrated that in our EC model RLCs are not involved in endothelial regeneration. Rather, recruitment of RLCs seems to be specific for the repair of the concomitantly damaged mesangium.

Interference with Gsα-Coupled Receptor Signaling in Renin-Producing Cells Leads to Renal Endothelial Damage.

Intracellular cAMP, the production of which is catalyzed by the α-subunit of the stimulatory G protein (Gsα), controls renin synthesis and release by juxtaglomerular (JG) cells of the kidney, but may also have relevance for the physiologic integrity of the kidney. To investigate this possibility, we generated mice with inducible knockout of Gsα in JG cells and monitored them for 6 months after induction at 6 weeks of age. The knockout mapped exclusively to the JG cells of the Gsα-deficient animals. Progressive albuminuria occurred in Gsα-deficient mice. Compared with controls expressing wild-type Gsα alleles, the Gsα-deficient mice had enlarged glomeruli with mesangial expansion, injury, and FSGS at study end. Ultrastructurally, the glomerular filtration barrier of the Gsα-deficient animals featured endothelial gaps, thickened basement membrane, and fibrin-like intraluminal deposits, which are classic signs of thrombotic microangiopathy. Additionally, we found endothelial damage in peritubular capillaries and vasa recta. Because deficiency of vascular endothelial growth factor (VEGF) results in thrombotic microangiopathy, we addressed the possibility that Gsα knockout may result in impaired VEGF production. We detected VEGF expression in JG cells of control mice, and cAMP agonists regulated VEGF expression in cultured renin-producing cells. Our data demonstrate that Gsα deficiency in JG cells of adult mice results in kidney injury, and suggest that JG cells are critically involved in the maintenance and protection of the renal microvascular endothelium.

Persistent and inducible neogenesis repopulates progenitor renin lineage cells in the kidney.

Renin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow-derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis (de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury.

Effect of antimicrobial peptides from Apis mellifera hemolymph and its optimized version Api88 on biological activities of human monocytes and mast cells.

Apidaecin peptides are produced by the honeybee Apis mellifera as a major part of its non-specific defense system against infections. Having verified that the peptides apidaecin 1b and Api88-a designer peptide based on the native apidaecin 1b sequence-are highly active against Gram-negative bacteria, we studied their ability to modulate biological activities of human monocytes and mast cells (MC), two important cell types of the human innate immune system. We could show that both peptides are nontoxic and fairly resistant to degradation in cell culture medium containing 10% FBS. Among the peptides tested we found Api88 to inhibit LPS-induced TNF-α production in a concentration-dependent manner. Resting monocytes did not respond to Api88. Whilst Api88 neither induced migration nor affected the phagocytic activity of monocytes it partially inhibited the generation of reactive oxygen intermediates produced in response to LPS. In human MC, however, Api88 triggered degranulation and the mobilization of intracellular Ca(2+)-ions. Taken together these data clearly indicate that Api88 is a multifunctional molecule that can modulate biological responses of human monocytes and MC in addition to its antimicrobial activity.