## 4 Discussion  
Keeping ex vivo podocytes alive will help to clarify the pathophysiological mechanisms involved in glomerular diseases to design new strategies for diagnosis and treatment. Because of this, the development of biocompatible materials that simulate the environmental conditions of specific cells could be useful in future studies. Our results indicate that swelling percentages of scaffolds CTS and PVA are of the order of 600%, decreasing with the increasing crosslinking type IV collagen and integrin \(\alpha_{3}\beta_{1}\). These results coincide with those of Caruapoma and Santiago [18] who report that scaffolds of CTS, PVA, and CTS/PVA present swelling percentages of the order of 600%, although they used 10 kilograys (kGy) of gamma radiation to obtain the scaffolds. [PERSON] et al. [13] reported a decrease in the degradation percentage of scaffold CTS/PVA by increasing of glutaraldehyde (GA) as a crosslinker agent. These results and ours indicate that cross-linking of the polymer chains maintains the stability of the supports and reduce degradation; this is confirmed by the results obtained from FTIR, indicating polymeric crosslinking of type IV collagen and integrin \(\alpha_{3}\beta_{1}\) through the group O=C-N of Chitosan. These findings coincide with the results reported by [PERSON] et al., where GA has higher affinity for amino groups (NH\({}_{2}\)) of Chitosan in comparison to hydroxyl groups (OH) of PVA. In patients with certain glomerular diseases, podocytes in urine have been detected, correlating with the loss of kidney function. Therefore, we obtained a higher number of podocytes in ISN patients (6000 cells) than HS (2000 cells). These results are consistent with those published by [PERSON] et al. [19], reporting the presence of viable podocytes in a urine a model of passive Heymann nephritis induced in rats (2500 cells), in contrast to controls where no presence of viable podocytes was observed. Our results are also consistent with those of [PERSON] et al. [20] that detected viable podocytes (0.8-5.3 cells/mL) in the urine of 50 diabetic patients and an absence of viable podocytes in the 10 HS. Evaluating the scaffold in culture, it was observed by IF that a higher number of podocytes adhered to the scaffolds (HS = 2, ISN = 3.5 and mouse = 2), in contrast to the control culture (HS = 1.75, ISN = 2.5 and 1.5 = mouse). These results agree with those of [PERSON] et al. [12] who reported greater adherence of fibroblasts taken from skin (5 cells per field) on a support of CTS/PVA and presence of  
Figure 12: Podocin and Podocalyxin expression in podocytes from urine of patients with INS. (**A**–D) Immunofluorescenterence of Podocalyxin used as marker of podocytes. (**C**–**F**) Merge of DAPI with Podocin and Podocalyxin in cultured podocyte (magnification \(40\times\)). microvilli, in contrast to a support of PVA where fewer cells adhered (3 cells per field) and without presence of microvilli. Expression of podocalyxin in podocytes is observed homogeneously both on the scaffold (HS = 10.3, INS = 10.3 and mouse = 11.4) and control cultures (HS = 9.4, INS = 9.4 and mouse = 9.4) however the expression of CD80 is increased in podocytes grown on the scaffold (HS = 7, INS = 7.3 and mouse = 7.4) in contrast to the control culture (HS = 3.9, INS = 3.9 and mouse = 4.5). There are reports that have observed that biomaterials promote increase in the expression of costtimulatory molecules (e.g., Ex. CD80, CD86, CD40) of primary culture cells [21]. Recent data also indicate that podocyte cells, under certain circumstances, can be induced to express CD80 (B7.1) [22]. So, we believe that CTS provides an active signaling pathway that leads to the expression of this receptor in podocytes. Cytotoxicity tests using cell cultures are the first step to determine the safety of biomaterials and identifying active compounds. Our results of cell viability show a greater LDH release in culture control in contrast with scaffold culture podocytes. These results agree those of [PERSON] et al.